Yeast Genetics Meeting August 22-26, 2018 Stanford University

Full text of Abstracts

Lightning Talks Remembering the Stressful Past: Elucidating Mutation-Independent Mechanisms of Drug Resistance. J. Xie, D. Garcia, C. Gill, D. Jarosz Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA. By poisoning rapidly dividing cancer cells, chemotherapy has been an effective means of extending the lifespans of many cancer patients. However, the prevalence of drug resistance in tumors – and collateral damage to healthy tissues – have been major road blocks to improving the efficacy of this treatment modality. Thus, understanding the mechanisms of drug resistance could shift the balance in this life and death struggle for survival in favor of healthy cells. Although much research has focused on identifying mutations that confer cancer drug resistance, epigenetic heterogeneity could be a hidden force promoting drug resistance. Previous studies have shown that both chromatin- and protein-based epigenetic states can be induced in response to stress. These heritable ‘molecular memories’ can confer a fitness advantage during future exposures to stress. We tested >300 isogenic yeast isolates against a panel of 54 stresses, including multiple chemotherapeutics. Many of these conditions induced adaptive epigenetic states at a very high frequency, orders of magnitude more frequent than adaptive mutations. In addition, they exhibited a non-Mendelian pattern of inheritance characteristic of yeast prions. One such epigenetic state was robustly induced by transient exposure to carmustine, an alkylating agent commonly used to treat recurrent glioblastoma. To identify the genes involved in the adaptive response to carmustine, we screened the non-essential yeast knockout collection and the DAmP collection of essential genes. We found that the molecular memory of carmustine is mediated by a complex network of genes involved in DNA replication, chromatin modification and metabolism. Further characterization of this epigenetic-based mechanism of drug resistance will have important implications for the design and delivery of chemotherapeutics and our fundamental understanding of adaptation.

Lightning Talks Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1. W.-L. Lee, S. Omer Department of Biological Sciences, Dartmouth College, Hanover, NH. Cortical dynein generates pulling forces via microtubule (MT) end capture/shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. Using live cell imaging technique, we show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture/shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit, but not the MT plus end depolymerase Kip3 or Kar3. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

Lightning Talks The trafficking and tracking of Gbg during the pheromone response in budding yeast. R. Abdul- Ganiyu, L. Venegas Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL. Directed cell growth in response to a chemical gradient (chemotropism) is necessary for cellular processes like axonal pathfinding, angiogenesis and yeast mating. Yeast mating is a well-studied chemotropic model. In mating mixtures, yeast cells interpret shallow pheromone gradients from cells of opposite mating type, polarize their growth toward the closest potential mating partner, and fuse to form zygotes. The G protein bg subunit (Gbg) is a key component of the mating pathway. Phosphorylated Gb (GbP) inhibits receptor phosphorylation and its subsequent internalization. This promotes receptor polarization on the up-gradient side of the cell, which contributes to gradient sensing. Our computational model predicts that with internalization of ≥3% GbP, pheromone-gradient stimulation does not induce receptor polarity. Therefore, we hypothesize that in response to pheromone, GbP is preferentially left on the plasma membrane (PM) while unphosphorylated Gb (GbP-) is internalized. Consistent with our hypothesis, in a gel-shift assay, we found that the PM of pheromone-treated cells mostly contained GbP whereas the untreated cells contained GbP and unphosphorylated Gb (GbP-) in a roughly equal proportion. To better understand the dynamics of GbP trafficking, we are developing an in-vivo GbP sensor. We screened a phage-display library of Forkhead Associated (FHA) Domain variants against a GbP peptide using ELISA as a readout. We identified four variants with high affinity to GbP peptide. We fused GFP to the highest binding FHA variant, to test its utility as an in vivo sensor. We found that this fusion protein (FHA3-GFP) localizes similarly to GFP-Gb in pheromone-treated cells. Interestingly, the FHA-GFP signal at the shmoo tip is more focused, consistent with the expected localization of GbP. Pheromone treatment of cells overexpressing Gb led to an increase in the FHA3-GFP PM signal. In mating cells, FHA3-GFP 1 redistributes from the default site to the eventual chemotropic site. These results are consistent with FHA3-GFP working as a GbP cell sensor. However, we also saw an FHA3-GFP signal in cells lacking Gb suggesting that FHA-GFP cross-reacts with other protein(s). To eliminate the possibility that our FHA reagent is binding to a protein that has similar localization as GbP, we performed a BiFC assay. Our results confirm that the FHA reagent binds to GbP and can be used as an in-vivo sensor to monitor the trafficking of GbP during yeast chemotropism.

Lightning Talks Evolutionarily conserved pathways prevent mislocalization of CENP-A for chromosome stability in yeast and human cells. Munira Basrai1, Lars Boeckmann1, Sultan Ciftci-Yilmaz1, Jessica Eisenstat1, Prashant Mishra1, Kentaro Ohkuni1, Austin Rossi1, Roshan Shrestha1, Tianyi Zhang1, Mahfuzur Rahman6, Michael Costanzo2, Anastasia Baryshnikova7, Chad Myers6, Peter Kaiser4, Dan Foltz5, Richard Baker3, Charlie Boone2 1) Dept Genetics, NCI/NIH, Bethesda, MD; 2) University of Toronto; 3) Univ. of Massachusetts Med. School ; 4) Univ. of California, Irvine ; 5) Northwestern Univ. Feinberg School of Medicine; 6) Univ. of Minnesota ; 7) Princeton Univ. Faithful chromosome segregation prevents chromosomal instability (CIN), a hallmark of aggressive tumors and other diseases. The evolutionarily conserved centromeric histone H3 variant CENP-A (Cse4 in budding yeast, Cid in flies) is essential for chromosome segregation. Overexpression of Cse4 or Cid causes its ectopic mislocalization to chromosome arms and promotes CIN in yeast and flies respectively. Overexpression and mislocalization of CENP-A have been reported in numerous cancers and is correlated with poor prognosis. To define pathways that prevent mislocalization of Cse4 we performed genome-wide synthetic dosage lethality (SDL) screens to identify mutants that exhibit growth sensitivity when Cse4 is overexpressed. Among the top hits are genes that encode the evolutionarily conserved ubiquitin ligase (SCF), replication dependent kinases (DDK) and the replication-independent histone chaperone (HIR) complexes. We determined that SCF- Met30, SCF-Cdc4, DDK and HIR proteins regulate proteolysis of Cse4 to prevent its mislocalization for chromosome stability. For studies with human cells we used a HeLa cell line stably overexpressing CENP-A and provide the first evidence to show that CIN results from mislocalization of overexpressed CENP-A in human cells. Depletion of the human homologs of HIR and SCF-Met30 show increased mislocalization of CENP-A and a CIN phenotype similar to our findings in yeast cells. In summary, we have identified evolutionarily conserved pathways that regulate proteolysis and localization of Cse4/CENP-A for genome stability in yeast and human cells. The results from the screen will offer insights into how CENP-A mislocalization contributes to aneuploidy in cancers and identify potential therapeutic targets for CENP-A overexpressing cancers.

Lightning Talks Viability in response to heat stress requires shelterin rearrangement and telomere shortening. T. Pohl, C. Webb, Y. Wu, V. Zakian Princeton University, Princeton, NJ. Telomeres are DNA-protein structures that protect the ends of linear chromosomes from degradation, end-to-end fusions, and the loss of genomic DNA that arises from incomplete replication. In all eukaryotes, telomeres consist of tandem copies of a non-coding short DNA repeat that is associated with a number of conserved proteins that together contribute to the capping or end protection functions of telomeres. In Schizosaccharomyces pombe, shelterin, the protein complex that protects telomere ends and regulates access of telomerase to telomere DNA is composed of six proteins; Taz1, Rap1, Poz1, Pot1, Ccq1, and Tpz1. Many of these proteins, especially Tpz1, are homologues of the shelterin components in humans. A large number of stresses are associated with human telomere shortening yet there is little mechanistic data to explain how this shortening occurs. We used fission yeast, whose shelterin-like telomere structure makes it a tractable model for humans to understand the mechanistic basis for reduction of telomere length in response to heat stress. A variety of genetic and molecular techniques were employed to show that S. pombe telomeres shorten by as much as 50% in response to heat stress. The extent of shortening increased with higher temperatures and had no negative effect on doubling time. Even the most extensive shortening was rapidly reversed by return to lower temperatures. Shortening was accompanied by reduced levels of two telomerase subunits, Est1 and Trt1 as well as in reduced abundance and telomere binding of Ccq1 and Tpz1, the ortholog of human TPP1, which is critical for telomerase recruitment and activity. All of these reductions occurred post-transcriptionally. Inability to reduce telomere bound Tpz1 correlated with telomere loss and cell death at high but not low temperatures. We propose that the ability to shorten telomeres by altering the stoichiometry of telomerase and shelterin complexes is an adaptation that makes these hard-to-replicate regions easier to maintain during environmental stress.

Lightning Talks Cdc48 regulates spliceosome assembly and nuclear protein sequestration during genotoxic stress. V. Mathew, K.Y. Jiang, A.S. Tam, P.C. Stirling Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, CA. Cdc48/VCP is a highly conserved ATPase chaperone that plays an essential role in the assembly or disassembly of protein- DNA complexes, in degradation of misfolded proteins, largely through endoplasmic reticulum-associated degradation. We find that Cdc48 relocalizes during cellular stress to intranuclear protein quality control (INQ) sites. Cdc48 function is required to suppress INQ formation under non-stress conditions. We previously linked the sequestration of a splicing factor, Hsh155 to INQ sites and here we find that Cdc48 physically associates with Hsh155 by co-immunoprecipitation and regulates the assembly of Hsh155 with its partner spliceosome proteins. Cdc48 mutants exhibit splicing defects and show diminished recovery from genotoxic stress treatments. Overall, this study links Cdc48 to the spliceosome assembly/disassembly for the first time, and describes a new role for Cdc48 in nuclear protein quality control aggregate regulation.

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Lightning Talks A toolkit for barcode-based combinatorial screening in yeast. X. Liu1,2, Z. Liu1,2, A. Dziulko1,2, D. Francois1,2, R. Morabito1,2, S. Levy2,3,4,5 1) Department of Biochemistry and Cell Biology, Stony Brook University, New York, NY; 2) Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY; 3) Joint Initiative for Metrology in Biology, Stanford, CA; 4) National Institute of Standards and Technology, Gaithersburg, MD; 5) Department of Genetics, Stanford University, Stanford, CA. High-throughput combinatorial screens have become an important tool with a wide variety of applications in basic and applied research. For example, they are commonly used to discover protein-protein and genetic interaction networks, and in protein design and engineering. Early screens, such as the yeast two-hybrid and synthetic genetic array technologies, require that combinations be generated and assayed one-at-a-time, limiting throughput and reproducibility. More recently, amplicon- sequencing-based screens have been developed, greatly improving throughput. For example, CRISPR screens introduce gRNA combinations into cells en masse and use pooled sequencing of gRNAs or associated barcodes as a readout. However, such screens have generally been limited to assaying combinations of small genetic elements that can fit on a single plasmid. Here, we developed a general combinatorial screening platform that uses tandem genomic integration of barcoded plasmids, pooled competitive growth, and double barcode sequencing to quantitatively assay genetic construct pairs. Importantly, genetic constructs can be of any size, from gRNAs to genomes, and, once barcoded, can be easily reused and mixed to form novel pairs. Barcode pairs can be generated either through sequential plasmid insertion or yeast mating, both of which reassemble a split selectable marker and can generate yeast pools containing >107 pairs. To expand the possible applications of this platform, we designed a set of split drug resistance markers (KanMX AI HygMX AI NatMXAI) that allow combinatoric screens to be performed in prototrophic yeast with various genetic backgrounds. We also generated a set of 15 plasmid libraries each containing >100,000 barcodes and two large barcoded yeast strains collections (~12,000 of both MATa and MATα) that can be used for REcombinase Directed Indexing of newly barcoded yeast strains. As a proof-of-principle, we generated a library of ~3000 double barcoded haploid and diploid yeast strains containing different pairs of auxotrophic rescue constructs (MET15, LEU2, HIS3, TRP1, LYS2), and competed cell pools in 12 different environments. We found that the plasmid size has a minimal impact on representation of a construct in our screen and fitness estimation is highly reproducible across barcode replicates and growth replicates. Additionally, this screen revealed new interactions between auxotrophic markers that likely stem from cross-talk in amino acid metabolic pathways. Current efforts are focused on standardizing barcode sequencing and improving the convenience of plasmid library construction by making barcoded plasmids GATEWAY-compatible.

Lightning Talks Machine Learning and Computer Vision Approaches for Phenotypic Profiling in Yeast. N. Sahin1,2, M. Mattiazzi Usaj2, C. Boone1,2, Q. Morris1,2,3, B. Andrews1,2 1) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 2) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada; 3) Department of Computer Science, University of Toronto, Toronto, ON, Canada. A powerful method to study the genotype-to-phenotype relationship is the systematic assessment of mutant phenotypes using high-content screening and automated image analysis. We have developed a combined experimental-computational pipeline for analysis of the effect of genetic perturbations on subcellular compartments in yeast. Our approach involves using Synthetic Genetic Array (SGA) analysis to introduce markers of various subcellular compartments into yeast mutant arrays to identify comprehensive lists of genes involved in the establishment and maintenance of proper subcellular morphology. Quantitative analyses of these large image datasets require computational approaches such as image recognition, feature extraction and machine learning. We describe a general single cell image analysis pipeline for the quantification of the effect of any genetic or environmental perturbation on subcellular morphology. For the identification of mutant phenotypes, the first step in the computational analysis involves mathematically learning the wild-type morphology of unperturbed cells, and subsequent classification of single cells as having either normal or abnormal morphology. Determining the percentage of cells with mutant morphology in an isogenic population thus allows us to asses the penetrance of a given genetic perturbation. We tested the performance of different outlier detection methods, both traditional machine learning as well as deep learning approaches, by comparing the detected mutant phenotypes with positive controls, and by validating the functional enrichment of high-penetrant genes. To validate the developed pipeline, we queried the S. cerevisiae genome for genes required for proper formation and maintenance of endocytic compartments. We determined the penetrance of approximately 5300 yeast mutant strains for each of the 4 screened endocytic markers. This analysis revealed that mutation of ~10% of the screened genes leads to a morphological phenotype with a penetrance of 50% or greater for at least one of the markers. Mutation of hundreds more genes, mostly connected to more distant bioprocesses, caused moderate but still significant defects in at least one of the major compartments involved in endocytosis. This type of quantitative analysis will allow for the identification of connections between biological processes, prediction of novel gene function, and generation of a clearer understanding of eukaryotic cell biology.

Lightning Talks Translation kinetics and co-translational proteostasis in health and disease. K.C. Stein, J. Frydman Department of Biology, Stanford University, Stanford, California. Maintaining proteostasis by generating functional and properly folded proteins is crucial to every cellular process. Disruption of protein quality control can lead to protein misfolding and aggregation, which are key hallmarks of aging and many human diseases. Understanding how cells synthesize and maintain a functional proteome is critical for elucidating the 3 mechanisms underlying aging and age-related diseases. At the heart of synthesizing a healthy proteome is the ribosome, centered at the interface of translation and protein quality control. The ribosome intricately coordinates mRNA translation with a network of ribosome-associated machinery, including molecular chaperones, to fold newly synthesized proteins or degrade aberrant translation products. This coupling of mRNA translation and nascent protein folding relies on precise regulation of translation kinetics. Increasing evidence indicates that the kinetics of translation elongation are an important determinant of protein fate: faster elongation can increase protein abundance, whereas slower elongation (ribosome stalling) can facilitate protein folding. Defects in translation kinetics and co-translational machinery can impair protein biogenesis and enhance the formation of protein aggregates. Yet, how translation kinetics and protein quality control machinery are coordinated to regulate co-translational proteostasis remains poorly defined.

We sought to determine the principles of translation elongation that dictate chaperone-mediated co-translational protein folding, and how aging might disrupt translation kinetics and the folding of nascent polypeptides. Using selective ribosome profiling with yeast, we have identified distinct mechanisms that influence the Hsp70 SSB and the essential chaperonin TRiC/CCT to associate with ribosome-nascent chain complexes. In addition, we have found that aging disrupts translation kinetics and overwhelms nascent polypeptide quality control to lead to the aggregation of newly synthesized proteins. By understanding how translation kinetics are regulated and dictate co-translational proteostasis, our work provides mechanistic insight into the principles that define both the maintenance and collapse of proteostasis, and the role that co-translational events have in the progression of aging and age-related protein misfolding disorders.

Lightning Talks Identification of novel genetic elements for improving acetic acid tolerance in an industrial yeast strain for second-generation bioethanol production. M. Stojiljkovic, M.R. Foulquié Moreno, J.M. Thevelein Center for Microbiology, KU Leuven - VIB, Heverlee, BE. The main requirements for conversion of biomass into second-generation bioethanol by yeast are high xylose fermentation capacity and high inhibitor tolerance. Acetic acid is among the most important inhibitors. Therefore, the goal of our work is to find novel alleles/SNPs conferring high acetic acid tolerance and introduce them in a strain for second-generation bioethanol production. In previous work, we used pooled-segregant whole-genome sequence analysis for QTL mapping and identified five genes linked to high tolerance to acetic acid (Meijnen et al. 2016). The K11 strain displays high acetic acid tolerance but contains all five inferior alleles. We transformed its genomic DNA into the inferior ER18 strain used in the previous work. Around 60 transformants with higher acetic acid tolerance were isolated. The most tolerant transformant, MS164, contained only 7 SNPs compared to its parent, and was tolerant to 0.9% acetic acid, instead of 0.6% for the parent (growth on YPD solid medium at pH 4.7). MS164 was crossed with the unrelated haploid acetic acid tolerant strain 16D and about 30 segregants isolated with high fermentation performance in liquid YPD medium with 1.2% acetic acid. They were pooled and subjected to pooled- segregant whole-genome sequence analysis for QTL mapping. This analysis revealed 4 major and 5 minor QTLs linked to either parent strain, indicating that both parents contain superior alleles for conferring acetic acid tolerance, but all QTLs were largely unlinked to the 7 SNPs. We are currently identifying the causative elements in these QTLs and testing all 7 SNPs for their effect on acetic acid tolerance. We can conclude that QTL mapping by pooled-segregant whole-genome sequence analysis is able to identify the genomic positions of novel causative genetic elements present in the superior parent strain and likely also in the inferior parent strain, irrespective of the presence of the SNPs introduced by the whole-genome transformation. Whole genome transformation itself turns out to be a powerful technique for strain improvement, although side-effects caused by spurious mutations cannot be ruled out. Once identified, the causative genetic elements from both approaches will be combined into a single industrial strain for second-generation bioethanol production to further improve acetic acid tolerance and further optimize the performance of the strain in hydrolysates with high levels of acetic acid.

Lightning Talks High-throughput phenomics for identifying concentration-dependent chemical interactions and understanding the mechanistic basis of the mixture toxicity. V. Mukherjee1,2, T. Backhaus 3, A. Blomberg2 1) Division of Industrial Biotechnology, Biology and Biological Engineering, Chalmers University of Technology, Göteborg, SE; 2) Department of Chemistry and Molecular Biology, University of Gothenburg, Lundbergslaboratoriet, Göteborg, Sweden; 3) Department of Biological and Environmental Sciences, University of Gothenburg, Göteborg, Sweden. Prevalence of mixtures of synthetic and natural chemicals in the environment is a growing concern for public health and environmental effects. Currently, most chemical legislations around the world are based on the risk assessments carried out on individual substances and theoretical estimates of combination effect. However, exposure to multi-component mixtures may stimulate unpredicted overall toxic response due to interactions in chemical mixtures, which in turn induce unpredictable adverse impacts. Therefore, it is increasingly important to implement a high-throughput experimental approach to identify and to characterize dose-responses of single and mixtures of chemicals. The final output would be to upgrade the current modeling approaches and to obtain more reliable risk assessment. In our project, we are investigating the frequency of interactions in mixtures of chemicals by employing high-throughput yeast phenomics involving high- resolution phenotyping techniques recently developed in our laboratory. Initially we are focusing on five compounds with relatively known specific mode of action. The baker’s/brewer’s yeast Saccharomyces cerevisiae and the marine 4 yeast Debaryomyces hansenii are used in this study as the model organisms to determine the single-substance and the mixtures dose-responses of the chemicals. Thus, we examine organisms at large evolutionary distance hoping to identify generic response of relevance to a vast array of organisms. Our results clearly suggest that both synergistic and antagonistic relationships exist among the tested chemicals and some of these relationships are concentration-dependent. We are also investigating the mechanistic causes of the mixture toxicity by RNA sequencing analysis and metabolomics.

Lightning Talks Eukaryotic acquisition of a bacterial operon. D.T. Doering1,3, J. Kominek1,2, D.A. Opulente1, X.-X. Shen4, X. Zhou4, J. DeVirgilio5, A.B. Hulfachor3, C.P. Kurtzman5, A. Rokas4, C.T. Hittinger1,2,3 1) Laboratory of Genetics, Genome Center of Wisconsin, Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, WI; 2) DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, WI; 3) Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI; 4) Department of Biological Sciences, Vanderbilt University, Nashville, TN; 5) Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, IL. Encompassing roughly 500 million years of evolution, the budding yeast subphylum (Saccharomycotina) displays a remarkable level of genetic, phenotypic, and metabolic diversity. Systematically exploring this diversity by whole-genome sequencing through the Y1000+ Project has led to the first-in-class discovery of a horizontal operon transfer event of a catecholate-class siderophore biosynthesis pathway. Through phylogenetic hypothesis testing, we demonstrate that the operon was transferred roughly 50 million years ago from an Enterobacteriaceae lineage into the ancestor of the Wickerhamiella/Starmerella clade, an under-studied group of yeasts that diverged from their common ancestor with Saccharomyces cerevisiae roughly 300 million years ago. Additionally, despite the fundamental differences between bacteria and eukaryotes in genome organization and Central Dogma processes, we show that all transferred and retained operon genes are actively expressed, exhibit both bacterial and eukaryotic transcriptional features, result in the production of the siderophore enterobactin, and enable the sequestration of iron from the environment. After transfer to the eukaryotic host, several genetic changes occurred, including structural rearrangements, insertion of additional eukaryotic genes, and secondary loss of the horizontally-acquired genes in some lineages. We conclude that the operon genes were transferred from the bacterial lineage into the yeast ancestor, underwent structural changes for eukaryotic gene expression, and were maintained by selection to adapt to a highly-competitive, iron-limited environment.

Lightning Talks The complex underpinnings of genetic background effects. M. Mullis, T. Matsui, R. Schell, R. Foree, I. Ehrenreich Molecular and Computational Biology, University of Southern California, Los Angeles, CA. Spontaneous and induced mutations commonly show different phenotypic effects across genetically distinct individuals. Although these background effects are known to result from epistasis between mutations and the polymorphisms already present in a population, their underlying genetic architecture remains poorly understood. Here, we genotyped 1,411 wild type and mutant segregants from the same budding yeast cross, and measured their growth in 10 environments. We then mapped genetic interactions between each of seven different gene knockouts and segregating loci. In total, we detected 1,086 interactions between a knockout and one or more loci. 95% of these interactions involved higher-order epistasis between a knockout and multiple loci. When collapsed into unique sets of epistatic loci, 65% of these interactions were found in only a single knockout background, while 35% were detected in multiple knockout backgrounds. Loci that were found in one knockout background almost exclusively exhibited larger effects in mutants than wild type individuals. In contrast, loci that were detected in multiple knockout backgrounds tended to show smaller effects in mutants than wild type individuals. Analysis of these loci across growth conditions revealed that most of the interactions between the knockouts and segregating loci were specific to a particular environment. Our results clarify the genetic complexity, types of epistasis, extent of pleiotropy, and role of the environment in background effects.

Lightning Talks Genetic dissection of an ancient divergence in yeast thermotolerance. Carly Weiss1, Jeremy Roop1,2, Rylee Hackley1,3,4, Julie Chuong3, Igor Grigoriev1,5, Adam Arkin1,6, Jeffrey Skerker1,6, Rachel Brem1,3 1) UC Berkeley, Berkeley, CA; 2) Fred Hutchinson Cancer Research Center, Seattle, WA; 3) Buck Institute for Research on Aging, Novato, CA; 4) Duke University, Durham, NC; 5) US Department of Energy Joint Genome Institute, Walnut Creek, CA; 6) Lawrence Berkeley National Laboratory, Berkeley, CA. Some of the most unique and compelling survival strategies in the natural world are fixed in isolated species. To date, molecular insight into these ancient adaptations has been limited, as classic experimental genetics has focused on interfertile individuals in populations. Here we use a new mapping approach, which screens mutants in a sterile interspecific hybrid, to identify eight housekeeping genes that underlie the growth advantage of Saccharomyces cerevisiae over its distant relative S. paradoxus at high temperature. Pro-thermotolerance alleles at these mapped loci were required for the adaptive trait in S. cerevisiae and sufficient for its partial reconstruction in S. paradoxus. The emerging picture is one in which S. cerevisiae improved the heat resistance of multiple components of the fundamental growth machinery in response to selective pressure. This study lays the groundwork for the mapping of genotype to phenotype in clades of sister species across Eukarya.

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Lightning Talks A high-throughput mutational scan of an intrinsically disordered acidic transcriptional activation domain. Max Staller1, Alex Holehouse2, Rohit Pappu2, Barak Cohen1 1) Edison Family Center for Genome Sciences and Systems Biology and Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO; 2) Center for Biological Systems Engineering and Department of Biomedical Engineering, Washington University in St. Louis, Saint Louis, MO. Transcription factors (TFs) activate gene expression by binding the genome with DNA binding domains (DBDs) and recruiting coactivators with activation domains. While DBDs are phylogenetically conserved, well structured, and bind to related DNA sequences, activation domains are poorly conserved, intrinsically disordered and bind structurally diverse coactivators. These features have made it difficult to identify the amino acid composition features that define activation domains. We have developed a method to measure the activities of thousands of transcriptional activation domains in parallel. We deployed a rational mutagenesis scheme that deconvolves the function of four activation domain sequence features — acidity, hydrophobicity, intrinsic disorder, and short linear motifs — by quantifying the activity of thousands of variants in vivo and simulating their conformational ensembles using an all-atom Monte Carlo approach. Our results with a canonical activation domain from the Saccharomyces cerevisiae transcription factor Gcn4, reconcile existing observations into a unified model of its function: the intrinsic disorder and acidic residues keep two hydrophobic motifs from driving collapse. Instead, the most active variants keep their aromatic residues exposed to the solvent.

In addition, during amino acid starvation, the specific activity of the Gcn4 activation domain increases, a phenomenon we call induction. This induction is in additional to well-established mechanisms of translational induction during amino acid starvation. Mutations that increase activity decrease induction. We propose that Gcn4 activation domain has been selected to have low intrinsic activity and high induction. Our results illustrate how the sequence-to-function relationship of TF activation domains can be revealed by high-throughput rational mutagenesis.

Lightning Talks Inferring the genetic architecture of expression variation from replicated high throughput allele- specific expression experiments. X. Zhang, J. Emerson Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA. Variation in gene expression contributes significantly to phenotypic variation. Consequently, natural selection targets not only protein-coding regions, but also gene regulatory elements. The genetic architecture of variation of gene regulation can be decomposed into cis variation and trans variation. Cis-regulatory variation affects the expression difference between two individuals in a non-diffusible manner (eg. a mutation on a promoter region), while trans variation affects the expression difference in a diffusible manner (eg. a coding region mutation on a transcription factor). The cis/trans contribution to expression differences between two individuals can be measured by allele-specific expression in the two individuals and their F1 hybrid. Previous experiments in strains of budding yeasts show that cis variation is the dominant source of expression differences. A popular explanation is that the larger deleterious effects of trans-mutations experience more purifying selection, and therefore are removed from the population.

Previous experiments suffer from two limitations 1) biological replication of gene expression measurement in parental and F1 hybrid strains is not sufficient to estimate over-dispersion, leading to upwardly biased estimates of cis/trans contribution. 2) The cis×trans interaction (eg. the binding of promoters and corresponding transcriptional factors) is not considered in previous models which implicitly assume that cis and trans act independently.

We measure the allele-specific expression in two strains of yeast (yps128: a woodland strain; rm11: a wine strain ) with 10 biological replicates using RNAseq. In order to eliminate the error caused by RNAseq mapping bias, we also assemble the two genomes de novo with long reads using a hybrid metassembly approach. We propose a new statistical model based on normalized allelic RNA-seq read counts in hybrid and parental strains instead of only the allelic expression ratio as in previous models.

We find a significant number of genes that show cis-trans interactions but are classified as lacking cis/trans variation or exhibiting only cis variation according to previous models, indicating that cis-trans epistatic interactions are common. This raises the possibility that hybrid incompatibility is nontrivial even between closely related strains. With a high number of biological replicates, we also explore the model's power and false discovery rate.

1 Regulating telomerase: mechanisms that limit its abundance. V. Zakian, P. Daniela Garcia . We used mass spectrometry (MS) to identify proteins that associate with the catalytic core of telomerase, which consists of three proteins (Est1, Est2, Est3) and TLC1, the telomerase RNA, which is the template for extending the G-rich strand of the telomere. Est2 is the catalytic subunit of telomerase. While Est1 has a recruitment function, its indispensable action for telomere lengthening is telomerase activation. In addition to the three Est proteins and seven Sm proteins, there were ~70 proteins that co-purified with telomerase by MS. About a third of these proteins were implicated by genetic studies as affecting telomere length. Previously unidentified telomere proteins include Pop1, Pop6 and Pop7, which are subunits of two multi-protein-RNA complexes, RNase P (processes tRNAs) and RNase MRP (processes rRNA). The association of the Pop proteins with telomerase was highly significant, DNase-resistant, and present throughout the cell cycle (Lin et al. Nature 6

Comm., 2015). The Wellinger lab showed that the three Pop proteins interact directly with a portion of TLC1 RNA that is near the Est1 binding site (Lemieux et al. Cell, 2016). To determine the effects of limiting cells for Pop proteins, we used temperature sensitive alleles of POP1, POP6 and POP7. Mutant phenotypes were assessed at permissive temperatures (24°C), where growth rates and protein levels, as well as Est1 and Est2 abundance, were similar in mutant and WT cells. Telomeres were about a third shorter that WT telomeres in the three mutants yet, surprisingly TLC1 RNA was more abundant. Both phenotypes were exacerbated at semi-permissive temperature (30°C). Although TLC1 RNA was more abundant in pop mutants, its ability to assembly into a holoenzyme and the binding of the holoenzyme to telomeres were highly impaired, defects that can explain the short telomere phenotype but not the elevated abundance of TLC1. The defect in TLC1 RNA was not due to a defect in its nuclear localization, hyper-methylation of its 5’ cap, or removal of it poly A tail. Using DMS-MaPseq (developed by S. Rouskin), we find that the secondary structure of TLC1 was altered in pop mutants. These data suggest that Pop proteins act as chaperones to ensure the proper folding of TLC1 RNA. Paradoxically, Pop protein-mediated folding seems to promote TLC1 degradation as TLC1 was more abundant in mutant cells. Thus, our work discovered a heretofore unknown connection between telomerase and proteins in two well-known and highly conserved RNase complexes.

2 Aggregation and analysis of the yeast knock-out phenome. Brianna Richardson1, Christie Chang2, Rose Oughtred2, Jennifer Rust2, Kara Dolinski2, Anastasia Baryshnikova3 1) University of Maryland, Baltimore County, Baltimore, MD; 2) Lewis- Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ; 3) Calico Life Sciences LLC, South San Francisco, CA. Since its official release in 2002, the yeast knock-out collection has catalyzed discoveries in all domains of yeast biology and has paved the way for functional genomic analyses in mammalian systems. Nearly 250 yeast laboratories have published over 350 studies, each examining ~4,800 non-essential knock-out mutants for one or more phenotypes (e.g., growth rate, cell morphology, abundance/localization of proteins/metabolites) under one or more experimental conditions (e.g., limiting nutrients, high/low temperature, chemical compounds). This data represents the single largest, richest and most systematic phenotypic description of an organism. Yet, its integrative analysis has been virtually impossible due to the sparseness and heterogeneity of data storage and formatting. Here, we describe the aggregation, annotation and analysis of all published genome-scale phenotypic screens of the knock-out collection which we collectively refer to as the yeast knock-out phenome. We have exhaustively curated the literature and compiled ~15,600 screens covering ~6,600 phenotypes and ~5,000 conditions. We examined data reproducibility by comparing independent experiments that measured the same phenotype in similar conditions and found that, while specific mutants may rank differently across experiments, the biological processes associated with each phenotype are strikingly reproducible. We observed clear relationships between phenotypes (e.g., cell size and replicative lifespan) that likely result from perturbing a common mechanism and confirmed that virtually all knock- out mutants, including those corresponding to functionally uncharacterized genes, manifest an extreme phenotype in at least one tested condition. Phenotypic profiles proved to be strong predictors of gene function and diversity of data types was critical for prediction accuracy. The yeast knock-out phenome, available in beta version at www.yeastphenome.org, enables myriads of systematic enquiries into the nature of gene-gene, phenotype-phenotype and gene-phenotype relationships, and is an important resource for the yeast community.

3 The sentinel interaction mapping (sim) approach: human disease gene functionalization by genetic interaction mapping in yeast. B. Young, K. Post, P. Ganguly, R. Dingwall, T. McDiarmid, W. Meyers, F. Meili, B. Callaghan, M. Belmadani, S. Rogic, P. Pavlidis, C. Rankin, T. O'Connor, S. Bamji, D. Allan, K. Haas, C. Loewen University of British Columbia, Vancouver, BC, CA. Currently, there are ~5,000 genes known to be associated with human diseases and this number is expected to continue to rise as a result of comprehensive genome sequencing of individuals. Hence, there is an urgent unmet need for fast, cost- effective, cell-based assays of human disease gene function for personalized medicine. Our goal is to develop a generic, quantitative approach that can be used to study the function of potentially any/all human disease genes. The development of synthetic genetic array (SGA) technologies in yeast has enabled rapid, unbiased, high-throughput quantitation of yeast genetic interactions, which can provide a comprehensive description of a gene’s function. Similarly, genetic interactions can be identified for a human disease gene expressed in yeast and constitutes a functional assay for that gene and a means to study the effects of disease-associated variants. This is possible because expression of the human gene in yeast likely affects an aspect of yeast cell physiology that will produce a unique fingerprint of genetic interactions detected by SGA. Hence, even human disease genes without yeast orthologs/paralogs should produce genetic interactions, making this a highly generalizable approach. We have called the yeast mutants that show genetic interactions with the human gene “Sentinels” because they report on the function of the human gene. A human gene variant associated with disease that alters its function will predictably not interact with these sentinels in the same way as the wild type gene, providing a quantitative difference between wild type and variant. An advantage of the SIM approach is that it has the potential to assign functions to human disease genes of unknown function, since the nature of the sentinels identified report on the activity of the human gene in yeast. We have used the SIM approach with the human tumor-suppressor gene PTEN and determined the functional impact of 102 missense variants. PTEN was expressed in ~6,000 yeast mutants using SGA and 8 sentinels were identified, which were used to quantitatively assess the effects of all 102 PTEN variants. SIM measurements indicated that the majority of disease- associated PTEN variants were loss of function, supporting PTEN’s pivotal role in cancer. SIM results also matched to a high

7 degree PTEN variant functional measurements from other independent in vivo assays, including D. melanogaster, C. elegans and rat hippocampal cultures.

4 Genetic dissection of the functional relevance of eukaryotic protein phosphorylation. C. Vieitez1, B. Busby1, M. Galardini2, D. Ochoa2, A. Jawed1, A. Mateus1, O. Wagih2, M. Shahraz1, M. Savitski1, A. Typas 1, P. Beltrao1,2 1) Genome Biology Unit, EMBL, Heidelgerg, DE; 2) EMBL-EBI, Hinxton, UK. Protein phosphorylation is a reversible signaling mechanism involved in all cellular processes. Phosphorylation has been shown to diverge quickly during evolution leading to the suggestion that some phosphorylation sites may not be functional. Over 10,000 phosphorylation sites have been identified in S.cerevisiae but their function and the extent by which they contribute to fitness remains unknown. Here, we constructed a library of 500 phosphodeficient mutants and screened them along with the yeast KO collection under 100 stress conditions measuring colony size as a proxy for fitness. Our results show that 40% of the phosphodeficient mutants show at least one phenotype. Using computational analysis we generated a phenotypic fingerprint for each individual mutant that allowed us to identify not only individual phenotypes but also novel functional associations. Our follow up studies using thermal proteome profiling and fluorescence microscopy approaches highlight phosphorylation sites responsible for protein stability, protein abundance and protein subcellular localization.

5 Genetically deciphering complex traits using engineered populations. Albi Celaj1,2,3, Marinella Gebbia1, Louai Musa2, Atina Cote2, Minjeong Ko2,6, Jamie Snider1, Victoria Wong1, Tiffany Fong5, Joe Mellor1, Gireesh Seesankar5, Maria Nguyen5, Shijie Zhou1, Igor Stagljar1,3,7, Nozomu Yachie4, Frederick Roth1,2,3,6 1) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada; 2) Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 3) Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada; 4) Synthetic Biology Division, Research Center for Advanced Science and Technology, the University of Tokyo, Tokyo, Japan; 5) McMaster University, Hamilton, Ontario, Canada; 6) Department of Computer Science, University of Toronto, Toronto, Ontario, Canada; 7) Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada. Combined genetic perturbations can lead to unexpected phenotypes (‘genetic interactions’) that can be critical for understanding biological systems. We devised an ‘engineered population profiling’ strategy for phenotypic analysis of high- order mutant combinations within a targeted gene set. We engineered and genotyped a uniquely-barcoded population of ~7,000 yeast strains (‘individuals’), each carrying deletions for a random subset of 16 ABC transporters involved in multidrug resistance. This engineered population was profiled for resistance to each of 16 drugs, yielding a complex environment- dependent genetic landscape which was used to computationally reconstruct a predictive genotype-to-phenotype model for a subset of ABC transporters. This model hypothesized many novel transporter activities and antagonistic transporter- transporter relationships. We further investigated a quadruple ABC transporter knockout combination that, rather than yielding drug sensitivity as might be expected, resulted in resistance to fluconazole and two other azolic PDR5 substrates, lending mechanistic insight and support for a model of complex mutual antagonism amongst a subset of ABC transporters.

6 A novel screen uncovers new functions of lysine polyphosphorylation in yeast. Amanda Bentley-DeSousa1,6, Charlotte Holinier1,6, Houman Moteshareie3,6, Yi-Chieh Tseng1,6, Sam Kajjo2,6, Christine Nwosu1,6, Federico Amodeo5, Emma Bondy-Chorney1,6, Yuka Sai1,6, Adam Rudner2,6, Ashkan Golshani3,6, Norman Davey4, Michael Downey1,6 1) Cellular & Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; 2) Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario, Canada; 3) Department of Biology, Carleton University, Ottawa, Canada; 4) Conway Institute of Biomolecular & Biomedical Research & UCD School of Medicine & Medical Science, University College Dublin; 5) Department of Basic Sciences, New York University, College of Dentistry; 6) Ottawa Institute of Systems Biology, Ottawa, Canada. Polyphosphates (polyP) are long chains of inorganic phosphates (3-1000s of residues in length) found in all cell types. These ubiquitous chains have diverse functions, but the mechanism of their action is unclear. In 2015, it was discovered that polyP chains can be attached to lysine residues as a non-enzymatic post-translational modification. This modification was identified on two proteins – Nsr1 and Top1 – where it was found to impact protein localization, interactions and the enzymatic activity of Top1 (Azevedo et al. 2015).

We wondered whether this modification was unique to these two proteins or whether it functions as a global modifier of protein function akin to other lysine-based PTMs such as acetylation or ubiquitylation. We recently carried out the first screen for polyphosphorylated proteins in yeast. So far we have uncovered 24 targets including a conserved network of proteins functioning in ribosome biogenesis (Bentley-DeSousa et al. 2018), as well as proteins involved in chromatin biology and cell cycle control. We also identify a regulatory network of yeast genes including phosphatases and vacuolar proteins that modulates polyphosphorylation by controlling the length of polyP chains. Finally, we have developed a synthetic biology approach to identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.

Here I present our latest work on polyphosphorylation with an emphasis on exploiting yeast genetic tools to uncover novel modes of regulation and function for polyphosphorylation as a conserved post-translational modification.

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7 An ancestral mitochondrial cell death pathway protects yeast gametes from lethal viral accumulation. Marc Meneghini, Jie Gao, Tina Zhou, Saif Hossain, Fuad Chowdhury, Sai Dhardawa, Sabrina Chau Molecular Genetics, Univ Toronto, Toronto, ON, CA. S. cerevisiae is chronically infected with RNA viruses that rely on mitotic transmission for their persistence. We have discovered that the yeast endonuclease G homolog, Nuc1, opposes the deleterious accumulation of these viruses in meiotic cells through an apparent ancestral cell death pathway. The most widely studied yeast viruses are the double stranded (ds) RNA virus L-A and its frequent “Killer” satellite viruses. The dsRNA L-A genome encodes gag and gag-pol fusion proteins that assemble into virus-like-particles that support L-A propagation. Varied forms of dsRNA “Killer” viruses have been documented as satellites of L-A. The most common is “M”, which encodes a processed and secreted toxin that kills neighboring cells lacking immunity, which is conferred in still unclear ways by the Killer genome itself. Building on our previous work, we have determined that Nuc1 evacuates mitochondria in a developmentally programmed manner during sporulation, directed by the master regulator of meioitc commitment, NDT80. Moreover, Nuc1 evacuation requires the mitochondrial Porin proteins Por1 and Por2 suggesting that yeast meiosis involves programmed mitochondrial outer membrane permeabilization similarly to what is observed in diverse forms of programmed cell death observed in animals. Using genetic and molecular methods we show that programmed release of Nuc1 functions to prevent the accumulation of L-A and Killer dsRNA. Perturbation of this Nuc1 pathway in strains infected with the Killer virus causes spore lethality if the developing spores also lack the well-known yeast antiviral SKI complex. Molecular, biochemical, and genetic analysis confirms that accumulating Killer virus within the spores causes this lethality, illuminating that this rudimentary cell death pathway therefore functions in an innate immune capacity that has an especially important role during gametogenesis. We are interested in the roles this ancestral death pathway may play for defense of the yeast “germline”, as well as of more familiar animal germlines, from widely found genetic parasites such as retrotransposons.

8 Evolutionary Rate Covariation as a Predictive Tool to Identify α-Arrestin-Cargo Pairs. David Macar1, Tova Finkelstein1, Abdullah Malik1, Uthman Fadu1, Alexiy Nikiforov1, Hilary Serbin1, Zelia Ferreria2, Nathan Clark2, Allyson O'Donnell1 1) Dept. of Biological Sciences, Duquesne University, Pittsburgh, PA; 2) Dept. of Computational and Systems Biology, Univ. of Pittsburgh, Pittsburgh, PA. Alpha-arrestins help cells survive environmental changes by controlling membrane protein trafficking. One hurdle to understanding α-arrestins is that few α-arrestin-cargo pairs have been identified. It is technically challenging to identify α- arrestin cargos due to their transient α-arrestin associations and the biochemical nature of their transmembrane cargo. To identify α-arrestin-regulated cargos, we used Evolutionary Rate Covariation (ERC), which uses sequence-based signatures to identify genes with similar evolutionary histories. We compared ERC rates for α-arrestins with cargos across 18 yeast species. Among the top co-evolving proteins were those previously defined as α-arrestin cargos. We are determining if the membrane proteins with the highest ERC values are α-arrestin cargos by assessing their localization and relative protein abundances in wild-type cells versus those lacking the α-arrestin. Mean fluorescent intensity was used to quantify the abundance and/or subcellular distribution of GFP tagged proteins. Statistically significant changes in the abundance or distribution of our GFP-tagged proteins between wild-type cells and those lacking α-arrestins suggests a dependence on these protein trafficking adaptors and makes these proteins good candidates as new α-arrestin-dependent cargos. Using this approach, we have quantitatively confirmed that 16 integral membrane proteins previously unassociated with α-arrestins display α-arrestin-dependent localization changes, and an additional 16 proteins appear to be regulated by α- arrestins based on qualitative analyses of the microscopy data for which we are currently completing the image quantification. This represents a dramatic increase in the α-arrestins regulatory network. In conclusion, the ERC approach is a powerful new tool that is able to define protein trafficking regulatory networks, which will undoubtedly be of interest to the cell biology community.

9 Deciphering mixed messages: Linking ubiquitin chains to protein quality control circuits. R.S. Samant, C.M. Livingston, D.R. Gestaut, J. Frydman Biology, Stanford University, Stanford, CA. Protein misfolding in the cell creates toxic species linked to an array of diseases. Therefore, protective cellular protein quality control (PQC) mechanisms evolved to triage misfolded proteins and limit their toxic effects. Molecular chaperones recognize misfolded proteins, while the ubiquitin-proteasome system (UPS) promotes their clearance through the attachment of ubiquitin chains. We previously identified a PQC pathway for spatial sequestration and clearance of misfolded proteins, conserved from yeast to humans, that is amplified when the UPS is impaired. However, the identity of the ubiquitination machinery involved in this pathway—and how it interacts with molecular chaperones—is unresolved. In yeast, where most proteasomes exist in the nucleus, whether or not cytoplasmic proteins require nuclear PQC circuits for clearance is especially controversial. Starting with a fluorescence microscopy-based genetic screen, we show that distinct chaperone and ubiquitination circuits cooperate in PQC of multiple misfolded proteins in the cytoplasm and nucleus. In contrast with the canonical model where Lys48-linked ubiquitin chains are sufficient for proteasomal targeting, we found that cytoplasmic misfolded proteins requires tagging with mixed ubiquitin chains that contain both Lys11 and Lys48 linkages to be cleared. Each type of linkage-specific ubiquitination requires a distinct combination of ubiquitin ligases and chaperones. Strikingly, unlike cytoplasmic PQC, proteasomal degradation of nuclear misfolded proteins only requires Lys48 ubiquitin linkages and is independent of Lys11-specific circuits. We conclude that cytoplasmic and nuclear PQC involves combinatorial recognition by 9 defined sets of cooperating systems. The distinct PQC requirements reveal underlying differences in nuclear and cytoplasmic proteome management, with important implications for our understanding of a wide range of diseases.

10 Developmental regulation of an organelle tether couples mitochondrial inheritance to the meiotic program. E.M. Sawyer1, P.R. Joshi1, L.E. Berchowitz2, E. Ünal1 1) Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA; 2) Department of Genetics and Development, Columbia University Medical Center, New York, NY. To generate haploid gametes, meiosis is accompanied by cellular differentiation where the cytoplasmic contents of the progenitor cell undergo dramatic remodeling. The underlying mechanisms responsible for the segregation of organelles into gametes remain poorly understood. Here we investigated how mitochondrial dynamics is regulated during meiotic differentiation in budding yeast. We found that mitochondria abruptly detach from the cell cortex at the onset of anaphase II. By analyzing a series of meiotic mutants, we determined that a meiosis-specific, CDK-like kinase, Ime2, is essential for mitochondrial detachment, even though the meiotic divisions themselves are dispensable entirely. Ime2 phosphorylates both subunits of the mitochondria-ER-cortical anchor (MECA), Num1 and Mdm36. MECA is the sole protein complex responsible for mitochondria-plasma membrane tethering, with the mitochondrial organization of num1∆ mitotic cells resembling wild-type meiosis II cells. Ime2 causes MECA assemblies to become dispersed and unstable, leading to mitochondrial detachment in meiosis II. Because the plasma membrane is not inherited by spores, we conclude that Ime2-directed loss of tethering potentiates mitochondrial segregation. Our study defines a cellular mechanism that coordinates mitochondrial morphogenesis with the landmark events of meiosis and demonstrates that organelle tethers, which are thought to be highly stable structures, can be developmentally regulated to alter cellular organization.

11 Systematic genetic analysis in the opportunistic yeast Candida albicans reveals evolutionary plasticity of cell size homeostasis mechanisms in eukaryotes. J. Chaillot, A. Sellam Infectious Diseases Research Centre (CRI), CHU de Québec Research Center (CHUQ), University Laval, Quebec, QC, CANADA. The basis for commitment to cell division in late G1 phase, called Start in yeast and the Restriction Point in metazoans, is a critical but still poorly understood aspect of eukaryotic cell proliferation. All eukaryotic cells must grow to a critical cell size before to pass Start. This size threshold couples cell growth to division and thereby establishes long-term size homeostasis. To probe the conservation of the extensive size homeostasis network, we performed quantitative genome-wide analyses of a systematic collection of gene deletion strains in the pathogenic yeast Candida albicans. Analysis of the size phenome uncovered 195 genes that markedly altered cell size. Our results revealed an unexpected high degree of divergence between genes that affect size in C. albicans versus the yeast model Saccharomyces cerevisiae. In addition to conserved size regulators previously identified in S. cerevisiae and metazoans, we uncovered previously undocumented regulatory circuits that govern critical cell size at Start. A comprehensive genetic connectivity, using suppressive-dosage and epistasis genetic interactions, were used to order the size regulatory network in C. albicans. This comprehensive analysis revealed a complex network of novel regulator of Start and cell size the most potent of which were the transcription factors Ahr1, Sfp1, Dot6, the AGC kinase Sch9 and the MAPK Hog1. In particular, we delineate a novel stress-independent function of the p38/HOG MAPK network in coupling cell growth to cell division. Our genetic and biochemical analysis suggests that the HOG module directly and interacts with central components of both the cell growth and cell division machineries. Furthermore, we identify a novel size network where the helix-loop-helix transcription factors Dot6 act as a major effector of the TOR pathway to modulate cell size at Start through control of both cytoplasmic and mitochondrial translation. We also showed that the transcription factor Ahr1 that modulates adhesion genes and virulence in C. albicans, control cell size of both yeast and hyphae forms. This work establishes the first systematic characterization of the mechanisms underlying regulation of growth and division in a pathogenic fungus and illuminates the evolutionary plasticity of the size control network in eukaryotes.

13 Transcription factors and nuclear pore proteins control the organization of the yeast genome at the nuclear periphery. Jason Brickner, Donna Brickner, Carlo Randise-Hinchliff, Marine LeBrun, Michael Sumner Molecular Biosciences, Northwestern University, Evanston, IL. Hundreds of yeast genes physically interact with nuclear pore proteins (NPC) and many inducible genes reposition to the nuclear periphery when they are activated. Interaction with the NPC is controlled by cis-acting targeting elements (or "DNA zip codes") and requires both NPC proteins and transcription factors (TFs). Here, we describe the mechanistic dissection of the function of TFs and transcriptional regulators/mRNA export factors in mediating targeting to the nuclear periphery. Using conditional inactivation as well as tethering, we find that although null mutations in factors associated with mRNA production and export block localization to the nuclear periphery, these effects are likely indirect. In contrast, conditional inactivation of transcription factors and nuclear pore basket proteins leads to rapid loss of peripheral localization. Likewise, light-induced recruitment of a transcription factor to an ectopic site in the genome is sufficient to cause rapid repositioning to the nuclear periphery. To test the generality of this phenomenon, we have performed a global screen of all ~200 yeast TFs/DNA binding proteins (DBPs). Each TF/DBP was tagged with the DNA binding domain from LexA and crossed against a strain having the LexA binding site integrated at a locus that normally localizes to the nucleoplasm. The position of this locus with respect to the nuclear envelope was scored using confocal microscopy. Greater than 65% of the 188 TFs/DBPs that were successfully tagged were sufficient to cause localization to the nuclear periphery. By mutating nuclear pore proteins in >100 diploid yeast 10 strains expressing LexA-tagged TFs/DBPs, we found inactivation of the nuclear pore protein Nup2 blocked peripheral targeting in >95% of these strains. Thus,the major pathway by which TFs/DBPs mediate targeting to the nuclear periphery is through interaction with the NPC. However, only a subset of these TFs/DBPs (~35%) required the nuclear pore protein Nup100. This suggests that there are two distinct mechanisms for TF/DBP-mediated targeting to the NPC based on the requirement for Nup100.

14 Rad5 recruits TLS DNA polymerases for mutagenic repair of ssDNA gaps on undamaged templates. David Gallo1,2, TaeHyung Kim1,3, Barnabas Szakal4, Xanita Saayman1,2, Ashrut Narula1,2, Yoona Park1,3, Dana Branzei4,5, Zhaolei Zhang1,3, Grant Brown1,2 1) Donnelly Centre, University of Toronto, Toronto, ON, Canada; 2) Department of Biochemistry, University of Toronto, Toronto, ON, Canada; 3) Department of Computer Science, University of Toronto, Toronto, ON, Canada; 4) Fondazione Istituto FIRC di Oncologia Molecolare, Milan, Italy; 5) Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche (IGM-CNR), Pavia, Italy. Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA lesions in both an error-free and an error- prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. Polyubiquitination of PCNA by the ubiquitin ligase Rad5 initiates error-free PRR, using the sister chromatid as a template for DNA synthesis in lieu of the lesion-containing strand. We find that Rad5 forms nuclear foci during normal S-phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the site of stressed DNA replication forks, where it recruits TLS polymerases to repair ssDNA gaps, preventing mitotic defects and chromosome breaks. Our data indicate that Rad5 is the central effector of PRR at stressed replication forks, where Rad5 promotes mutagenic repair of undamaged ssDNA that arises during physiological and exogenous replication stress.

15 Labeling and tracking single nucleosomes through the cell cycle. Gavin Schlissel, Jasper Rine UC Berkeley, Berkeley, CA. Nucleosomes are assumed to store and transmit epigenetic information between mitotic generations. This assumption requires that nucleosomes maintain their genomic position throughout the cell cycle, despite the large-scale reorganization of chromatin that occurs during transcription or DNA replication. We developed a method to biotinylate nucleosomes at defined genomic loci by fusing the E. coli biotin ligase BirA to the heterochromatin-associated protein Sir4. Using biotin pulse- chase experiments, we established that heterochromatic nucleosomes retain their position throughout the cell cycle and through DNA replication, suggesting that nucleosomes can transmit information about local heterochromatin environment through mitosis. We extended our approach to study euchromatic loci, leveraging the bacterial DNA-binding proteins TetR and LacI to biotinylate nucleosomes adjacent to single-copy operator sequences. Using this approach, we can label ~1-2 nucleosomes at an arbitrary locus, and we can control the duration of labeling using small molecule ligands. The results from these experiments resolve whether nucleosomes retain their position during transcription, and whether nucleosomes can transmit memory of an accessible chromatin configuration through mitosis.

16 Multisite substrate recognition in Asf1-dependent acetylation of histone H3 K56 by Rtt109. L. Zhang1,2,5, A. Serra- Cardona3,5, H. Zhou3, M. Wang1, N. Yang4, Z. Zhang3,6, R. Xu1,2,6 1) National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China; 2) University of Chinese Academy of Sciences, Beijing, China; 3) Institute for Cancer Genetics, Departments of Pediatrics and Genetics and Development, Irving Cancer Research Center, Columbia University, New York, NY; 4) State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin, China; 5) These authors contributed equally to this work; 6) Corresponding author. During the S phase of the cell cycle both parental and newly synthesized histones must be deposited on the replicated DNA in a process termed replication-coupled nucleosome assembly. Newly synthesized histones have to be shuttled from the cytoplasm into the nucleus while preventing their aggregation and their spurious interaction with DNA. This is achieved by a complex interplay between histone chaperones, histone modifying enzymes, and histone modifications. Prior to deposition on the DNA, new H3-H4 dimers associate with histone chaperone Asf1. This Asf1-H3-H4 complex is recognized by lysine acetyltransferase Rtt109 which acetylates lysine 56 of histone H3 (H3K56ac), a modification critical for replication-coupled nucleosome assembly and genome stability. However, how Rtt109 achieves its substrate specificity and what role does Asf1 play in it is largely unknown. In order to understand the molecular mechanism of H3K56ac we have determined the crystal structure of the Rtt109-Asf1- H3-H4 complex. The critical residues found in the structure were further validated in vivo in Saccharomyces cerevisiae and using in vitro histone acetyltransferase assays. We have found that the region where K56 resides, which normally adopts a helical conformation in nucleosomal histone H3, unwinds in the Rtt109-Asf1-H3-H4 complex. This is most likely a necessary step for this residue to interact with the substrate-binding channel of Rtt109. Surprisingly, even though Asf1 is essential for the acetylation of H3K56, the direct contacts between this histone chaperone and Rtt109 are inconsequential for the acetylation activity. Instead, Asf1 is important for the stabilization of the C-terminal β–strand of H4, which interacts extensively with Rtt109 and is required for H3K56ac. Furthermore, we found that the central histone fold of H3, located far away from H3K56, is also directly contacting Rtt109. Specifically, glutamate 94 of histone H3 interacts with two arginines of Rtt109, and this acidic-basic contact is important for the correct acetylation of H3K56. Our work reveals Asf1’s role in reshaping the H3-H4 complex for H3K56 acetylation, unearthing a new dynamic function of the versatile histone chaperone in 11 addition to its traditional capacity as a histone escort. Moreover, we describe the involvement of histone H3 residues proximal to H3K56 along with distal residues at the central histone fold domain in the correct acetylation of H3K56. This unprecedented multiprotein, multisite substrate recognition mechanism among histone modification enzymes provides mechanistic understandings of Rtt109 and Asf1 in H3K56 acetylation, as well as new insights into substrate recognition by histone modification enzymes in general.

17 Persistent DNA-break potential near telomeres promotes a higher density of meiotic recombination on small chromosomes. V. Subramanian1, T. Markowitz1, L. Vale-Silva1, P. San-Segundo2, N. Hollingsworth3, A. Hochwagen1 1) Department of Biology, New York University, New York, NY. ; 2) University of Salamanca, Salamanca, Spain. ; 3) Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY. Infertility, spontaneous fetal loss and birth defects in humans result mainly from chromosome segregation errors during meiosis [1,2]. Faithful chromosome segregation requires induction of numerous programmed DNA double-strand breaks (DSBs), and their repair as crossovers [3]. To avoid excessive DNA damage, feedback mechanisms must down-regulate DSBs. In Saccharomyces cerevisiae, this regulation requires the removal of the conserved DSB-promoting protein Hop1/HORMAD during chromosome synapsis, assembly of a higher-order chromosome structure intimately linked to crossover repair initiation [4,5]. Allocation of a higher density of DSBs/crossovers to the smaller chromosomes of the genome (e.g. Chr 21) is critical to avoid errors like Trisomy 21 that causes Down syndrome. Despite its clinical significance, we have only a meager understanding of how DSBs are apportioned to the small chromosomes. Here, we identify privileged domains spanning roughly 100 Kb near all telomeres that escape negative regulation by synapsis and continue to break in pachynema, well after chromosomes are fully synapsed. These end-adjacent regions (EARs) retain Hop1 despite normal synapsis, indicating that synapsis is necessary but not sufficient for Hop1 removal. EAR enrichment requires the AAA+-ATPase, Pch2/TRIP13, which preferentially removes Hop1 from telomere-distant sequences. This enrichment is enhanced in a sir2 deletion where reserves of Pch2 are delocalized from the nucleolus, potentially hyper activating Pch2. On the other hand, the nuclear pore component Nup2 positively regulates Pch2 to establish the EAR domains. Importantly, the uniform size of EARs between chromosomes contributes to disproportionately high DSB and repair signals on small chromosomes in pachynema [6], suggesting that EARs partially underlie the curiously high recombination rate of small chromosomes [7].

References 1. Hassold T, Hunt P (2001). Nat Rev Genet 2: 280-291. 2. Jones KT, Lane SI (2013). Development 140: 3719-3730. 3. Subramanian VV, Hochwagen A (2014). Cold Spring Harb Perspect Biol 6: a016675. 4. Subramanian VV, MacQueen AJ, Vader G, et al. (2016). PLoS Biol 14: e1002369. 5. Thacker D, Mohibullah N, Zhu X, Keeney S (2014). Nature 510: 241-246. 6. Subramanian VV, Markowitz TE, Vale-Silva LA, San-Segundo P, et al. (2017). bioRxiv. 7. Kaback DB, Guacci V, Barber D, Mahon JW (1992). Science 256: 228-232.

18 A combination of transcription factors mediates inducible interchromosomal pairing. Seungsoo Kim1, Maitreya Dunham1, Jay Shendure1,2 1) Department of Genome Sciences, University of Washington, Seattle, WA; 2) Howard Hughes Medical Institute, Seattle, WA. The three-dimensional organization of the genome is dynamically remodeled in response to cell type differentiation and extracellular signals. However, the molecular mechanisms driving cell type- or condition-specific changes in chromosome conformation are poorly understood, especially interchromosomal contacts, which remain controversial. We previously discovered condition-dependent homolog pairing between HAS1pr-TDA1pr alleles in saturated cultures of diploid Saccharomyces yeasts, a rare example of a robust inducible interchromosomal contact. As this pairing requires a 1 kb intergenic region, we hypothesized that it might be mediated by conditionally expressed transcription factors (TFs). However, it is unknown which, if any, TFs regulate chromosome conformation at any given locus. To address this challenge, we developed an assay to systematically dissect a chromosomal contact or loop of interest. Our method allows us to test hundreds of perturbations in parallel, by coupling saturation mutagenesis with the chromosome conformation capture (3C) assay followed by sequencing. Using this pooled approach, we tested 861 subsequences for pairing activity, and identified a 125 bp region sufficient for homolog pairing. Next, we tested all substitutions within this narrowed region to identify the exact base pairs required for pairing, which corresponded to TF motifs for three TFs: Leu3, Rgt1, and Sdd4 (Ypr022c). We confirmed that these TFs are required in trans, by using the existing yeast deletion collection to test nearly all non-essential site-specific TF gene deletions for homolog pairing. Zooming in even further, we tested mutant TFs for pairing activity to identify a domain of Rgt1 that negatively regulates this pairing activity. As expected, Rgt1 and Sdd4 are upregulated in saturated cultures, explaining the condition-specificity of HAS1pr-TDA1pr pairing. Finally, by looking for motif clusters for these TFs, we identified homolog pairing between HXT3 promoters. Together, our results demonstrate how a combination of transcription factors can mediate condition-specific chromosomal contacts and thereby contribute to the dynamic nature of chromosome conformation.

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19 A central metabolic regulator that can undergo reversible and irreversible aggregation. Koby Simpson- Lavy, Martin Kupiec School of Molecular Cell Biology and Biotechnology, Tel Aviv Univ, Tel Aviv, IL. Utilization of non-fermentable carbon sources requires the activity of the Snf1 protein kinase (yeast AMPK ortholog). Snf1 is active in the absence of glucose and regulates the expression and activity of proteins involved in respiration. We have identified two new regulators of Snf1 activity in S. cerevisiae (Sip5 and Vhs1). These new regulators control the sequestration of the Snf1 activator Std1 from the nucleus to the cytoplasm in response to glucose by a reversible aggregation mechanism. Under these conditions, Std1 forms liquid droplets that require the chaperones Hsp104 and Hsp70, usually involved in the aggregation of amyloidogenic or misfolded proteins (such as huntingtin or Ubc9-1). The glucose response, however, takes place under normal, non-stressed conditions. Interestingly, Std1 also responds to genotoxic stress; however, upon DNA damage, Std1 localizes to a different cellular compartment in an irreversible fashion. This aggregation requires a different set of chaperones (Hsp40 and Hsp42). This suggests that protein aggregation mechanisms are a normal, non-pathological physiological state that can be used to regulate central metabolic processes, and that proteins can switch states to reversible or irreversible aggregates depending on the growth conditions. These results shed light on the evolutionary role of protein aggregation in eukaryotes and have implications for our understanding of cancer and neurodegenerative diseases.

20 Regulating the metabolic flow of fatty acids – a greasy line between life and death. S.D. Kohlwein, D. Liebelt, G.N. Rechberger, H.F. Hofbauer, F. Sarkleti Institute of Molecular Biosciences, BioTechMed-Graz, University of Graz, Graz, AT. “Fat” is on everyone's lips. The conspicuous consequences of malnutrition and a sedentary lifestyle have sparked great biomedical interest in understanding the metabolic flow of fatty acids as key constituents of lipids, and their impact on organelle structure and function during cellular growth and development. Using stable isotope labeling with 13C-glucose and mass-spectrometry we have developed a method to trace the fate of de novo-synthesized fatty acids into various lipid classes in growing yeast cells. The dynamic appearance of intermediate lipid species that harbor a combination of labeled and newly synthesized acyl chains follows distinct differences between lipid classes, challenging the general view of common precursor- product relationships. Whereas newly synthesized phosphatidylinositol obtains its fatty acids from lipid turnover (e.g. triglyceride breakdown), phosphatidylcholine is preferentially remodeled by exchange of its acyl-chains with de novo synthesized fatty acids. Any nutritionally or genetically induced imbalance in cellular fatty acid composition has major consequences for organelle structure and function, may trigger the unfolded protein response, impairs secretion, and ultimately leads to cell death. A genome-wide suppressor screen has uncovered the RIM101 pathway – implicated in the cellular response to alkaline stress – as a central player in cellular lipid balance and fatty acid-mediated lipotoxicity.

21 Probing primary metabolism with a yeast secondary metabolite gene cluster. D.J. Krause1,2, J. Kominek1,2, D.A. Opulente1,2, X.X. Shen3, X. Zhou3, J. DeVirgilio4, A.B. Hulfachor1, C.P. Kurtzman4, A. Rokas3, C.T. Hittinger1,2 1) Laboratory of Genetics, Genome Center of Wisconsin, Wisconsin Energy Institute, J.F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, WI; 2) DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, Madison, WI; 3) Department of Biological Sciences, Vanderbilt University, Nashville, TN; 4) Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, IL. The budding yeast subphylum Saccharomycotina harbors a wealth of genotypic and phenotypic diversity. The Y1000+ Project is interrogating this diversity by sequencing the genomes of type strains of all known budding yeast species. Here we use this rich dataset to identify and characterize the first yeast secondary metabolite gene cluster, which is responsible for production of the siderophore pulcherrimin. Specifically, using phylogenomic footprinting on this rare trait and targeted gene replacement in Kluyveromyces lactis, we show that a four-gene cluster is responsible for production of pulcherrimin and utilization of its bound iron. We also found partial gene clusters in several yeast species corresponding to pulcherrimin utilization genes in the absence of biosynthetic genes. One of these species is Saccharomyces cerevisiae, and the two-gene partial cluster is composed of previously uncharacterized genes that we have named PUL3 (YNR062C) and PUL4 (YNR063W). Through phylogenetic topology testing of the PUL genes and their homologs, we conclude that the gene cluster was ancestral to budding yeasts and was lost in part or in whole in most lineages. Therefore, the partial gene cluster is a derived genotype that confers a public goods cheater phenotype that allows cells to utilize the iron captured by pulcherrimin without incurring the costs of production. Since pulcherrimin is deep red and synthesized from two leucine molecules, it may also have the potential to serve as a visual flux reporter for the branched-chain amino acid (BCAA) biosynthesis pathway, which is shared with production of the advanced biofuel isobutanol. Heterologous expression of the pulcherrimin biosynthesis genes yields red strains of S. cerevisiae, and we are using this system to probe metabolic flux through these pathways.

22 The genetic basis for obese yeast: From forward genetic screens to genome analysis of fatty acid-overproducing mutants. S. Rana1,3, B. Vidrine2,3, E. Boggess3,4, P. Ammodt1,3, K. Radkhe1, B. Nikolau2,3, J. Dickerson3,4, M. Yandeau- Nelson1,3 1) Dept. of Genetics, Development & Cell Biology, Iowa State University, Ames, IA; 2) Dept. of Genetics, Development & Cell Biology, Iowa State University, Ames, IA; 3) Dept. of Electrical and Computer Engineering, Iowa State University, Ames, IA; 4) NSF Engineering Research Center for Biorenewable Chemicals, Iowa State University, Ames IA. Fatty acid metabolism is critically important for downstream cellular functions, including energy storage, membrane composition and signaling. Fatty acids can also be produced as oleochemicals that serve as precursors for industrial products (e.g. soaps, lubricants, and biofuels) and are promising alternatives for petroleum-based chemicals. Recent synthetic biology 13 and metabolic engineering approaches have targeted fatty acid biosynthesis and downstream pathways within S. cerevisiae for enhanced fatty acid production, however, a limited understanding of downstream lipid metabolic regulation remains a barrier to microbial chemical production on an industrial scale. A forward genetics screen was employed to identify haploid yeast mutants with increased lipid content, specifically neutral lipids (triacylglycerols; TAG). Fatty acid- overaccumulating (fao) mutants were identified from an EMS-mutagenized population subjected to a two-step screen: buoyant density-based selection followed by identification of high-lipid strains via staining with a lipophilic dye. From a pilot study, seven unique fao strains were identified and backcrossed to remove non-causal mutations. TAG levels were generally higher in the fao mutants and fatty acid accumulation was doubled as compared to the parental strain. To identify putative causal mutations, each fao strain was sequenced; 50 of the 123 unique mutations identified were non-synonymous or nonsense substitutions. Candidate mutations were introduced individually into the parental strains using CRISPR technology and several of these mutations have yielded modest increases in fatty acid accumulation. For example, a mutation introduced into the PIB2 gene, which encodes a phosphatidylinositol kinase, increased total fatty acid accumulation by ~60% within the BY4742 parent. Because segregation of the high-oil phenotype in the backcrossing series suggests multiple causal mutations within each fao strain, candidate mutations are being evaluated both individually and in combination. Other mutations being pursued include targets with putative functions in TAG degradation, GPI-anchor remodeling, and phosphatidylinositol binding and regulation. This work identifies novel genetic interventions that can be integrated into efforts to engineer S. cerevisiae as a microbial oleochemical factory.

23 Natural variation in a cellular adhesin suggests the possibility of kin-recognition in Saccharomyces cerevisiae. H.A. Murphy, Z.J. Oppler, M.E. Parrish Biology, William and Mary, Williamsburg, VA. Cooperative and altruistic behaviors have long fascinated and puzzled evolutionary biologists. Most examples of altruism can be explained by kin selection, with kin recognition common in many systems. In microbes, cooperative behaviors involving cells adhering to one another generally rely on “kind” recognition where a single locus or trait, known as a greenbeard, signals cooperation. In behaviors that require microbes to locate one another, rare examples of kin- and self- recognition have been reported; in these cases, membrane-associated proteins with variable extracellular domains confer discrimination. However, in non-motile microbes, such systems of self-recognition are not expected, as growth generates patches of identical clones. In Saccharomyces cerevisiae, Flo11 is a membrane-anchored adhesin required for most social phenotypes (i.e., biofilms, mats, pseudohyphal growth), and has been hypothesized to play a role in recognition. Variegated expression of this highly regulated protein has also been hypothesized to be an adaptation for resource utilization. We amplified and sequenced the regulatory and coding regions of FLO11 in 78 environmental isolates that vary in their social phenotypes, and generated de novo assemblies of the locus. Population genetic analyses suggest evidence for selection on both recognition and expression level. The precise regions implicated in cell-cell adhesion exhibit a signature of positive selection, while the rest of the gene is under purifying selection. In the promoter region, there is evidence for balancing selection at a binding site for the Rpd3L complex, whose activity leads to epigenetic silencing. Furthermore, phenotypic assays demonstrate that different natural FLO11 alleles generate diverse biofilm architectures in an otherwise constant genetic background, with certain alleles conferring a competitive advantage. Unlike in motile microbes where cheater avoidance is likely driving the evolution of recognition, competition among clones may be driving the evolution of homophillic binding and expression level in S. cerevisae. Thus, the interplay between inter-clone competition and intra-clone cooperation in spatially structured microbial communities may potentially lead to recognition systems with specific expression patterns.

24 Human isolates of baker's yeast display MLH1-PMS1 incompatibilities that contribute to a broad range in mutation rate. V. Raghavan1, D. Bui1, N. Al-Sweel1, A. Friedrich2, J. Schacherer2, C. Aquadro1, E. Alani1 1) Molecular biology and Genetics, Cornell University, Ithaca, NY; 2) Université de Strasbourg, Centre National de la Recherche Scientifique, Génétique Moléculaire, Génomique, Microbiologie, Unité Mixte de Recherche, Strasbourg, France. Laboratory baker’s yeast strains bearing an incompatible combination of the MLH1 and PMS1 mismatch repair genes are mutators that can adapt more rapidly to stress but do so at the cost of long-term fitness (Bui, PLoS Genetics 11, e1005407; Bui, Genetics 205, 1459). Previously we identified 18 baker’s yeast isolates from 1011 yeast isolates surveyed (Peter, Nature 556, 339) that contain the incompatible MLH1-PMS1 genotype in a heterozygous state. Surprisingly, the incompatible combination from two human clinical diploid isolates, YJS5885 and YJS5845, contain the exact MLH1 (S288c derived) and PMS1 (SK1 derived) open reading frames originally shown to confer incompatibility. While both of these isolates were non-mutators, their meiotic spore progeny displayed mutation rates that varied over a 340-fold range. This range was ~30- fold higher than seen between compatible and incompatible combinations of laboratory strains. Genotyping analysis indicated that MLH1-PMS1 incompatibility was the major driver of mutation rate in the isolates. The variation in the mutation rate in both incompatible and compatible spore clones appears to be due to suppressors and enhancers in the genetic backgrounds. Our data are consistent with the variance in mutation rate providing a bet-hedging strategy for spore progeny that can provide an adaptive advantage to environmental challenges (e.g. in a human host) that is buffered by subsequent mating to prevent long term fitness costs.

25 Population-scale diallel cross reveals the impact of rare variants on the phenotypic landscape. Teo Fournier1, Jackson Peter1, Jing Hou2, Joseph Schacherer1 1) UMR7156, University of Strasbourg, Strasbourg, FR; 2) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, CA. 14

Understanding the rules laying behind the natural phenotypic variation has been a key point of modern genetics for decades. However, it is still difficult to precisely address and dissect the molecular bases underlying complex traits. Today, a better understanding of the genetic architecture of traits requires a precise estimation of genetic components governing phenotypes at a species-wide level. In this context, we took advantage of the large set of 1,011 natural Saccharomyces cerevisiae isolates that we completely sequenced. We selected a set of 55 isolates as genetically diverse as possible to generate a diallel cross panel of 3,025 hybrids. These hybrids were then phenotyped on 49 stress related traits resulting in 148,225 cross/trait combinations. The results clearly showed that although phenotypic variance is mostly governed by additivity, 30% of this variance can be explained by non-additive phenomena. This is confirmed by the fact that a majority of complete dominance is observed in 25% of the traits. The dataset we generated also allowed us to perform genome-wide association studies (GWAS) to uncover variants responsible for the tested phenotypes. Interestingly, 2,156 significantly associated variants were found and among them 12% are present in less than 5% of the 1,011 population. It clearly shows that those so-called rare variants represent an important source of phenotypic variance and can be mapped using GWAS on a diallel panel. To complete this view, we are currently looking at the phenotypic distribution and segregation in the progeny to uncover the phenotypic expressivity variation across genetic backgrounds.

26 Understanding Adaptation and Fitness Trade-offs in Yeast. Yuping Li1, Sandeep Venkataram1,5, Atish Agarwala2, Barbara Dunn3, Daniel Fisher4, Dmitri Petrov1, Gavin Sherlock3 1) Department of Biology, Stanford; 2) Department of Physics, Stanford; 3) Department of Genetics, Stanford; 4) Department of Applied Physics, Stanford; 5) Division of Biology, University of California, San Diego. Few studies have quantitatively probed how adaptive mutations result in increased fitness. Even in microbial evolution experiments, with full knowledge of the underlying mutations and specific growth conditions, it is still challenging to determine where within a growth-saturation cycle those fitness gains occur. I have characterized thousands of evolved yeast clones, each carrying a unique DNA barcode and quantified their fitness gains that result from different phases of the growth cycle by measuring their fitness under conditions where lengths of fermentation, respiration and stationary phases were systematically varied. A common implicit assumption is that most benefits derive from an increased exponential growth rate. However, I instead found that while all adaptive lineages gained similar and modest benefits from fermentation, most of the benefits came instead from respiration where there is little cell division. From pairwise fitness competitions for a dozen of these clones, I determined that the benefits accrued during respiration are realized later as a shorter duration of lag phase in the following growth cycle. These results reveal hidden complexities of the adaptive process even under ostensibly simple evolutionary conditions, and the sensitivity of fitness to subtle quantitative changes of conditions. This sensitivity of fitness to the growth cycle conditions suggests that adaptation would proceed differently under conditions with a different growth-saturation cycle. To study this, I evolved barcoded yeast population under conditions modified from the previous Original Evolutionary Condition (OEC, which includes lag, fermentation and respiration phases). The Modified Evolutionary Conditions (MECs) have either a shorter growth cycle, without respiration, or a longer cycle with a prolonged stationary phase. I have found, by contrast to adaptation in the OEC, adaptive clones from the MECs have 1) a different genetic basis, e.g. mutations in the HOG pathway instead of the RAS/PKA pathway in OEC, and 2) different adaptive strategies, gain and loss of fitness benefits from different growth phases. My study of adaptation under systematically varied conditions has enabled the dissection of regulatory networks and intrinsic evolutionary constraints underlying yeast growth. This approach provides the opportunity to better understand how organismal evolution can be constrained by a changing environment.

27 Humanized yeast - an evolution guided approach to engineeing biology. A.H. Kachroo1,2, J.M. Laurent3,4, A. Akhmetov3, R.K. Garge3, M. Adbullah1,2, E.M. Marcotte3 1) Department of Biology, Concordia University, Montreal, Quebec, CA; 2) Center for Applied Synthetic Biology, Concordia University, Montreal, Quebec, CA; 3) Centre for Systems and Synthetic Biology, The University of Texas at Austin, Austin, Texas, USA; 4) Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University Langone Health, NY, USA. The most deeply evolutionarily conserved human genes encode essential cellular machinery whose failures are linked to diverse diseases, from cancer to cardiovascular disease. Recent systematic studies have discovered extensive genetic polymorphism in these genes yet studying how these variations contribute to cellular function and overall human health remains a challenge. The remarkable extent to which protein-coding genes are still functionally equivalent between humans and yeast emphasizes the power even of a distant organism for studying human gene function. Previously, we have already created >250 humanized strains (Science, 2015). Surprisingly, as many as 47% of the human genes can complement a lethal growth defect conferred by loss of the corresponding yeast gene with little or no effect on growth. Humanizability is not well- explained by sequence similarity between the human and yeast genes but is instead a property of specific protein complexes and pathways. We also discovered that a large proportion (>60%) of the bacterial genes tested can successfully replace their yeast orthologs and complement an otherwise lethal loss of an essential yeast gene. As with our systematic tests of yeast humanization, we observed genes in the same pathway and complex to be similarly replaceable or not, showing heme biosynthesis as a near universally swappable pathway by orthologs from E. coli, plants and humans in yeast (eLife, 2017). We will present our work involving the entire set of shared essential genes (>400 human genes) in yeast that have 15 several co-orthologs in humans assaying for functional complementation. By expanding humanization assays to include those yeast genes that have more than one human ortholog, we have successfully added 90 new human genes to our tested set (a 73% increase). We find that duplicated human genes tend to differentially replace their yeast ortholog, rarely observing broad ability to replace within gene families. These results suggest that within-species paralogs do indeed diverge in function at a higher rate than between-species orthologs. Finally, while we have thus far tested human gene replaceability as one-gene-at-a-time in yeast, we will show our extensions of this work to humanize yeast biological processes in their entirety.

28 Origins, diversity and evolution of industrial Saccharomyces interspecific hybrids. J.L. Gordon1,2,3, B Gallone1,2,3,4,5, J Steensels1,2,3, S Mertens1,2,3, R Wauters1,2,3, F Thesseling1,2,3, F Bellinazzo1,2,3, V Saels1,2,3, B Herrera1,2,3, M Hutzler6, P Malcorps7, B Souffriau7, L Daenen7, G Baele8, S Maere4,5, K.J. Verstrepen1,2,3 1) VIB – KU Leuven Department of Microbiology, VIB, Leuven, Belgium; 2) CMPG Laboratory of Genetics and Genomics, Department M2S, KU Leuven, Gaston Geenslaan 1, B-3001 Leuven, Belgium; 3) Leuven Institute for Beer Research (LIBR), Gaston Geenslaan 1, B-3001 Leuven, Belgium; 4) Ghent University, Department of Plant Biotechnology and Bioinformatics, (Technologiepark 927,) 9052 Ghent, Belgium; 5) VIB Center for Plant Systems Biology, (Technologiepark 927,) 9052 Ghent, Belgium; 6) Research Center Weihenstephan for Brewing and Food Quality, TU München, 85354 Freising, Germany; 7) AB-InBev SA/NV, Brouwerijplein 1, B-3000 Leuven, Belgium; 8) Department of Microbiology and Immunology, Rega Institute, KU Leuven – University of Leuven, Herestraat 49, 3000 Leuven, Belgium. The Saccharomyces species complex is made up of closely related species that are able to cross and form hybrids. Hybridization is thought to be a fast route to the development of new phenotypes by bridging together different genetic combinations in one genome and by inducing profound changes in genome structure. Despite a rapidly increasing body of work revealing how frequently Saccharomyces hybrids occur, especially in industrial settings, we still know relatively little about their origin and diversity. From a collection of more than >200 hundred yeasts from different environments (beer/brewery, wine, bread, bioethanol, sake, cider etc.) we identified and characterised around 60 Saccharomyces interspecific hybrids with different combinations of Saccharomyces parental species. We show that the formation of these hybrids happened within the industrial environment and is niche-specific: beer and wine hybrids have different origins. We also describe how hybridization induces severe aneuploidies, chromosomal rearrangements and chromosomal conversion between the sub-genomes, which lead to unique genome structures in each hybrid combination and environment. To further explore the genome structure and short-term evolution of a beer hybrid in an industrial setting we performed PacBio sequencing of clones and Illumina sequencing of a population sample from an initial frozen stock as well as after one year of fermentation. Together these analyses shed light on the diversity and evolution of Saccharomyces hybrids.

29 The ecology and evolution of yeast hybrid speciation in the wild. Guillaume Charron1, Jean-Baptiste Leducq2, Lou Nielly-Thibault1, Chris Eberlein1, Mathieu Hénault1, Souhir Marsit1, Christian R. Landry1 1) Institut de Biologie Intégrative et des Systèmes (IBIS), Université Laval, Québec City (Québec), Canada; 2) Département des Sciences Biologiques, Pavillon Marie- Victorin, Université de Montréal, Montréal (Québec), Canada. Hybridization is a powerful mechanism to generate novelty and, ultimately, biodiversity through speciation. However, well- supported cases of homoploid hybrid speciation are limited to plants and animals, which indicates that either unicellular eukaryotes have been overlooked or that this mechanism is restricted to multicellular organisms. Data from experiments and brewing facilities show that yeast hybrid species can be formed, suggesting that it could occur in natural settings and thus contribute to yeast diversity. Using population genomics, experimental evolution and fitness assays, we show that homoploid hybrid speciation took place recently in natural populations of Saccharomyces paradoxus inhabiting the North American temperate forests. The hybridization between the two indigenous North-American lineages left an introgressed lineage at the overlap between the two parental distribution. We find (1) reproductive isolation between hybrid and parental species, (2) evidence of hybridization in the genome, and (3) a link between hybridization and reproductive isolation. The hybrid species also displays specific growth phenotypes which might reflect a new ecological niche. Experimental crosses also suggest that early generations hybrids, under experimental conditions, display heterosis which should promote hybridization. On the other hand, these hybrids also suffer from low fertility, which could significantly reduce the impact of hybridization or even prevent it through reinforcement. Using experimental evolution, we show that neutral mitotic proliferation can be sufficient to overcome these reproductive barriers, allowing for successful hybrids to diversify and diverge through sexual reproduction. Our work provides insights on the evolution of hybrid lineages and on the processes underlying the formation of introgressed species after hybridization.

30 Rewiring of cell cycle-regulated gene expression by calcineurin. C.M. Leech, H.E. Arsenault, M.J. Flynn, J. Ou, H. Liu, L.J. Zhu, J.A. Benanti Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA. Upon exposure to environmental stresses, cells activate pathways that promote adaptation and restore homeostasis. During this process, cells transiently arrest the cell cycle. The mechanisms that coordinate these diverse responses are not well understood. We previously found that the stress-activated phosphatase calcineurin (CN) dephosphorylates and inactivates the S-phase transcription factor (TF) Hcm1, which is a key regulator of genome stability in budding yeast. Here, we show that CN also inactivates additional TFs in the cell cycle-regulatory network, leading to widespread changes in cell cycle- regulated gene expression. In addition to targeting Hcm1, CN promotes the dephosphorylation of the downstream TFs Fkh2 and Ndd1, displacement of Ndd1 from G2/M promoters, and downregulation of Fkh2/Ndd1 target genes. Moreover, CN 16 activation downregulates genes that are expressed at the G1/S transition and required for entry into the next cell cycle. In contrast to late cell cycle TFs, inhibition of G1/S-regulatory TFs is indirect and occurs through CN-dependent activation of the osmostress-activated MAPK Hog1. We also find that CN activation contributes to a G1/S cell cycle arrest. In sum, our results reveal an unexpected crosstalk between the CN and Hog1 stress response pathways and demonstrate that CN functions to coordinate the stress response with a transient cell cycle arrest.

31 SCARSeq, a method for rapidly assessing transcriptional changes during replicative aging, reveals highly divergent aging patterns across environments. J. Rogers, D. Gottschling Calico Life Sciences LLC, South San Francisco, CA. The replicative potential of S. cerevisiae is segregated asymmetrically; mother cells accumulate several phenotypic hallmarks of aging over their lifespan (~15-30 cell divisions), yet they give birth to rejuvenated daughter cells that exhibit full replicative potential. Despite an ever-growing list of genetic and environmental factors that affect replicative lifespan, we still lack a unified model that explains how cells age and how the natural aging process is affected by perturbations that alter lifespan.

Here we introduce SCARSeq (Sorting Cells by Age, followed by RNA Sequencing), a FACS-based method to separate a mixed population of cells into its constituent ages for RNA sequencing. SCARSeq rapidly generates a pseudo- timecourse transcriptional profile of yeast cells during their first ~10 divisions. Because cells are sorted from a single batch culture, SCARSeq avoids the need for lengthy aging timecourses which are prone to time-dependent batch effects. Furthermore, it avoids cellular stresses commonly associated with standard old cell enrichment and isolation protocols. Finally, it enables easy subsorting of age-matched cells on secondary reporters, such as those for stress response.

Using SCARSeq, we are characterizing the aging process across a range of environmental conditions and genetic backgrounds. Several themes have emerged: First, aging is a global phenomenon; consistent with previous studies, over 2000 genes significantly change expression as cells age. Second, aging starts early; even populations of cells that have divided only ~5 times exhibit age-related changes that become much stronger in older cells, including an upregulated stress response, subtelomeric gene expression, and loss of rDNA silencing. Third, environment trumps genetics; two genetically different strains grown in the same medium exhibit remarkably similar age-related transcriptional changes (cor ~ 0.8), whereas the aging transcriptomes of the same strain grown in different media are often highly divergent (for YNB vs. SC, cor ~ 0.4). Experiments are ongoing to use biological age reporters (time until death) to transcriptionally profile cells that are the same replicative age yet are closer to or farther from death, providing a crucial step forward in identifying the aging factors that ultimately limit lifespan.

32 Mutations that alter the DNA entry-exit site of the nucleosome impair transcription termination. A. E. Hildreth, K. M. Arndt Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA. In eukaryotes, transcription is controlled by factors that remove or modify nucleosomes, allowing RNA polymerase II (Pol II) to contact otherwise occluded DNA. The mechanisms by which this occurs are well understood in regard to transcription initiation and elongation. Despite a few studies showing that transcription-coupled histone modifications and chromatin remodelers are important for proper transcription termination at a few genes, little else is known about the role of chromatin at this final step. To fill this knowledge gap, we conducted a genetic screen in Saccharomyces cerevisiae for histone mutants with termination defects. We identified histone residues which, when altered, cause read-through of a well-characterized reporter containing the termination element of a Pol II-transcribed snoRNA gene. Interestingly, many of these residues reside in or near the DNA entry-exit site of the nucleosome. This protein surface, which includes portions of histones H3 and H2A, is important for regulating the stability of the nucleosome. Genome-wide analysis of our termination-defective histone mutants reveals altered nucleosome occupancy. RNA sequencing data from two of the H3 mutants reveal increased and decreased levels of many mRNAs and terminator read-through of most Pol II transcribed snoRNAs, likely explained by their altered nucleosome occupancy. Led by previous studies, we investigated Pol II elongation rate and elongation-coupled histone modifications in these mutants, but defects in these processes do not correlate with the termination defects. Another likely hypothesis, in line with our nucleosome sequencing and data from other labs, is that a stable nucleosome is required within the termination region to act as a physical roadblock to the polymerase. We have tested this hypothesis by integrating a nucleosome superbinder sequence that strongly binds histone proteins to form a stable, site-directed nucleosome. Nucleosome occupancy, as observed by histone ChIP, increases at the targeted location in a DNA entry-exit site mutant harboring the superbinder sequence. Insertion of this sequence suppresses terminator read-through of a candidate snoRNA to the level of a wildtype control. Together, these data implicate the DNA entry-exit site as an important player in maintenance of chromatin organization that supports proper transcription termination.

33 Memory of stress response in S. cerevisiae. Zacchari Ben Meriem1,2, Pascal Hersen1, Emmanuelle Fabre2 1) Laboratory of Complex Matter and Systems, University Paris VII, France; 2) Laboratory of Pathologiy and Molecular Virology, Saint-Louis Hospital, Paris, France. Cells undergoing external stress respond by dynamically activating the expression of specific genes. In budding yeast, better tolerance or faster transcriptional response have been described for successive osmotic or nutritive stresses respectively. 17

Various hypotheses have been proposed to explain the mechanisms behind those different effects such as the influence of the chromatin or the involvement of specific cytoplasmic factors. However it remains unclear how such effects are established. To address these questions we have investigated the response to repeated hyperosmotic stresses in S. cerevisiae. We used a microfluidic system to expose the cells to short repeated stresses while making single-cell measurements of a fluorescence reporter under the control of the STL1 promoter. The STL1 locus is endogenously located in the subtelomere of the right arm of chromosome IV, a domain of low transcriptional activity in absence of stress. Many other stress response genes are also located at subtelomeres. Our results show that at the single-cell level, cells display a dynamical variability in their response to repeated stresses. We have classified this variability according to five typical single-cell profiles. The more predominant profile consists in a decrease of the amplitude of the genetic response upon stress. We define this phenomenon as “memory effect”. Transcriptional inhibition experiments show that this memory effect does not require de novo protein synthesis, suggesting an influence of the chromatin context. We therefore moved the promoter of interest to a distinct, centromeric, chromatin domain. This led to a decrease in the activity of the promoter upon stress and the loss of the memory effect. This suggests that a specific level of transcriptional activity, which depends on genomic location, is required for the emergence of the memory effect. We also observed that some cells did not respond to all pulsed stresses. As shown by stochastic simulations, this occurrence can be explained by a variable delay among cells between stress sensing and the start of the transcriptional response. Altogether our study shows that osmotic stress memory depends on chromosomal position and on the level of transcriptional activity. Evolutionary organization of the stress response genes in the nucleus might be involved in the emergence of events such as the memory effect.

34 Homotypic cooperativity and collective binding are determinants of bHLH specificity and function. Christian Shively, Jiayue Liu, Xuhua Chen, Michael Wilkinson, Rob Mitra Genetics and Center for Genome Sciences and Systems Biology, Washington University of St ouis, St Louis, MO. Eukaryotic cells often express transcription factors (TFs) that bind to very similar DNA sequences in vitrobut bind at different genomic loci and perform different functions in vivo. To understand how this in vivospecificity is achieved, we analyzed two yeast bHLH proteins, Cbf1 and Tye7, which have nearly identical binding preferencesin vitro, but bind at almost completely non-overlapping target loci in vivo. We dissected the determinants of specificity for these two proteins by making a number of chimeric TFs where we swapped different domains[RM1] of Cbf1 and Tye7 and determined the effects on in vivobinding and cellular function. From these experiments, w[RM2] e learned that the Cbf1 dimer achieves its specificity by binding cooperatively with other Cbf1 dimers bound nearby. This type of interaction, known as homotypic cooperativity, allows Cbf1 to outcompete Tye7 at most promoters that contain their shared consensus binding site. In contrast, we found that Tye7 achieves its specificity by binding cooperatively with three other DNA binding proteins, Gcr1p, Gcr2p, and Rap1p. Tye7 binds in an “all-or-none” fashion with its partner TFs, and most promoters (63%) that are bound by Tye7 do not contain a consensus Tye7 binding site. Instead, different Tye7-bound loci contain different subsets of Gcr1/2, Rap1, and Tye7 motifs, and these motifs display no fixed spacing or orientation. These features are the hallmarks of a “TF Collective”, a recently described but poorly understood model of TF cooperativity. Using this information, we were able to build simple models to accurately discriminate bound and unbound genomic loci for both Cbf1p and Tye7p (AUROCs of 0.92 and 0.96, respectively) and to assign bound targets to the correct TF. To demonstrate the generality of these findings, we show that Usf1p, a human homologue of Cbf1 that is a therapeutic target for obesity and metabolic disease, also utilizes homotypic cooperativity to achieve its function. These results demonstrate that the genome-wide binding targets of paralogous TFs can be discriminated using sequence information alone, and provide lessons about TF specificity that can be applied across the phylogenetic tree.

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35 Transcript isoform toggling: a novel and widespread gene regulatory mechanism in budding yeast meiosis. G.M. Otto1, Z. Cheng1, E.N. Powers1, A. Keskin2, P. Mertins3, S.A. Carr3, M. Jovanovic2, G.A. Brar1 1) Department of Molecular and Cell Biology, UC Berkeley, Berkeley, CA; 2) Department of Biological Sciences, Columbia University, New York, NY; 3) Broad Institute of MIT and Harvard, Cambridge, MA. Decoding of genetic information from DNA to protein is crucial for the establishment and maintenance of cell identity, particularly during times of cellular change. To better understand the gene regulatory processes governing developmental change, we collected parallel, genome-wide measurements of mRNA, translation, and protein abundance through meiosis in budding yeast. We were surprised to find hundreds of genes for which mRNA and protein abundance were anti-correlated over time, which cannot be explained by traditional models of gene regulation. We show that for at least 380 genes, this regulation arises from developmentally timed switching between the production of a canonical, well-translated isoform and a 5' extended isoform that is poorly translated and does not result in protein production. Through this mechanism, a single transcription factor can drive up- or down-regulation of protein production from its target loci, a distinction that depends not on how much transcript is produced, but rather on the type of transcript produced. I will discuss the interesting consequences of this mechanism for how we think about gene regulation, as well as evidence that this mechanism is employed in contexts of cellular change beyond that of meiotic differentiation. 18

36 A high-throughput yeast assay to test the activity of CYP2C9 variants. C. Amorosi1, E. Wong1, K. Sitko1, M. McDonald2, A. Rettie2, D. Fowler1, M. Dunham1 1) Genome Sciences, University of Washington, Seattle, WA; 2) Medicinal Chemistry, University of Washington, Seattle, WA. The field of pharmacogenomics is currently overwhelmed by the huge amount of genetic variation being discovered by new sequencing efforts. One key limitation is the lack of corresponding functional annotation of these gene variants that would allow the field to link them to clinically actionable drug interactions. We are addressing this problem in a particularly important pharmacogene: CYP2C9. CYP2C9 encodes an enzyme responsible for metabolizing many different drugs including warfarin, a widely-prescribed oral anticoagulant with a narrow therapeutic window. Efforts to comprehensively characterize CYP2C9 and other pharmacogene variants have been hindered by the low-throughput nature of classic biochemical assays. Instead, we have developed a yeast-based activity assay that can test variants at high-throughput in a pooled manner. This assay, which uses activity-based protein profiling, is able to recapitulate the activity of known variants in both individual and pooled tests. Briefly, yeast cells expressing a single CYP2C9 variant are bound in an activity-dependent manner by a modified CYP2C9 inhibitor and are then labeled with a fluorophore for cell sorting and sequencing. Key improvements to the assay came from yeast strain background engineering. This included yeast humanization by adding other human metabolism enzymes, screening different yeast strain backgrounds, and making targeted strain background modifications, all with the goal of increasing the activity of CYP2C9 expressed in yeast. We found that the greatest increases in activity corresponded to strain humanization, followed by strain background choice. Surprisingly, we discovered that expressing CYP2C9 in a humanized sake stain produced five-fold higher levels of active CYP2C9 than the humanized lab strain. We are in the process of testing a library of all 9,800 single amino acid variants of CYP2C9 with our yeast-based assay. We will use this data to classify unknown variants and ultimately create a sequence-function map of CYP2C9 variants. Our approach will lead to advances in adverse drug response prevention by providing CYP2C9 clinical guidance for patients carrying both currently known and yet-to-be discovered alleles.

37 A global assessment of molecular fluctuations associated with cell cycle progression in yeast. B.T. Grys1,2, O.Z. Kraus1,3, H. Friesen1, M.T. Couvillion4, L.S. Churchman4, B.J. Frey1,3, C. Boone1,2, B.J. Andrews1,2 1) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada; 2) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 3) Department of Electrical and Computer Engineering, University of Toronto, Toronto, ON, Canada; 4) Department of Genetics, Harvard Medical School, Boston, MA, USA. Gene and protein expression, turnover, and localization are all subject to regulation throughout the cell division cycle. However, there has been no systematic study of multi-level regulatory events throughout the cell cycle in eukaryotes. To address this problem, we have developed a pipeline for tracking changes in protein localization and abundance over the course of the cell cycle in the budding yeast, Saccharomyces cerevisiae. This pipeline combines Synthetic Genetic Array (SGA) technology, high-throughput fluorescence microscopy of the ORF-GFP fusion collection, and sophisticated deep learning techniques to generate and analyze cell cycle-specific image-data for ~75% of the yeast proteome. Using this pipeline we have identified 825 proteins that fluctuate in abundance with cell cycle progression, and 692 proteins that change in localization. Clustering of these data has allowed us to explore putative functions of numerous unknown proteins and place them in a cell cycle context based on their proximity to established regulatory proteins. To further our analyses, we have combined these proteomic data with cell cycle-specific gene expression and translational efficiency data generated by RNA sequencing and ribosome profiling, respectively. In accordance with previous work, we identified >1200 genes regulated by transcription, and a significantly smaller portion (213) that are translationally regulated. Taken together, our systematic analyses have uncovered hundreds of genes and proteins that are regulated by one or more processes over the course of cell cycle progression, offering a unique resource for exploring the cellular motivation to regulate different groups of genes and proteins in unique ways.

38 The Plastic „Essentialome“ of Yeast. A. H. Michel, S. van Schie, B. Kornmann Institute of Biochemistry, ETH Zurich, Zurich, CH. Yeast genetic screens are instrumental to our understanding of cell biology. Yet they remain tedious and oftentimes incomplete. We present a versatile, time- and labor-efficient method to functionally explore the S. cerevisiae genome, using saturated transposon mutagenesis, coupled to high-throughput sequencing [1]. Like earlier work in S. cerevisiae [2] and insertion profiling in S. pombe [3], the method relates to Tn-seq in bacteria [4]. The principle is to saturate the yeast genome with independent insertions of the miniDs transposon [5]. Transposons cannot insert in loci that are essential in a given condition. Deep-sequencing of the transposon-genome junctions of the whole library identifies the locations that tolerate the presence of the transposon and allows to deduce those that cannot, revealing at once the corresponding set of essential genetic loci. The strength of the method relies on its ease and versatility, but more importantly on the depth and kind of information it delivers. SAturated Transposon Analysis in Yeast (SATAY) (1) reveals positive and negative genetic interactions in single and multiple mutant strains, (2) can identify drug targets. Moreover, contrary to genome-wide deletion library-based screens, SATAY (3) detects not only essential genes but also essential protein domains, (4) generates not only null but also other informative alleles, and (5) can be adapted to other yeast strains and species [6]. The initial SATAY procedure however had bottlenecks. Here, we present an improved protocol that massively increases SATAY’s throughput, boosting transposition efficiency by a factor ten. This, in combination with multiplex DNA sequencing, 19 allows us to perform the first head-on comparison of two common laboratory backgrounds, BY4741 and W303. In addition to genes expected to be differentially needed in both backgrounds, we identify new pathways essential in BY4741, but dispensable in W303. Interestingly, besides a few expected well-identified genes, no factor appeared to be required for the growth of W303 only. We discuss the potential genetic variations that may explain these differences. [1] Michel et al. (2017) eLife, 6, e23570 [2] Gangadharan et al. (2010) PNAS, 107, 21966 [3] Guo et al. (2013) Genetics, 195, 599 [4] Kwon et al. (2016) Appl Microbiol Biotechnol, 100, 31 [5] Weil & Kunze (2000) Nat Genet 26, 187 [6] Coradetti et al. (2018) eLife, 7, e32110

39 Compositional and Time-course Aware Genetic Analysis (CTG): A novel analysis platform for high-throughput functional genetic interaction screens. Brenton Munson, John Paul Shen, Samson Fong, Amanda Birmingham, Roman Sasik, Jason Kreisberg, Prashant Mali, Trey Ideker University of California San Diego, La Jolla, CA.

Genetic interactions are the unexpected deviation in an observed phenotype of a double mutant from the multiplicative combination of the corresponding single mutant effects. The budding yeast, Saccharomyces cerevisiae, has proved to be a valuable model to quantitatively measure the growth defects of double gene knockout mutants in large format arrays, where individual strains are spatially separated, and their fitness quantified through time-course imaging. To meet the demand of genome-scale genetic interaction screens in yeast, as well as, their environmental dependence, array formats as large as 6,144 colonies per plate and competitive pooled growth assays are becoming more common. These high-throughput experimental approaches, however, present several unique bioinformatic challenges. Here we present the Compositional and Time-course aware Genetic analysis (CTG) package, a novel analysis platform specifically designed to quantify the fitness of both single and double knockouts, while robustly estimating their underlying genetic interactions.

The CTG analysis model accounts for the compositional effects inherent in pooled growth experiments by performing an initial log-ratio transformation of strain abundances and leverages time course data to improve estimation of the growth rate of individual strains. The accuracy of single gene knockout growth rates is increased by imputing fitness as the latent variables in an iterative least squares fitting of all dual knockout growth rates simultaneously. Accurate quantification of fitness results in robust interaction scores, estimated using numerical Bayes sampling, which are significantly more correlated across biological replicates than previous methods (Pearson r=0.73 vs. 0.30). Furthermore, the CTG method is shown to be superior at recovering known genetic interactions by fold enrichment. To facilitate the use of the CTG method to the greater informatics community, CTG is distributed as a python package, available on GitHub.

40 Measuring the functional effect of amino acid substitutions proteome-wide using mistranslation. S.M. Zimmerman1, R.A. Rodriguez-Mias1, J. Villen1, S. Fields1,2 1) Department of Genome Sciences, University of Washington, Seattle, WA; 2) Howard Hughes Medical Institute, University of Washington, Seattle, Washington, USA 98195. Millions of missense mutations have been observed in human genomes, but functional annotation of these variants has lagged far behind their discovery. To bridge this gap, we are developing a method to measure the effects of amino acid substitutions on the physical properties and function of all proteins simultaneously. First, we engineer a low level of mistranslation in cells that inserts an incorrect amino acid at instances of a given codon. The result of the mistranslation is a complex proteome with many variants of each protein. Second, we impose biochemical selections that separate variants by function or physical property, such as stability, solubility, or activity. Finally, we use mass spectrometry to quantify each variant relative to wild type before and after selection. Depletion after selection indicates that a variant is deleterious.

To generate variants, we engineered inducible serine tRNAs with all possible anticodon sequences and expressed these tRNAs in yeast. We identified tRNAs that cause 2-12% serine substitutions at the expected codons. Surprisingly, the level of proteome-wide mistranslation does not correlate well with toxicity; some amino acid exchanges are lethal, but others cause little toxicity even at high levels. We subjected proteins from yeast expressing mistranslating tRNAs to selections for solubility and thermal stability. We measured the effect of serine substitutions at multiple amino acid sites in hundreds of proteins, and identified and validated positions where substitution causes loss of function. We are currently developing selections that interrogate other aspects of protein function, such as protein-protein interactions. Ultimately, our goal is to measure the effects of many substitutions at all sites in the proteome to discover clinically and biologically relevant variants.

41 Rerouted PKA signaling coordinates sugar and hypoxia responses for anaerobic xylose fermentation in yeast. K.S. Myers1, N.M. Riley2, M.E. MacGilvray3, T.K. Sato1, M. McGee1, J. Heilberger1, J.J. Coon2,4,5,6, A.P. Gasch1,3,4 1) Great Lakes Bioenergy Research Center, University of Wisconsin - Madison, Madison, WI USA 53704; 2) Department of Chemistry, University of Wisconsin - Madison, Madison, WI USA 53704; 3) Laboratory of Genetics, University of Wisconsin - Madison, Madison, WI USA 53704; 4) Genome Center of Wisconsin, University of Wisconsin - Madison, Madison, WI USA 53704; 5) Department of Biomolecular Chemistry, University of Wisconsin - Madison, Madison, WI USA 53704; 6) Morgridge Institute for 20

Research, Madison, WI USA 53715. Microbes, including Saccharomyces cerevisiae, can be engineered for novel metabolism to produce valuable biofuels and chemicals, but rerouting metabolic flux toward products remains a major hurdle. To dissect flux regulation in yeast, we used multi-omic network analysis, combining transcriptomics, proteomics, phosphoproteomics, and metabolomics, to examine strains with progressively improved anaerobic fermentation of xylose, a sugar abundant in cellulosic biomass. Active Protein Kinase A (PKA) signaling coupled with Snf1 phosphorylation enables anaerobic xylose fermentation by triggering a cascade of events coordinating hypoxia and sugar responses. Further, we discovered that deleting the PKA regulatory subunit, Bcy1, decouples division and metabolism, revealing phosphorylation events related to xylose-dependent growth versus fermentation. Remarkably, simply tagging the regulatory subunit of PKA generated a strain with increased rates of anaerobic xylose utilization and of ethanol production compared to the strain that had been evolved for anaerobic xylose utilization. Integrative modeling of transcriptomic, proteomic, phosphoproteomic, and metabolomic data presents the metabolic logic behind high flux of a non-native sugar under anaerobic growth conditions.

42 Interactions between two transcription factors modulate positive feedback in a eukaryotic bistable system. N. Ziv, A. Johnson UCSF, San Francisco, CA. Transcription circuits, defined as transcription regulators and the DNA cis-regulatory sequences they bind, control the expression of genes and define cellular identity. Some networks are bistable, meaning they can toggle between two stable steady states. Bistable networks are responsible for such varied processes such as irreversible decisions during cell-cycle progression and embryonic stem cell differentiation. Bistable systems must contain feedback regulation and non-linearity (such as cooperativity) within the feedback circuit, which can convert graded inputs into discontinuous switch-like outputs. A major challenge to investigating the functional role of the multiple feedback loops present in complex eukaryotic circuits is the ability to independently manipulate the different components. A bistable transcriptional network in the human commensal yeast Candida albicans controls an epigenetic switch between two distinct cell types, termed white and opaque. This network shows many features of those in higher eukaryotes including long regulatory regions and a high degree of cell type stability. Using transcriptional reporters and synthetic inducible systems, we have investigated the function of the main autoregulatory feedback loop regulating white/opaque switching. We can isolate specific regulatory interactions by recapitulating aspects of the bistable system in a heterologous cellular environment. We find that both inhibitory and activating interactions are possible between two transcription factors dependent on particular relative concentrations. Quantitative molecular analysis of bistability in the white-opaque circuit can serve as a model for the general understanding of complex circuits.

43 Epigenetic memory banks: prions that modify RNA. D.M. Garcia, E.A. Campbell, C.C. Gill, Y. Chen, C.M. Jakobson, D.F. Jarosz Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA. Cell size and the rate of cell division can both strongly impact organismal lifespan. At the molecular level, protein synthesis is linked to all of these parameters. In most cases the decision to live fast and die young, or live slow and die old is set genetically. In fluctuating environments, however, switching between these two strategies could be advantageous. We discovered a prion-like epigenetic state precipitated by a deeply conserved RNA-modifying enzyme (RME) that causes yeast to proliferate, and age, rapidly. These cells are larger in size, have increased protein synthesis, and have an altered response to TOR pathway inhibition, consistent with their growth traits. This state thus provides a means by which cells can epigenetically and reversibly adopt different proliferation and aging programs. We extended our investigation to ask whether any other RMEs can precipitate analogous epigenetic, stress-responsive growth states. We conducted a screen of all yeast RMEs, by altering their expression transiently under dozens of stresses, and then testing for the initiation of long-lasting, stress- responsive “memories” many generations later. We discovered eight more examples, arising from RMEs with diverse biochemical activities. Our work suggests an unanticipated capacity for RNA modifying enzymes to initiate epigenetic changes that sharply modify cell growth across different environments. This enables exploration of how protein-based inheritance can channel environmentally-shaped epigenetic changes onto RNA.

44 Recycling of 40S ribosomes revealed by ribosome profiling of large and small subunits. David Young, Nicholas Guydosh National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD. Ribosome recycling leads to the removal of ribosomes from mRNAs after the completion of peptide synthesis and is essential for allowing the reuse of ribosomes in new rounds of translation. The ribosome dissociates from the mRNA in two stages: the 60S subunit is removed first by the ATPase Rli1 (ABCE1) and the 40S subunit is removed second in a mechanism thought to involve the proteins Tma64, Tma20, and Tma22 (eIF2D/ligatin, MCT-1, and DENR). Previous data showed these factors promote 40S removal from mRNAs in vitro and, therefore, prevent un-recycled 40S ribosomes from reinitiating new translation downstream of stop codons. However, direct in-vivo evidence of un-recycled 40S subunits persisting on stop codons in strains missing these factors is lacking. To address this, we performed 40S ribosome profiling (TCP-seq) on tma64∆/tma20∆ and tma64∆/tma22∆ strains using an enhanced protocol to increase read depth and eliminate contamination from ribosomal RNA. The data revealed widespread accumulation of 40S ribosomes on stop codons at the end of annotated coding sequences, establishing these proteins as 40S recycling factors in the cell. As expected, we also found increased 40S footprints in 3’UTRs as un-recycled 40S subunits reinitiated translation downstream. Surprisingly, we also noticed widespread accumulation of 40S ribosomes at out of frame stop codons internal to main coding sequences. To 21 determine where these ribosomes had initiated translation, we performed 40S ribosome profiling on a rpl11b∆ strain that is known to be deficient in 60S subunit production and therefore likely to slow the recruitment of 60S subunits to 40S subunits waiting at start codons. As expected, data from this strain exhibited 40S footprint accumulation on start codons of annotated coding sequences but also revealed many footprints on start codons internal to coding sequences. This result implies that “leaky scanning,” a process where 40S ribosomes fail to initiate at the main start codon and instead initiate at a downstream start codon (internal to the coding sequence), occurs widely. This phenomenon could impact the stability of these transcripts through surveillance pathways, such as nonsense mediated decay, since these small ORFs terminate near the 5’ end of the transcript. Our approach of identifying the “bookends” that demarcate the start and stop codons of open reading frames serves as a powerful tool for identifying novel translation sites throughout the transcriptome.

45 Genetic Evidence that Spt5 Overcomes a Nucleosomal Barrier to Transcription Elongation. M.J. Doody1, T.K. Quan1, A. Ortiz1, L. Valenzuela1, R. Shelansky1, S. Katzman2, H. Boeger1, G.A. Hartzog1 1) Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, California; 2) Center for Biomolecular Science and Engineering, University of California Santa Cruz, Santa Cruz, California. The eukaryotic genome is organized as a DNA/protein complex known as chromatin. The principle repeating component of chromatin, the nucleosome, restricts access to the underlying DNA, and is composed of a (H3-H4) 2 histone tetramer and two (H2A-H2B) histone dimers. Interestingly, nucleosomes over gene promoters are dynamic — coming on and off of DNA, thereby, permitting access to regulatory factors, whereas those over the transcribed body (ORF) of a gene are relatively static. This suggests that there are distinct mechanisms for overcoming the nucleosome barrier during transcription initiation and elongation by RNA polymerase II (pol II). To date, the molecular mechanism(s) that pol II employs to faithfully transcribe through ORF chromatin remains unclear.

Spt5 is a conserved and essential transcription factor that associates with pol II and interacts with many proteins that modulate chromatin structure either directly or in-directly. We have identified a novel class of histone H3 alleles as genetic suppressors of a transcription elongation defective spt5 mutant. These H3 alleles display strong ORF disruption phenotypes (cryptic initiation) and are genetically distinct from the sin, bur, and, lrs class of H3 mutations. S. cerevisiae only has one non- centromeric isoform of histone H3, but a sub-class of our H3 alleles disrupts the H3 surface that is distinct between H3.3 and H3.1 in other eukaryotes. Genome-wide mapping of MNase digested mononucleosomes of this sub-class suggests that nucleosome positioning and phasing are unaltered with respect to the transcription start-site, but nucleosome occupancy at the 3’ end of the genes is reduced. Single molecule analysis of the PHO5 gene is consistent with a transcription dependent 3’ end nucleosome loss and not random loss throughout the ORF. Other subclasses of spt5 suppressor mutations alter H3 residues that are predicted to contribute to nucleosome stability or that are known to be post-translationally modified. We propose that Spt5 normally helps to overcome the nucleosomal barrier to elongating pol II, and that the histone mutations described here decrease that barrier to allow transcription when Spt5 is defective.

46 Internal polarization landmarks interfere with gradient sensing in S. cerevisiae. A. Colman-Lerner1,2, G. Vasen1,2 1) Department of Physiology, Molecular, and Cell Biology, School of Exact and Natural Sciences, University of Buenos Aires, Buenos Aires, Argentina; 2) IFIBYNE, University of Buenos Aires - National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina. Polarity decisions are central to many processes, including mitosis and chemotropism. In S. cerevisiae, budding and mating projection (MP) towards a partner engage the same polarization machinery, centered on Cdc42. To bud, the Rsr1 localizes Cdc42 at the bud site following a complex system of protein landmarks/cues (Axl2 and Bud9 at the proximal pole and Bud8 at the distal pole). Which cues are used depend on cell type (haploid vs diploid) and on environmental conditions, but Δrsr1 or Δbud8Δbud9Δaxl2 bud randomly. In the absence a pheromone gradient, classic experiments had suggested that MPs are formed using the internal budding cues. Here we revisited this topic using fluorescence microscopy of the Cdc42 activator proteins, such as Bem1. We found three mechanisms for MP site selection, whose relative influence depends on the position in cell cycle at the time of pheromone stimulation. One mechanism, evidenced in yeast that were in G1 at the time of stimulation, depends on competition between the canonical cues, with the following strength order: Axl2>Bud8>Bud9. A second mechanism becomes evident when cues are inactivated. Despite their random budding pattern, Δrsr1 or Δbud8Δbud9Δaxl2 yeast make distal pole MPs. This distal bias depends on the activity of pheromone-specific landmark proteins. Inactivation of canonical budding cues together with pheromone-specific ones, results in a mutant that forms random mating projections when stimulated in G1. The third mechanism results in proximal MPs and it operates in yeast already committed to cell division at the time of stimulation, even in Δbud8Δbud9Δaxl2. During cytokinesis, the polarization machinery is at the bud neck. When cells enter G1, this machinery disassembles; however, we found that in the presence of pheromone it is not. Instead, cells directly initiate a mating projection next to the septum (ie, at the proximal site) using the pre-polarized structure. Do these biasing mechanisms affect MP site selection in a pheromone gradient? We compared wild- type and the landmark-free mutants in pheromone gradients using microfluidic devices. We found that yeast without landmarks aligned with the gradient significantly better than wild-type. Thus, the internal cues interfere with the extracellular gradient. In summary, these results suggest that the system of internal landmarks in response to pheromone is more complex than originally thought and that it can interfere with gradient sensing.

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47 Guanidine hydrochloride reactivates an ancient alternative septin hetero-oligomer assembly pathway in S. cerevisiae. Courtney Johnson1, Andrew Weems1, Anum Khan2, Aurelie Bertin3,4, Amy Gladfelter2, Michael McMurray1 1) Cell and Developmental Biology, Univ Colorado Anschutz Medical Campus, Aurora, CO; 2) Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC; 3) Laboratoire Physico Chimie Curie, Institut Curie, PSL Research University, Paris, France; 4) Sorbonne Universite?, Paris, France. All cellular processes require macromolecular assemblies in which distinct polypeptides interact in precise ways. The efficient assembly of these higher-order structures in the crowded cellular milieu relies on complex, but poorly understood, regulatory mechanisms that ensure the proper folding and incorporation of individual subunits. The highly-conserved septin family of cytoskeletal proteins represents a powerful model to address multisubunit complex assembly in vivo. Septins were first identified in budding yeast and found to comprise a ring-shaped array of filaments at the bud neck. The five mitotically expressed septins are highly similar at the structural level yet assemble into hetero-oligomers with strict organizational rules. Two distinct linear, non-polar, hetero-octameric protofilaments, differing in their “terminal” subunits, co-exist in the cytoplasm and polymerize end-on-end into membrane-associated filaments. We recently identified the stepwise assembly pathway that produces the two species of hetero-octamers, and found that a slow monomeric septin GTPase activity couples the ratio between the two species to the cytosolic GTP:GDP ratio. Here we explore an additional assembly pathway. We provide new evidence to support a model in which, during evolution of the fungal lineage including S. cerevisiae, the loss of GTPase activity by one septin drove incorporation of the central homodimer within hetero-octamers. Conversely, persistent GTPase activity in other fungal lineages may promote the assembly of hetero-hexamers lacking the central homodimer. Strikingly, addition of the small molecule guanidine hydrochloride to the medium promotes hexamer formation in vivo. Our data suggest that guanidinium ion binds directly at a septin-septin interface, mimicking an arginine sidechain that was lost during evolution, and thereby reactivates an ancient, alternative septin hetero-oligomer assembly pathway.

48 Mating yeast cells become competent to sense pheromone gradients by assembling signaling and trafficking proteins at the default polarity site. X. Wang, D. Stone Biological Science, University of Illinois at Chicago, Chicago, IL. Chemotropism and chemotaxis are fundamental processes required for a broad range of biological phenomena. The mating process of budding yeast (Saccharomyces cerevisiae) is the best-studied example of chemotropism to date. In mating mixtures, haploid cells interpret pheromone gradients, chemotrope toward the closest partners, and fuse to form diploid zygotes. The molecular machinery required for polarized growth has been well characterized, but how cells accurately position the polarity machinery towards the pheromone source is unclear. In vegetative cells, the pheromone receptor and its G protein are uniformly distributed on the plasma membrane (PM) throughout most of the cell cycle. In gradient-stimulated cells, it is commonly thought that the receptor and G protein polarize directly to the chemotropic site (CS), where they recruit Far1-Cdc24 and polarity proteins. In our published model of yeast gradient sensing, Gβγ-inhibition of receptor phosphorylation and internalization promotes receptor and G-protein polarization toward the source of pheromone. Despite many advances in understanding yeast chemotropism, it is still not clear how the cell switches away from the strong default polarity site (DS) pre-marked during cytokinesis and accurately positions the CS in response to shallow and dynamic physiological gradients. Here we show that in mating cells, the initially uniform receptor and G protein first concentrate at the DS, then redistribute along the PM until they reach the CS. This redistribution depends on a treadmilling mechanism: exocytosis and endocytosis are biased to the leading and lagging edges of the receptor/G-protein crescent, respectively. When the receptor/G-protein crescent reaches the CS, the endocytic machinery surrounds the growth site. We also show that polarization of Gβ to the DS is dependent on the Far1-Cdc24 interaction. In addition, we show that the receptor polarizes as multiple patches at random sites on the PM of bud1Δ cells before redistributing to the CS. In some cases, bud1Δ cells mate with more than one partner. Based on these data, we propose the following model of yeast gradient sensing: Signaling, polarity, and trafficking proteins are concentrated and spatially organized at the DS to generate what we call the gradient tracking machine (GTM). Gradient-induced asymmetric activation of the receptor and G protein triggers feedback mechanisms that separate exocytosis and endocytosis, thus enabling redistribution of the GTM upgradient. The strong intrinsic polarity of the DS ensures that only one GTM is assembled, which promotes mating fidelity.

49 Motifs related to RNA recognition motifs activate a MAPK. Chong Wai Tio, Timothy Phillips, Abhimannyu Rimal, Edward Winter Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA. Smk1 is a meiosis-specific mitogen activated protein kinase (MAPK) that controls the post-meiotic program of spore formation in S. cerevisiae. Although Smk1 is activated by phosphorylation of a threonine and tyrosine in its activation loop, it is not phosphorylated by a dual specificity MAPK kinase. Instead, Smk1 is activated by a sequential, two-step mechanism, as different stages in meiosis are taking place. Step 1 occurs during meiosis I, when the CDK activating kinase, Cak1, phosphorylates Smk1’s activation-loop threonine. This produces a diffusely-localized pool of monophosphorylated MAPK that is primed for activation. The second step requires Ssp2, a meiosis-specific protein that binds the MAPK and triggers the intramolecular autophosphorylation of Smk1’s activation loop tyrosine. Ssp2 is itself tightly spatiotemporally regulated and it forms a complex with Smk1 at the membranes that surround the haploids formed during meiosis II. Here we show that a fragment of Ssp2 that is sufficient to activate Smk1 contains two segments that are similar to RNA Recognition Motifs (RRMs). Mutations in either of these motifs eliminated Ssp2’s ability to activate Smk1. In contrast, the segment of Ssp2 linking the RRM-like motifs tolerates substantial mutational perturbation and can even be replaced with a flexible linker without eliminating functionality. Moreover, the two motifs can activate Smk1 when produced as separate proteins in a heterologous 23 expression system. Incorporation of non-natural cross-linkable amino acids into different positions of the Smk1 and Ssp2 proteins shows that both RRM-like motifs bind Smk1 and that one of them binds near the ATP-binding pocket of the MAPK. These findings suggest that RRMs have been repurposed during evolution to directly activate protein kinases.

50 Complex interplay of phosphorylation events regulate the Mth1 co-repressor in the Sensor/Receptor-Repressor glucose signaling pathway. J. B. Pierce, M. Johnston Biochemistry and Molecular Genetics, University of Colorado - Anschutz Medical Campus, Aurora, CO. The Snf3/Rgt2-Rgt1 (Sensor/Receptor-Repressor, or SRR) pathway detects extracellular glucose through the Snf3 and Rgt2 glucose sensors and induces expression of the HXT genes encoding glucose transporters, enabling cells to import glucose. In the absence of glucose, Mth1 acts in the nucleus as a co-repressor with the Rgt1 DNA-binding protein to repress HXT gene expression. Mth1 interacts with the glucose sensors at the cell membrane, where it becomes phosphorylated by Yck1/2 when cells encounter glucose. Mth1 is consequently ubiquitinated and then degraded, relieving repression of HXT gene expression. We observed that shuttling of Mth1 between the nucleus and the cytoplasm is not regulated by glucose. Previous studies identified eight serine residues in conserved Yck1/2 consensus phosphorylation sequences in Mth1 required for its degradation. We found that phosphorylation of only three of these eight serine residues – Ser133 and Ser126 or Ser130 – is required for degradation of Mth1. However, an additional glucose induced event is necessary to trigger Mth1 degradation in response to glucose. Furthermore, we discovered that phosphorylation of Ser133 is required for the interaction between Mth1 and the SCF ubiquitin ligase F-box protein Grr1. Our results reveal a complex interplay of glucose-induced phosphorylation events that regulate the Mth1 co-repressor.

51 Sfp1 regulates the SAGA component Tra1 during proteotoxic stress in Saccharomyces cerevisiae. Y. Jiang1, M.D. Berg2, J. Genereaux2, K. Ahmed1, M.L. Duennwald1,3, C.J. Brandl2, P. Lajoie1 1) Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, CA; 2) Biochemistry, The University of Western Ontario, London, Ontario, CA; 3) Pathology and Laboratory Medicine, The University of Western Ontario, London, Ontario, CA. Proteotoxic stress triggers transcriptional changes that allow cells to cope with aberrant accumulation of toxic misfolded proteins. Several signaling events, including chromatin remodeling, control gene expression in response to the accumulation of misfolded polyQ expansions associated with Huntington’s disease (HD). Tra1 is an essential component of both the SAGA/SLIK and NuA4 transcription co-activator complexes and is linked to multiple cellular processes associated with misfolded protein stress. Due to Tra1 incorporation in both SAGA and NuA4, cells with compromised Tra1 activity display phenotypes that are not identical to deletions encoding components of either complex. Thus, Tra1 potentially has unique regulatory roles in the cellular response to protein misfolding. Here, we used a yeast model of HD to define the relationship between Tra1 expression and function and misfolded polyQ expansion toxicity. We found that deleting genes encoding SAGA, but not NUA4 components exacerbates polyQ toxicity. Cells carrying a mutant Tra1 allele similarly displayed increased sensitivity to expanded polyQ. Interestingly, polyQ expansion also upregulated the expression of TRA1 and other genes encoding SAGA components, thus revealing a feedback mechanism that maintains SAGA integrity. Moreover, deleting the TORC1 effector SFP1 specifically abolished upregulation of TRA1 upon expression of misfolded polyQ. We also found that Sfp1 regulates Tra1 nuclear localization. While Sfp1 is known for adjusting ribosome biogenesis and cell size is response to stress, we identified a new role for Sfp1 in the control of Tra1, linking TORC1 and cell growth regulation to functions of the SAGA acetyltransferase complex during misfolded protein stress.

52 Inhibitory codon pairs are conserved in budding yeast. DH Ghoneim1,2, X Zhang1,2, CE Brule1,2, DH Mathews1,2, EJ Grayhack1,2 1) Department of Biochemistry and Biophysics, University of Rochester, Rochester, NY; 2) Center for RNA Biology, University of Rochester, Rochester, NY. Nonrandom usage of synonymous codons and codon pairs (adjacent codons) has been observed in all domains of life and can be a mechanism for translational regulation. There is evidence that codon bias is generally driven by selection for optimal translation, however, translational selection on codon pairs has not been previously established. We calculated the conservation of all codon pairs across the Saccharomyces sensu stricto yeasts and found that nine codon pairs associated with significantly reduced expression and translation rate in vivo (Gamble et al. 2016) are more conserved than expected based on the conservation of their constituent codons. Seven of these nine inhibitory codon pairs are the most highly conserved ways of encoding their dipeptides, even compared to their synonymous optimal codon pairs. The degree of high conservation of these codon pairs correlated with ribosome enrichment at the pair, consistent with a selection for slow translation. Furthermore, genes in which these inhibitory pairs are conserved were found to be depleted from polysomes, (Heyer and Moore 2016). We similarly observed higher than expected conservation of a subset of codon pairs in Candida species, fungi that diverged from Saccharomyces ~270 million years ago. The subset of highly conserved codon pairs in Candida is enriched for codons decoded by wobble interactions, similar to highly conserved Saccharomyces inhibitory codon pairs. The selection for inhibitory codon pairs suggests a conserved role for suboptimal codon pairs in translation.

Gamble CE, Brule CE, Dean KM, Fields S, Grayhack EJ. 2016. Adjacent Codons Act in Concert to Modulate Translation Efficiency

24 in Yeast. Cell 166: 679-690. Heyer EE, Moore MJ. 2016. Redefining the Translational Status of 80S Monosomes. Cell 164: 757-769.

53 High-throughput suppressor analysis identifies molecular surfaces and contacts that likely control activation of the B complex spliceosome. D.A. Brow Dept Biomolecular Chemistry, Univ Wisconsin, Madison, WI. Selection of suppressor mutations that correct growth defects caused by a substitution in an RNA or protein is a fruitful strategy for identifying important molecular structures and interactions in living cells. This approach is particularly useful for complex biological pathways involving many proteins and/or RNAs, such as pre-messenger RNA splicing. When a large enough number of suppressor mutations is obtained, it is possible to map functional molecular interfaces in proteins and RNAs (Montemayor et al. 2014). However, the laborious and expensive task of identifying suppressor mutations in whole- genome selections limits the utility of this approach. Here I show that the identification of mutations in the Saccharomyces cerevisiae genome that overcome a block in the pre-mRNA splicing cycle can be greatly accelerated by the use of a custom targeted sequencing panel that interrogates 112 genes encoding splicing factors. The identified suppressors implicate specific residues of the splicing proteins Brr2, Prp8, Prp31, Sad1, and Snu114, as well as the recently identified spliceosome cofactor inositol hexakisphosphate, in the transition from the catalytically inactive spliceosomal B complex to the partially activated Bact complex. This approach should be generally useful for identifying large numbers of suppressor mutations in complex biological assemblies or pathways for which most or all of the constituent macromolecules have been identified. Montemayor, E.J., et al. 2014. Nat. Struct. Mol. Biol. 21: 544-551.

54 Functional Analysis of Disease Mutations Identified in RNA Exosome Genes using Budding Yeast. Milo Fasken1, Sara Leung1, Liz Enyenihi1,2, Laurie Hess1, Jillian Losh4, Maria Sterrett1,2,3, Munira Basrai5, Ambro van Hoof4, Anita Corbett1 1) Department of Biology, Emory University, Atlanta, GA; 2) Emory Initiative to Maximize Student Development, Laney Graduate School, Emory University, Atlanta, GA; 3) Graduate Program in Biochemistry, Cell, and Developmental Biology, Laney Graduate School, Emory University, Atlanta, GA; 4) Department of Microbiology and Molecular Genetics, University of Texas Health Science Center-Houston, Houston, Texas; 5) Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institute of Health, Bethesda, Maryland. The RNA exosome is an evolutionarily conserved, riboexonuclease complex that processes/degrades numerous classes of non-coding RNA. The 10-subunit core exosome forms an ring-like structure composed of an upper ring of three S1/KH cap subunits (yeast Rrp4/40/Csl4-human EXOSC2/3/1), a lower ring of six PH-like subunits (yeast Rrp41/42/43/45/46/Mtr3-human EXOSC4/7/8/9/5/6), and a 3’-5’ riboexonuclease subunit, Rrp44/DIS3, at the base. The nuclear exosome contains an eleventh, cap-associated riboexonuclease subunit, Rrp6/EXOSC10. RNAs are targeted directly to Rrp6 or threaded through the central channel of the exosome to Rrp44 for processing/degradation. Recently, mutations in four exosome subunit genes, EXOSC2/RRP4, EXOSC3/RRP40, EXOSC8/RRP43, and EXOSC9/RRP45 have been linked human diseases. Mutations in EXOSC2 cause a novel syndrome characterized by retinitis pigmentosa, hearing loss, and mild intellectual disability. In contrast, mutations in EXOSC3 and EXOSC8 cause pontocerebellar hypoplasia type 1b and 1c, respectively - autosomal recessive diseases characterized by cerebellar hypoplasia and neuronal degeneration with early mortality - and mutations in EXOSC9 cause cerebellar atrophy. To gain insight into the functional consequences of the mutations in EXOSC2/3/8/9 identified in patients, we generated the corresponding mutations in the S. cerevisiae orthologs, RRP4/40/43/45, respectively, and examined their function in budding yeast. We find that rrp variants cause differential effects on cell growth and RNA exosome function, which could shed light on why impairments in different exosome subunits cause distinct and tissue- specific phenotypes. We show that an rrp40 variant corresponding to the severe EXOSC3 variant in PCH1b patients is unstable and does not associate efficiently with the RNA exosome in the presence of wildtype Rrp40, suggesting cells possess a mechanism to discriminate between wildtype and variant exosome subunits. We have complemented these functional studies with RNA-Seq analysis to identify specific RNA targets that are differentially affected by the changes in RNA exosome subunits that model human disease as well as a high copy suppressor screen. To extend our functional studies of EXOSC2 and EXOSC3 mutations to mammalian cells, we have performed a proteomic analysis of variant and wildtype EXOSC2 and EXOSC3 proteins expressed in neuronal cells. These data support a model where differential interactions with RNA exosome cofactors could underlie disease-specific phenotypes.

55 Dealing with excess: Mating-induced stabilization of Kar4p by down-regulation of the E3-ubiquitin ligase Ubr1p. J. Kim1,2, J. Alcantar2, W. Yun2, M.D. Rose1,2 1) Georgetown University, Washington, DC; 2) Princeton University, Princeton, NJ. Proliferation and differentiation must be tightly coordinated because many proteins that are active during differentiation would interfere with the normal progression of the cell cycle. During mating, Kar4p is highly induced by Ste12p; Kar4p then acts together with Ste12p to regulate karyogamy, via the transcriptional induction of the minus-directed kinesin motor proteins, Kar3p and Cik1p, and other proteins. Kar4p is expressed in two forms, a constitutive long form, and the pheromone-induced short form, which is initiated from an internal AUG. Kar4p-short is stable during mating, but is rapidly degraded as cells return to mitosis. Here, we demonstrate that the stability of Kar4p during mating is due to inhibition of the Ubr1p E3-ubiquitin ligase. In mitotic cells either the ubr1Δ or a RING-domain mutation stabilized Kar4p-short to the level of pheromone-treated cells. Stabilization is not limited to Kar4p; other known Ubr1p substrates, including Scc1p and Ste6p, are also stabilized during mating. Using an allele of Kar4p that does not bind Ste12p, we found that stabilization is dependent on 25 transcriptional induction by Kar4p. Screening of Kar4p dependent genes identified Srl4p as being required for stabilization during mating; in pheromone-treated srl4Δ cells, the turnover rate of Ubr1p substrates was similar to the rate in mitotic cells. Ubr1p is an N-end rule ligase that normally requires the canonical E2-conjugating enzymes Ubc2p/Rad6p. However, mutation of Ubc2p and Rad6p did not affect Kar4p stability. However, deletion of Ubc6p did cause mitotic stabilization of Kar4p similar to ubr1Δ, suggesting that Kar4p is degraded by an alternate pathway. Immunoprecipitation and LC-MS of Srl4p- associated proteins in pheromone treated cells identified both Kar4p and Ubr1p. No E2-ligases, including Ubc6p, were detected. We propose that Kar4p-induced Srl4p might block access of the E2-conjugating enzyme to Ubr1p, thereby causing down-regulation of Ubr1p and stabilization of Kar4p during mating.

56 From Petri plates to Petri Nets, and beyond. S. Oliver Cambridge Systems Biology Centre & University of Cambridge. Classical genetics starts with variant functions (mutant phenotypes) and follows their patterns of inheritance to define the genes that specify those functions (genotypes). The availability of complete genome sequences turned that approach on its head. The first complete sequence of a chromosome revealed that only 20% of the protein-encoding genes had been defined by classical genetics and that it would now be necessary to go from genotype to phenotype, rather than the other way round. To achieve this, methods of functional analysis were required that were every bit as comprehensive as the genome sequences that preceded them – transcriptome, proteome, metabolome, etc. Such comprehensive data sets offered the prospect of understanding the workings of living cells on a systems level. That meant not only defining the functions of individual genes, but also how those genes and their protein, or RNA products, interact to determine phenotype. Such interaction networks often have a bewildering complexity that can only be understood by employing computational models.

Among the first such models to be constructed were those for metabolic networks. Both logical and constraint-based models of the yeast metabolic network have been available for some time and are continually being improved by the research community. While such models are very successful at predicting the impact of deleting single genes, the prediction of the impact of higher order genetic interactions is a greater challenge. Initial studies of limited gene sets provided encouraging results. However, the availability of comprehensive experimental data for the interactions between genes involved in metabolism demonstrated that, while the models were able to predict the general properties of the genetic interaction network, their ability to predict interactions between specific pairs of metabolic genes was poor. I will examine the reasons for this poor performance and demonstrate ways of improving the accuracy of the models by exploiting the techniques of machine learning and robotics.

The utility of these metabolic models rests on the firm foundations of genome sequencing data. However, there are two major problems with these kinds of network models – there is no dynamics, and they do not deal with the uncertain and incomplete nature of much biological data. To deal with these problems, we have developed the Flexible Nets (FNs) modelling formalism. FNs were inspired by Petri Nets and can deal with missing or uncertain data, incorporate both dynamics and regulation, and also have the potential for model predictive control of biotechnological processes.

57 Quantifying how yeast cells respond to small perturbations yields evolutionary insights. K.A. Geiler-Samerotte, G. Kinsler, D. Petrov Stanford University, Palo Alto, CA. Problem: Recent experiments evolved thousands of barcoded yeast lineages to adapt to a glucose-limited environment. These thousands of replicates offer insights into Stephan Jay Gould’s famous question: will evolution happen the same way every time? It is possible to imagine many ways yeast cells might evolve to contend with glucose depletion, e.g., consuming initial glucose greedily, improving survival once glucose runs out, utilizing other resources. But what methods are available to determine the phenotypes involved in adaptation, and whether evolution happens by a similar phenotypic change every time? Though previous work sequenced 100’s of the barcoded yeast lineages, discovering 135 adaptive mutations that largely fall into 10 genes representing 2 glucose-sensing pathways, the molecular and cellular phenotypes influenced by these mutations remain difficult to discern. Understanding the collection of phenotypes involved in adaptation is relevant to practical questions in evolutionary medicine (e.g. if you prevent one phenotypic change underlying drug resistance, will others emerge?). Approach: We approach this problem by (1) subtly perturbing the environment from the original glucose-limited condition, (2) precisely re-measuring the fitness of adaptive yeast lineages in the new environment, and (3) clustering adaptive lineages based on their responses across many subtle perturbations. We re-measured the fitness of 500 adaptive yeast lineages (100 of which are sequenced) in 60 subtly different environments. For the most recent 33 environments, we are live tweeting the experiments at #1BigBatch. We use Fisher’s geometric model to determine how many unique phenotypes underlie mutant behavior, and to make predictions about how mutants will behave in new environments. Our model makes accurate predictions for most mutants when assuming a small number of phenotypes (only 2) contribute to adaptation in glucose- limitation. This validation of our method opens up novel questions, e.g. does adaptation usually happen via a limited number of phenotypic changes, or are there a larger number of unique phenotypic solutions to certain evolutionary challenges? More generally, our experiments represent a step forward in our ability to identify the molecular and cellular effects of genetic change (i.e. mapping genotype to phenotype).

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58 The dynamics of adaptive genetic diversity during the early stages of clonal evolution. Jamie Blundell1,2,3, Katja Schwartz4, Danielle Francois3, Daniel Fisher1, Gavin Sherlock4, Sasha Levy3,4,5,6 1) Department of Applied Physics, Stanford University, Stanford, CA ; 2) Department of Oncology, University of Cambridge, Cambridge UK; 3) Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY; 4) Department of Genetics, Stanford University, Stanford, CA; 5) Joint Initiative for Metrology in Biology, Stanford, CA; 6) National Institute of Standards and Technology, Gaithersburg, MD. The dynamics of genetic diversity in large clonally-evolving cell populations are poorly understood, despite having implications for the treatment of cancer and microbial infections. Here, we combine barcode lineage tracking, sequencing of adaptive clones, and mathematical modelling of mutational dynamics to understand diversity changes during experimental evolution. We find that, despite differences in beneficial mutational mechanisms and fitness effects between two environments, early adaptive genetic diversity increases predictably, driven by the expansion of many single-mutant lineages. However, a crash in diversity follows, caused by highly-fit double-mutants fed from exponentially growing single-mutants, a process closely related to the classic Luria-Delbruck experiment. The diversity crash is likely to be a general feature of clonal evolution, however its timing and magnitude is stochastic and depends on the population size, the distribution of beneficial fitness effects, and patterns of epistasis.

59 Fitness estimation of pooled amplicon sequencing studies. F. Li1,2, M. Salit3,4,6, S. F. Levy2,3,4,5 1) Department of Applied Mathematics and Statistics, Stony Brook University, Stony Brook, NY; 2) Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY; 3) Joint Initiative for Metrology in Biology, Stanford, CA; 4) National Institute of Standards and Technology, Gaithersburg, MD; 5) Department of Genetics, Stanford University, Stanford, CA; 6) Department of Bioengineering, Stanford University, Stanford, CA. Amplicon sequencing-based phenotyping of complex cell pools is a widely-used high-throughput technique, with applications in CRISPR screening, deep mutational scanning, transposon mutagenesis screening, deletion collection screening, protein-protein interaction screening, and genetic interaction screening. Standard practice for phenotyping complex cells pools is to use the fold-enrichment of genotype-specific amplicons after a period of competitive growth as a proxy for genotype fitness. Here, we show that fold-enrichment is a biased proxy for fitness because it depends on the time of growth, the mix of genotypes in the pool, and how the fitness distribution of those genotypes changes across environments. That is, enrichment measures are incomparable across environmental perturbations or between independent experiments that contain partially overlapping genotype pools. We develop an alternative fitness estimation method (Fit-Seq) that calculates relative fitness by tracking genotype abundances over several time points. Using numerical simulations, we show that Fit-Seq is accurate across different experimental designs and robust to changes in the fitness distribution. By simulating various experimental regimes, we dissect the factors that contribute to Fit-Seq errors for various fitness distributions and how to minimize these errors. Finally, we show that experimental protocols with more time points are more robust to changes in the fitness distribution.

60 Host-virus genome coevolution in laboratory populations of yeast. S. Buskirk, G. Lang Biological Sciences, Lehigh University, Bethlehem, PA. Nearly all genomes contain genetic parasites that replicate selfishly, often at a cost to the host genome, leading to evolutionary arms races between selfish genetic elements and their hosts. Most genomes, including the human genome, exhibit clear signatures of past intragenomic conflicts. Yet our understanding of intragenomic conflict is limited in that few systems exist to study the coevolution of multiple genomes in high temporal resolution.

The yeast Saccharomyces cerevisiae is host to a selfish intracellular “Killer” virus. The Killer virus is an encapsulated double- stranded RNA virus that encodes both a Killer toxin and its corresponding immunity component. An infected host secretes the toxin, which kills non-Killer-containing cells. We recently discovered that our laboratory strain — a strain that we have used extensively in experimental evolution — contains the Killer virus.

Previously we evolved ~600 replicate populations of our Killer-containing laboratory strain for 1,000 generations. We find that Killer toxin production is lost in about half of our populations. Of those populations that lose killing ability, about half develop sensitivity to the toxin. Using RT-PCR, we amplified and sequenced the viral genomes from 70 populations. We correlate changes in Killer phenotype to mutations arising in both the host and the viral genomes. We find that both genomes coevolve in response to toxin production, acquiring mutations in host genes involved in toxin entry and in the virally-encoded toxin itself. Our results establish a tractable laboratory system for the study of host-virus genome coevolution.

61 Coding and noncoding variants underlie trans-acting hotspots of gene expression variation. Sheila Lutz, Margaret Kliebhahn, Christian Brion, Frank Albert Genetics, Cell Biology, & Development, University of Minnesota, Minneapolis, MN. Genetic variation among individuals shapes traits ranging from human disease to yeast fitness. In particular, polymorphisms that alter gene expression are a major source of phenotypic variation. Different isolates of Saccharomyces cerevisiae carry dozens of loci that each influences the expression of hundreds of genes in trans. However, the molecular nature of these “hotspot” loci is largely unknown. Here, we identified the causal variants at three hotspot loci in a cross between a laboratory (BY) and a wine strain (RM). To 27 rapidly dissect these loci, we developed a genome engineering method termed CRISPR-swap. In this method, genomic regions are replaced or flanked with commonly used pFA6 series knock-out cassettes. A single guide RNA that is compatible with all pFA6 cassette(s) is then co-expressed with Cas9 to cleave and exchange the cassette(s) with the desired allele as specified by a co-transformed linear repair template. Based on computational analyses we selected a candidate causal gene at each of the three hotspot loci: OAF1, a transcription factor that activates genes involved in b-oxidation of fatty acids, RGT2, a low affinity glucose receptor, and OLE1, an essential Δ9 fatty acid desaturase. We systematically narrowed each locus to the causal variant by CRISPR-swap using a series of recombinant alleles, and by quantifying the effect of each allele on the expression of a GFP-tagged gene affected in trans by the given hotspot locus. The identified variants in OAF1 and RGT2 are within the coding region of these genes. The variant coding for Oaf1(L63S) is located near a zinc-finger DNA-binding motif. The derived BY allele is rare among yeast isolates and is almost exclusively found in this laboratory strain. By contrast, the variant coding for Rgt2(V539I) is located in a trans-membrane domain and is present in many yeast isolates (~25% frequency of the derived RM allele). Its effect on trans gene expression is sensitive to the amount of glucose in the media. The final variant is located in the fatty-acid regulated promoter region of OLE1. This variant is common (~55% frequency of the derived RM allele), exerts a subtle effect on the expression of OLE1-GFP, and a stronger effect on the expression of GFP-tagged genes in trans. Our results demonstrate that rare and common single nucleotide variants in coding and non-coding regions of the genome can alter the expression of hundreds of genes in trans.

62 Survival of the fittest: The fight for dominance in wine fermentation and the genomic adaptations that underpin yeast strain performance. Simon Schmidt, Radka Kolouchova, Jane McCarthy, Angus Forgan, Anthony Borneman Wine Biosciences, The Australian Wine Research Institute, Urrbrae, South Australia, AU. The complex interaction between yeasts and their environment is brought sharply into focus by the numerous stresses encountered during industrial fermentations. Retrospective analysis of fermentation inefficiency or failure is difficult or impossible due to the multitude of factors (and their complex interplay) that can manifest as undesirable commercial outcomes. In winemaking, these factors include the commercial yeast strain(s) used, the nutrient composition of the specific grape juice and various winemaker interventions, such as the use of sulfite or other additives. To begin to address the complex interplay between genetics and the industrial environment, a multi-faceted genomics approach was adopted to elucidate the interactions between yeast strains and wine production.

A population genetic survey was performed by whole-genome sequencing over two hundred wine strains of the yeast Saccharomyces cerevisiae, which included both commercial and environmental isolates. Following this, a broadly representative set of ninety-four strains were selected and genomically-tagged with unique DNA barcodes to enable high- throughput functional profiling under competitive growth conditions. Competition experiments were used to evaluate differential fitness in response to environmental challenges enabling the parallel determination of fitness profiles in a range of industrially relevant conditions. While environmental variables such as sugar concentration and temperature were not discriminating factors of yeast strain fitness, levels of copper and nitrogen were powerful contributors to variations in fitness between wine yeast strains.

In order to elucidate the exact genetic determinants of copper tolerance that were observed in the wine yeasts strains, bulk segregant analysis was employed on different combinations of sensitive- and resistant parental strains. Pooled genome sequencing of the F1 progeny of these crosses has identified at least two major genetic loci that contribute to these differential responses to copper. Interestingly, selection at one of these loci is predicted to have occurred as a suppressive response to copper sensitivity that was induced as a secondary phenotypic consequence to an unrelated, primary adaptive mutation which provided S. cerevisiae with increased fitness under common industrial fermentation conditions.

63 Counteracting Evolution in 200,000 liter Industrial-Scale Fermentations. A. Khankhoje, H. Jiang, P. Chua, A. Meadows Amyris Inc, Emeryville , CA. At Amyris, we engineer S. cerevisiae strains to manufacture a variety of anabolic fermentation products that can be used for an array of renewable specialty chemicals, commodity chemicals, and fuels —replacing ingredients currently sourced from non-renewable or environmentally damaging sources. We currently have several fermentation products on the market with more in the pipeline. To reduce the cost of production, we engineer strains to perform at 200,000 L Industrial-Scale with high titer, yield, and productivity for many days or weeks. A key challenge during the dozens of cell divisions required to grow biomass for these large scale fermentations is that, statistically, every single nucleotide will be mutated. Evolution can wreak havoc on high-level and long-term production as fast-growing, non-producing mutants sweep the population. This has necessitated development of approaches to counteract evolution at the industrial scale. We will describe one successful approach, which has been to develop novel, scalable, and robust genetic expression switches for industrial S. cerevisiae strains.

64 Quantifying fitness in periodically fluctuating environments. F. Abdul-Rahman, D. Tranchina, D. Gresham Biology, New York University, New York, NY. In natural environments, organisms frequently face variable conditions that fluctuate periodically. While evolutionary 28 dynamics in static environments have been well characterized using experimental evolution, the effect of fluctuating environments on selection remains less well understood. The length of the period of fluctuation has been shown to have a significant effect on evolutionary dynamics and fitness. Partly due to the challenges in experimental design, the focus of previous studies has been on periodic fluctuations that exceed generation time. However, multiple organisms have rapid physiological responses to transient changes in the environment, indicating that organisms have been evolutionarily tuned to respond to intragenerational fluctuations in the environment. Here, we characterize selection on budding yeast growing in continuous culture using various fluctuation regimes. We use BARseq, a pooled fitness assay of the gene deletion library, to assay fitness across ~4000 genotypes in three distinct selective regimes: 1) static nutrient levels, 2) transient nutrient fluctuations periodically occurring at intragenerational intervals, and 3) periodic nutrient fluctuations occurring at multigenerational intervals. Using an expanded model of the chemostat we defined, and experimentally verified, nutrient fluctuations. We quantified the distribution of fitness effects for each genotype in each selective condition. We find that multigenerational fluctuations result in decreased fitness variance among genotypes and as a result maintain greater genetic diversity than intragenerational fluctuations and static environments. Using genome- wide fitness profiles we identified cellular processes that contribute to fitness effects unique to each condition. Our study highlights the importance of dynamic environments in the evolutionary history of budding yeast.

65 On the biosynthetic mechanism coupling cell growth to division. Jan M. Skotheim, Devon Chandler-Brown, Kurt Schmoller, Jon Turner, Mardo Koivomagi, Matthew Swaffer Biology, Stanford University, Stanford, CA. While cells of a given type span a large range of sizes, most proteins and mRNA are maintained at constant, size independent, concentrations. This raises the question of how cells can achieve size-dependent signals coordinating growth and division. Recently, we showed that budding yeast size control results from cell size-independent synthesis of the cell cycle inhibitor Whi5 and size-proportional synthesis of the cell cycle activator Cln3. Larger cells have a higher ratio of cell cycle activator to inhibitor, which triggers division. This raised two key questions: (1) To what extent do individual genes’ expression deviate from constant concentration? (2) What are the molecular mechanisms that determine whether gene expression depends on cell size? To address these questions, we examined the yeast GFP-fusion library. We identified approximately 200 proteins whose abundance does not scale with cell size. Many of these genes are involved in membrane transport and DNA- templated processes, which do not scale proportionally with cell size. A targeted analysis of WHI5 and histone genes suggest cells employ both transcriptional control and protein degradation for coordinating gene expression with size. Thus, our work demonstrates a functional role for differential size-dependency of protein synthesis and gives insights into the underlying molecular mechanisms.

66 Translational control of MPS1 couples cell growth with the chromosome segregation machinery and the timing of initiation of cell division. H. Blank1, A. Alonso2, M. Winey2, M. Polymenis1 1) Texas A & M University, College Station, TX; 2) University of California-Davis, Davis, CA. Protein synthesis underpins cell growth and the rate at which cells proliferate, both from the production of bulk proteins required for an increase in total mass as well as the production of specific cell cycle regulators. Recently, our lab performed a genome-wide ribosome profiling study in budding yeast to find mRNAs that are under translational control during a cell cycle in which cells are not arrested but instead maintain their normal coupling of division with growth. Of the 17 mRNAs identified in the study, one encodes the functionally conserved kinase Mps1p, which is essential for spindle pole body (SPB) assembly and duplication in yeast. We found that MPS1 has a uORF 17 bases upstream of the main start codon. The position of the uORF is conserved in fungi. The presence of uORFs can regulate translation of the downstream main ORFs, allowing cells to respond to changes in the ribosome content by nutrients. Hence, we mutated the uORF of MPS1 (uORFm) and measured its effect on basic cell proliferation parameters. We found that overall cell size was reduced in MPS1 uORFm cells, but there was no effect on cell growth. We next examined the role of the MPS1 uORF in the kinetics of cell cycle progression. We collected small, early G1 cells and followed their budding index (BI), size, and Mps1p levels as they progressed through a synchronous cell cycle. We found that while Mps1p levels oscillated in uORF+ and uORFm cells, uORFm cells initiated a new round of cell division at a significantly reduced size (~10% smaller), accompanied with a rise in Mps1p levels . Since the critical size is reduced in uORFm cells, without any effects on birth size or the rate at which cells increase in size, we concluded that START is accelerated and the G1 shortened in cells lacking the MPS1 uORF. Next, since Mps1p is required for SPB satellite formation, the first step in SPB duplication in yeast, MPS1 uORF+ and uORFm cells containing fluorescently tagged SPB components SPC110 and SPC42 were used to monitor satellite formation in a synchronous cell cycle. As with Mps1p protein levels, we found satellite formation to occur earlier in cells with a mutated Mps1p uORF. These results suggest unanticipated growth inputs into the SPB cycle, modulating when cells commit to another round of cell division. We propose that growth/nutrient inputs, through translational control of MPS1, directly impact the chromosome segregation machinery and the timing of initiation of cell division.

67 Nuclear PP2A-Cdc55 promotes DNA replication and inhibits spindle elongation. Amy Ikui, Shoily Khondker, Jasmin Phillip Brooklyn College, The City University of New York, Brooklyn, NY. 29

DNA replication stress results in DNA damage and activates Intra-S checkpoint which utilizes a sensor kinase Mec1 to create a signaling cascade with multiple targets, most notably anaphase inhibitor Pds1, securin. Mad2-mediated Spindle Assembly Checkpoint (SAC) also plays a role in Pds1 stability during the replication stress. The Pds1 inhibits Esp1, a protease that cleaves the cohesin complex component Scc1 leading to metaphase arrest. It has been shown that Pds1 phosphorylation, at CDK-consensus sites at the C-terminal region, is crucial for interaction between Pds1 and Esp1 which is necessary for recruitment of Esp1 into the nucleus. At anaphase onset, Esp1 has two independent functions: Scc1 cleavage and spindle elongation; both are regulated by Pds1. It has been extensively studied how Instra-S checkpoint is activated by phosphorylation events by kinases, but it is less known how phosphatase is involved during the replication stress response. Here we show a role for PP2ACdc55 in replication stress in S. cerevisiae. The CDC55 gene encodes the yeast homolog of the B regulatory subunit of protein phosphatase PP2A. Cdc55 function is dependent upon its localization in S. cerevisiae. The cytoplasmic Cdc55 activates CDK and promotes mitotic entry and nuclear Cdc55 inhibits mitotic progression. The cdc55-101 mutant has been shown to exhibit Cdc55 accumulation in the cytoplasm and exclusion from the nucleus. We found that Pds1 is hyperphosphorylated in the cdc55-101 mutant during replication stress by hydroxyurea (HU). Pds1 stabilization was observed both in wild type and cdc55-101 mutant after HU treatment. Interestingly, our findings also show that the both wild type and cdc55-101 mutant inhibited sister chromatid segregation, but that spindle elongation rate was accelerated in cdc55-101 mutant compared to the wild type. We conclude that nuclear PP2ACdc55 promotes Pds1 dephosphorylation during replication stress and inhibits spindle elongation in response to replication stress. We propose a model that Pds1 dephosphoryaltion by PP2ACdc55 disrupts its ability to recruit Esp1 to the spindle, thus inhibiting spindle elongation. Our genetic analysis further showed that the PP2ACdc55 dependent signaling pathway during replication stress acts separately from known Mec1- and Mad2-mediated pathways, therefore it serves as a novel intra-S checkpoint signaling circuit targeting Pds1.

68 LINC complex assembly and homeostasis in budding yeast. Hong-Guo Yu Biological Science, Florida State University, Tallahassee, FL. The linker of nucleoskeleton and cytoskeleton (LINC) complex bridges the inner and outer nuclear membranes and can transmit mechanical forces from the cytoskeleton to regulate chromosome movement and gene expression. The canonical LINC complex is composed of an inner-nuclear-membrane (INM)-localized SUN protein, which binds to the outer-nuclear- membrane (ONM)-localized KASH protein. In budding yeast, Mps3 is the sole SUN-domain protein, whereas Mps2 and Csm4 have been considered as two KASH-like proteins, with Mps2 acting in mitosis and Csm4 specifically in meiosis. We show here that Csm4, Mps2, and Mps3 form a functional heterotrimeric LINC complex in budding yeast meiosis. The ONM-localized Mps2 serves as a scaffold that mediates the assembly of the yeast LINC complex, which modulates nuclear morphology, telomere movement and meiotic recombination. We also show that for LINC complex disassembly, Mps3 is proteolytically cleaved. Furthermore, LINC components are subject to the regulation by the ER-associated protein degradation (ERAD) pathway. Our findings therefore reveal (1) the formation of a heterotrimeric LINC complex and (2) an unexpected role of EARD in maintaining LINC homeostasis in budding yeast.

69 What determines the nuclear:cell volume ratio? Insights from growth-arrested budding yeast cells. A. Walters1, K. Amoateng1, L. Matai1, R. Wang2, J. Chen3, G. McDermott3, S. Tollis4, M. Tyers4, C. Larabell3, O. Gadal2, O. Cohen-Fix1 1) Nation Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD; 2) Laboratoire de Biologie Moléculaire Eucaryote, Centre de Biologie Intégrative (CBI), Université de Toulouse, France; 3) Molecular Biophysics and Integrated Bioimaging, Lawrence Berkeley National Laboratory, Berkeley, CA ; 4) Institute for Research in Immunology and Cancer, University of Montreal, Montreal, Canada. Most cell types exhibit a constant ratio between nuclear volume and cell volume. The mechanism(s) dictating this constant ratio, and the nuclear component(s) that scale with cell size, are not known. Formally, the size of an organelle can be regulated by limiting the size of its surface area (i.e. the size of the container) or limiting the amount of its content (such as in a balloon). To determine which of these two possibilities applies to the budding yeast nucleus, we examined what happens to nuclear size and shape when cell expansion, but not cell cycle progression, is inhibited by down-regulating components of the secretory pathway. In wild type cells, nuclear envelope expansion occurs throughout the cell cycle and it accelerates as cells approach mitosis. We find that under conditions that inhibit cell size increase but allow for cell cycle progression, the nucleus becomes bilobed, with the bulk of the DNA in one lobe and the nucleolus in the other. Bilobed nuclei formation is dependent on fatty acid and phospholipid synthesis, suggesting that the nuclear membrane continues to expand despite the inhibition to cell growth. Bilobed nuclei appeared predominantly after spindle pole body separation, suggesting that nuclear envelope expansion follows cell cycle cues, rather than cell size. Importantly, cells with bilobed nuclei had the same nuclear:cell volume ratio as cells with round nuclei, despite having a greater nuclear surface area. Taken together, our data suggest that (a) nuclear envelope expansion is not linked to cell size, and (b) that nuclear volume, which is coupled to cell size, is not determined by nuclear envelope availability but likely by one or more nucleoplasmic factors. Newly isolated budding yeast mutants with altered nuclear:cell volume ratios suggest a role for protein homeostasis in this process.

70 Role of phosphoinositides and individual subunits in the assembly, localization and function of Saccharomyces cerevisiae TORC2. Maria Nieves Martinez Marshall, Anita Emmerstorfer-Augustin, Biyun Li, Jessica Bonnar, Kristin Leskoske, 30

Françoise Roelants, Melissa Locke, Jeremy Thorner Division of Biochemistry, Biophysics and Structural Biology, Dept. of Molecular and Cell Biology, Univ. of California, Berkeley, CA 94720-3202 USA. During the past year, cryo-EM structures have been published for both yeast (S. cerevisiae) [1] and human [2] TORC2, a plasma membrane (PM)-associated, multi-subunit protein kinase complex. However, these static pictures reveal little about the biogenesis, localization, dynamics, and function of TORC2. Core components of TORC2 have been conserved from yeast to man [reviewed in 3]. We have shown that, in yeast, TORC2 plays an essential role in sensing the status of the PM, especially when subjected to various stresses, and in controlling downstream reactions that ensure PM lipid homeostasis and that modulate PM protein composition via regulation of clathrin-mediated endocytosis at several levels [reviewed in 4]. We have constructed a comprehensive library of functional fluorescently-tagged derivatives of all of the relevant components of yeast TORC2, including: Tor2 (mammalian ortholog is mTOR), Lst8 (mammalian ortholog is mLST8), Avo1 (mammalian ortholog is mSIN1), Avo3 (mammalian ortholog is RICTOR), Bit2 and Bit61 (mammalian counterparts are likely PROTOR1/PRR5 and PROTOR2/PRR5L), and Avo2, Slm1 and Slm2 (which appear to be restricted to fungi). Likewise, we have constructed versions of each of these same proteins tagged with the auxin-inducible degron, which has allowed us to remove systematically one or more of each of these constituents, as well as to eliminate selected enzymes of yeast phosphoinositide synthesis and interconversion, and thereby test the effect of their absence on the stability, subcellular distribution, and integrity of this multi-component ensemble. The results of this analytical approach will be described. 1. Karuppasamy M et al. (2017) Cryo-EM structure of Saccharomyces cerevisiae target of rapamycin complex 2. Nat. Commun. 8: 1729. 2. Stuttfeld E et al. (2018) Architecture of the human mTORC2 core complex. Elife 7: e33101. 3. Gaubitz C et al. (2016) TORC2 structure and function. Trends Biochem. Sci. 41: 532-545. 4. Roelants FM et al. (2017) The TORC2-dependent signaling network in the yeast Saccharo-myces cerevisiae. Biomolecules 7: E66.

71 The trafficking and tracking of Gbg during the pheromone response in budding yeast. R. Abdul-Ganiyu, L. Venegas Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL. Directed cell growth in response to a chemical gradient (chemotropism) is necessary for cellular processes like axonal pathfinding, angiogenesis and yeast mating. Yeast mating is a well-studied chemotropic model. In mating mixtures, yeast cells interpret shallow pheromone gradients from cells of opposite mating type, polarize their growth toward the closest potential mating partner, and fuse to form zygotes. The G protein bg subunit (Gbg) is a key component of the mating pathway. Phosphorylated Gb (GbP) inhibits receptor phosphorylation and its subsequent internalization. This promotes receptor polarization on the up-gradient side of the cell, which contributes to gradient sensing. Our computational model predicts that with internalization of ≥3% GbP, pheromone-gradient stimulation does not induce receptor polarity. Therefore, we hypothesize that in response to pheromone, GbP is preferentially left on the plasma membrane (PM) while unphosphorylated Gb (GbP-) is internalized. Consistent with our hypothesis, in a gel-shift assay, we found that the PM of pheromone-treated cells mostly contained GbP whereas the untreated cells contained GbP and unphosphorylated Gb (GbP-) in a roughly equal proportion. To better understand the dynamics of GbP trafficking, we are developing an in-vivo GbP sensor. We screened a phage-display library of Forkhead Associated (FHA) Domain variants against a GbP peptide using ELISA as a readout. We identified four variants with high affinity to GbP peptide. We fused GFP to the highest binding FHA variant, to test its utility as an in vivo sensor. We found that this fusion protein (FHA3-GFP) localizes similarly to GFP-Gb in pheromone-treated cells. Interestingly, the FHA-GFP signal at the shmoo tip is more focused, consistent with the expected localization of GbP. Pheromone treatment of cells overexpressing Gb led to an increase in the FHA3-GFP PM signal. In mating cells, FHA3-GFP redistributes from the default site to the eventual chemotropic site. These results are consistent with FHA3-GFP working as a GbP cell sensor. However, we also saw an FHA3-GFP signal in cells lacking Gb suggesting that FHA-GFP cross-reacts with other protein(s). To eliminate the possibility that our FHA reagent is binding to a protein that has similar localization as GbP, we performed a BiFC assay. Our results confirm that the FHA reagent binds to GbP and can be used as an in-vivo sensor to monitor the trafficking of GbP during yeast chemotropism.

72 The Replication Factor A2 N-Terminus is Required for Proper Progression through Meiotic Divisions. A.M. Adsero1,2, Stuart Haring1,2 1) Chemistry and Biochemistry, North Dakota State University, Fargo, ND; 2) Cellular and Molecular Biology, North Dakota State University, Fargo, ND. During the process of homologous recombination used to repair the meiotic double-strand breaks (DSBs), single-stranded DNA (ssDNA) is generated, resulting in the recruitment of Replication Factor A (RFA). This ssDNA binding complex protects the ssDNA from degradation during repair and coordinates the process of homologous recombination through recruitment of proteins such as Rad52 and Rad51. RFA is composed of three subunits, Rfa1 (70 kDa), Rfa2 (32 kDa), and Rfa3 (14 kDa), and Rfa1 has already been shown to be required during meiosis. An rfa1-t11 mutant has been shown to display reduced sporulation, low spore viability, and reduced meiotic recombination. During mitosis, Rfa2 is phosphorylated on the N- terminus (NT) in response to prolonged DNA damage. Rfa2 NT phosphorylation has also been shown to occur during meiosis, but interestingly the Rfa2 NT phosphorylation occurs regardless of whether or not the DNA has been damaged by programmed DSBs. Using rfa2 NT mutants, we investigated the role the Rfa2-NT may play during meiosis, as it has yet to be clearly determined. Our data suggest the Rfa2-NT is playing a significant role in cells proceeding through meiosis, as our mutant missing the N- 31 terminus (rfa2-ΔNx) displays reduced sporulation efficiency with a nearly 1:1 ratio of dyads to tetrads (1:10 ratio in wild-type cells) and reduced spore viability. This suggests that the Rfa2 NT is required for proper meiotic divisions, and this study rules out defective DNA replication and homologous recombination as the culprit. Phosphorylation of the domain may be dispensable during an otherwise normal meiosis as our mutant that cannot be phosphorylated (rfa2-Ax) is not distinctly different from wild-type cells. However, a phospho-mimetic mutant (rfa2-Dx) shows reduced viability. During mitosis, the Rfa2 NT phospho-state appears to play a significant role in regulation of Rad53 checkpoint activity. Although Rad53 is not normally active during meiosis, Rad53 appears to be differentially modified during meiosis in non-phosphorylatable Rfa2 NT mutant cells. Taken together, the Rfa2 N-terminus is important for meiosis, and its phospho-state may be regulating cell cycle progression in both mitosis and meiosis through regulation of Rad53.

73 eIF5A depletion impairs oxidative phosphorylation. N. M. Barbosa1, M. M. Santoni1, A. A. Pinheiro1, V. M. P. dos Santos1, A. H. Klippel1, L. C. Alberici2, J. Frydman3, B. J. Andrews4, C. F. Valentini1, C. F. Zanelli1 1) São Paulo State University, UNESP, SP, Brazil; 2) University of Sao Paulo, USP, SP, Brazil; 3) Stanford University, CA, USA; 4) University of Toronto, Ontario, Canada. Eukaryotic translation factor 5A (eIF5A) is a highly conserved protein and contains the unusual and essential posttranslational modification that generates the hypusine residue. Although eIF5A was originally described as a translation initiation factor, more recent data have demonstrated that eIF5A interacts with elongating ribosomes and facilitates the translation of specific tripeptide motifs and also stimulates translation termination. eIF5A has been associated with several cellular functions, but it is still not clear the impact of eIF5A function upon different protein levels and to which extent the depletion of some proteins is related to eIF5A mutants phenotypes. In order to better understand the influence of eIF5A on the translational control of gene expression, it was performed a large-scale proteomic screening using the ORF-GFP collection of Saccharomyces cerevisiae and the eIF5A mutant hyp2-3, using the Synthetic Genetic Array methodology. Fluorescence intensities from each individual colony were assayed using a scanning fluorimager to reveal the differential GFP expression. Gene Ontology analysis of the proteomic profile identified enrichment of cellular processes previously involved with eIF5A, such as regulation of cell cycle and translation. Interestingly, an important downregulation of specific proteins of mitochondrial complexes was also observed. Finally, we demonstrate that yeast eIF5A mutants have defect in mitochondrial oxidative phosphorylation. eIF5A might be regulating the translation of specific mitochondrial proteins, which mostly are encoded in the nucleus, synthesized in the cytoplasm and posttranslationally imported into mitochondria as unfolded peptide or cotranslationaly translocated into mitochondria.

74 Dissecting Roles for Cdc42p Adaptors in Regulating a Differentiation-Type MAPK Pathway in Yeast. Sukanya Basu1, Boyang Li1, Heather Dionne1, Garrett Kimble1, Keith Kozminski 2, Paul Cullen1 1) Department of Biological Sciences, University at Buffalo, Buffalo, NY ; 2) Departments of Biology and Cell Biology University of Virginia School of Medicine, Charlottesville, VA. Rho GTPases regulate cell polarity and signaling to control morphogenetic behaviors during development and in response to extrinsic cues. The Rho GTPase Cdc42p is a master regulator of cell polarity and signaling that controls morphogenetic responses in many settings. How Cdc42p induces a specific response in a particular setting is an important question. In yeast, Cdc42p and the p21 activated kinase (PAK) Ste20p regulate three MAPK pathways that detect and respond to different stimuli. Cdc42p also has essential functions in polarity establishment and protein trafficking (exocytosis). To define how Cdc42p differentially regulates its effector pathways, a system was developed to examine multiple Cdc42p-dependent outputs in the same cell. The system provided information on differences in MAPK pathway kinetics and uncovered pathway- specific features surrounding the filamentous growth MAPK (fMAPK) pathway. Using a library of cdc42 alleles, residues were identified that were specifically required for Cdc42p function in the fMAPK pathway. A previously identified pathway-specific regulator of the fMAPK pathway, the SmgGDS-type adaptor Bem4p, interacts with Cdc42p. Bem4p associated with the third alpha helix of Cdc42p (switch 3 region) to regulate the fMAPK pathway and control aspects of polarity establishment. We also show that the polarity scaffold Bem1p regulates the fMAPK pathway. Using point mutations that impact steps in its regulatory cycle, we show that Bem1p interacted with effectors, like Ste20p, localized to the PM, and cycled between an open and closed conformation to regulate the fMAPK pathway. Therefore, two scaffold-type adaptors (Bem4p and Bem1p) interact with the same polarity complex (Rsr1p, Cdc24p, and Cdc42p) to regulate the fMAPK pathway. Genetic suppression tests showed that the proteins operate in an ordered sequence (Rsr1p -> Bem4p -> Bem1p). Different aspects of Bem1p’s regulatory cycle played different roles in different pathways. We also show that an intact septin ring, which is required for cytokinesis and for maintaining a barrier between mother and daughter cells was required for proper regulation of the fMAPK pathway. Collectively, the study provides insight into aspects of a regulatory mechanism for the activation of a GTPase in a particular MAPK pathway. Aspects of Cdc42p regulation may extend to Rho GTPase regulation in other systems.

75 Phosphatase activity is required for Replication Factor A2 N-terminal phosphorylation mediated checkpoint exit. Trevor Baumgartner, Stuart Haring Chemistry & Biochemistry, NDSU, Fargo, ND. Cells are continually under mutagenic pressures resulting in DNA breaks or lesions that can lead to permanent DNA mutations if dealt with improperly. Mutations ultimately have the potential for disrupted gene function or dysregulation responsible for several cellular diseases, including cancer. When DNA breaks occur, a host of cellular machinery is recruited to the site of the break. One category of machinery is that used for break repair. A second category, of equivalent importance, 32 is that which works to halt the cell cycle to prevent catastrophic events while DNA repair is occurring. This arrest is known as a cell cycle checkpoint. For example, the G2/M checkpoint occurs prior to mitosis to allow the repair proteins time to fix broken DNA before chromosomes are separated and allocated to the daughter cell.

It is well established that single-stranded DNA (ssDNA) intermediates resulting from processing of DNA lesions leads to recruitment of Replication Factor A (RFA), which then recruits Mec1. The result of Mec1 kinase activity is the activation of the checkpoint through phosphorylation of Rad53. It is equally important for exit from these checkpoints to occur, however beyond Rad53 being dephosphorylated by multiple phosphatases, the mechanism(s) controlling this process remains poorly understood.

It has previously been shown that Replication Factor A2 (Rfa2) phosphorylation drives checkpoint exit in all mutants tested defective for checkpoint adaptation. Therefore, we hypothesized that Rfa2 phosphorylation stimulates phosphatase activity to promote checkpoint exit. In these studies, we show that the promotion of checkpoint exit by Rfa2 phosphorylation requires the presence of some activity of phosphatase genes (PPH3, PTC2, and PTC3) to catalyze the inactivation of Rad53. Furthermore, in the absence of one phosphatase, Rfa2 phosphorylation appears to stimulate the activity of the remaining phosphatase acting on Rad53.

Support: This work was supported by a National Science Foundation grant (NSF-CAREER-1253723) awarded to SJH. Corresponding Author’s Email: [email protected]

76 Genes involved in curing of [URE3] prion by Btn2 and Cur1 proteins. E. Bezsonov, R. Wickner Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD. [URE3] is a prion based on an amyloid form of the nitrogen catabolism regulating protein Ure2. This prion can be cured by overproduction of Btn2p (also involved in regulation of protein trafficking from late endosome to Golgi) or by overproduction of Cur1p (a paralog of Btn2). During curing of [URE3] by overproduced Btn2-RFP, Ure2N-GFP aggregates are collected at one site in the cell which colocalizes with Btn2-RFP. This colocalization of Ure2p aggregates with Btn2p strongly suggests a prion seed sequestration mechanism of curing (Kryndushkin et al., 2008). Malinovska et al. (2012) found that overproduction of Btn2p or Cur1p leads to reduction of cytosolic levels of Sis1p chaperone which is required for [URE3] propagation suggesting curing mechanism by depletion of Sis1p accessible for prion. Indeed, we found that Sis1p overproduction inhibited curing of [URE3] upon Btn2p, Cur1p or Hsp42p overproduction. However, we found that a majority of [URE3] variants generated in a double mutant of btn2 and cur1 were cured upon restoration of normal levels of Btn2p and Cur1p (Wickner et al., 2014). These variants all had low seed number, supporting the seed sequestration model. The fact that normal levels of Btn2p and Cur1p cure [URE3], and the dramatically lower levels of these two proteins compared to Sis1p argues against the Sis1p depletion model of curing. Sis1p depletion also cannot explain the co-localization of Btn2p and Ure2p amyloid during curing. Rather, the overproduction of Sis1p may sequester Btn2p and Cur1p lowering their effective concentration and preventing curing. Among other factors affecting curing efficacy by Btn2p overproduction is the Hsp42p chaperone which is required for Btn2- curing and also is able to cure [URE3] upon its own overproduction. Proteasomal activity is not directly involved in [URE3] curing by Btn2p or Cur1p overproduction since treatment of cells with proteasomal inhibitor MG132 does not influence significantly on this process. Btn2p and Cur1p are not required for each other for overproduction curing of [URE3], but Cur1p is required for Hsp42p curing. Using a genetic screen based on transposon insertional mutagenesis we found several other genes affecting Btn2-induced curing of [URE3]. Thus, among the main factors affecting [URE3] curing by the Btn2p and Cur1p antiprion systems are prion seed number and the Hsp42 chaperone.

77 Regulation of the glucose-sensing pathway by a phosphotyrosine phosphatase. T. Biswas, Mark Johnston Biochemistry and Molecular Genetics, University of Colorado Denver, Aurora, CO. Glucose, the primary carbon source for S. cerevisiae, activates the SRR (Snf3-Rgt2/Rgt1 or Sensor-Receptor/Repressor) glucose sensing pathway, which leads to expression of HXT genes encoding hexose transporters. Binding of extracellular glucose to the Snf3 and Rgt2 glucose sensors leads to phosphorylation of the Mth1 and Std1 corepressors, which directs them to ubiquitin-mediated degradation, relieving repression of the HXT genes. Because protein kinases are often paired with protein phosphatases in signaling pathways, we sought to identify phosphatases involved in the SRR glucose sensing pathway. We tested catalytic and regulatory subunits of protein phosphatases for their ability to induce or repress HXT1 expression when overexpressed. We found that over-expression of Ptp2 (phosphotyrosine phosphatase) inhibits Mth1 degradation and HXT1 and HXT3 expression in cells growing on glucose. Ptp2-mediated repression of HXT expression is dependent on Mth1 and Std1 corepressors and their DNA-binding partner, Rgt1, suggesting Ptp2 plays a direct role in the SRR pathway. However, deletion of Ptp2 has no effect on signaling. Our results implicate Ptp2 in regulation of the SRR glucose sensing pathway.

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78 Screening of the ScUbI yeast deletion library for modifiers of Aly1- or Aly2-mediated resistance to rapamycin in an undergraduate lab course. Ray Wesley Bowman, Mike Hall, the 2017 class of BIOL 371w, Allyson F. O'Donnell Biology, Duquesne University, Pittsburgh, PA. Cells respond to cues in their extracellular environment by selectively redistributing proteins. This reorganization is imperative for cell survival and is regulated, in part, by α-arrestins. How then is α-arrestin-mediated trafficking controlled? We know that modification by ubiquitination plays a role in modifying α-arrestin function. To help us identify specific α-arrestin regulators, we generated and utilized a unique yeast gene deletion library called the Saccharomyces cerevisiae Ubiquitin Interactome (ScUbI) library. ScUbI contains all the non-essential genes annotated as important for ubiquitination/ubiquitin interaction. We used this library as part of the BIOL 371w Cell & Molecular Biology laboratory course to screen for gene deletions that altered the growth phenotypes associated with over-expression of α-arrestins. Specifically, the undergraduates transformed the ScUbI library with plasmids over-expressing α-arrestins Aly1 and Aly2 and then assessed these transformants for gene deletions that either increased or decreased cells sensitivity to rapamycin, an inhibitor of TORC1 function that mimics nitrogen starvation. Three replicate screens of the ScUbI library were evaluated for changes in rapamycin sensitivity when over-expressing Aly1 or Aly2 identified 45 and 39 hits respectively as having >1 or <-1 Z-scores. Of these, 11 are overlapping between Aly1 and Aly2. We then verified candidates using growth assays and PCR confirmation of deletion backgrounds. From this, Atg7 was chosen as a strong candidate and further assessed. We show that in atg7∆ cells, Aly1 and Aly2 mobility is altered, and demonstrate that this is due to a change in phosphorylation status of the α-arrestins. Additionally, deletion of α-arrestins results in impaired autophagic flux of GFP-Atg8 following treatment with rapamycin. These data suggest that α-arrestins have a role to play in autophagy and we are currently working to define that role.

79 Superoxide dismutase activity, oxidative stress and Sod1 agglomerates in cell models expressing fALS Sod1 heterodimers. Aline. Brasil1, Mariana Carvalho1, Daniela Queiroz1,2, Marcos Pereira1, Tiago Outeiro2, Elis Eleutherio1 1) Laboratório de Investigação de Fatores de Estresse (LIFE) - Department of Biochemistry IQ / Federal University of Rio de Janeiro (UFRJ) - Rio de Janeiro, Brazil; 2) Department of NeuroDegeneration and Restorative Research - Center for Nanoscale Microscopy and Molecular Physiology of the Brain UMG - Goettingen, Germany. Among the inherited cases of familial Amyotrophic Lateral Sclerosis (fALS), 20% are attributed to heterozygotic mutations in the SOD1 gene that encodes for Cu,Zn-superoxide dismutase (Sod1). Although much has been known about fALS-Sod1 homodimers, the investigation of Sod1 heterodimerization remains poorly unexplored in the literature. In this study, an imaging approach named Bimolecular Fluorescence Complementation (BiFC) was used to analyze the heterodimeric combination of WT and the mutant Sod1 proteins (A4V, L38V, G93A and G93C) as well as to investigate the effects of Sod1 heterodimers on aggregation, antioxidant activity and dynamics properties. The BiFC allows us to image and quantify the inclusions formed by WT/mutant heterodimers by fluorescence microscopy in human neuroglioma cells (H4) and in the budding yeast Saccharomyces cerevisiae, containing either one copy of WT hSod1-VN and one copy of mutant hSod1-VC. In this study, sod1Δ yeast cells were specifically used to better evaluate the effect of the human Sod1 expression, without the endogenous Sod1 influence. Moreover, yeast cells have long served as an advantageous model to study oxidative stress response. Exponential-phase glucose-grow yeast cells only ferment and, consequently, show low levels of reactive oxygen species (ROS), which increase in chronological aged cells. In both experimental models, we confirmed the intracellular WT/mutant heterodimer formation by observing the Venus fluorescence intensity. In mutant Sod1 heterodimers, a larger number of inclusions per cell were observed in comparison to WT and mutant homodimers. This effect was particularly strong for the A4V mutant, which significantly increases in the percentage of cells with inclusions. Moreover, by using Fluorescence Recovery After Photobleaching (FRAP), the inclusions formed by the Sod1 WT/A4V showed a reduced mobility and the most stable aggregate in comparison to the other Sod1 mutants analyzed. The presence of BiFC-tagged human Sod1 inclusions were also observed and quantified in sod1Δ cells submitted to chronological aging. In addition, our results in yeast cells showed the WT/mutants hSod1 activity significantly decreased after aging compared to the cells containing the WT homodimer. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics and antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

80 An intermediate step of cohesin’s ATPase cycle allows cohesin to entrap DNA. K.K. Carlborg, G. Camdere, D. Koshland Molecular and Cell Biology, University of California Berkeley, Berkeley, CA. Cohesin is a four-subunit ATPase in the family of Structural Maintenance of Chromosomes (SMC). Cohesin promotes sister chromatid cohesion, chromosome condensation, DNA repair, and transcription regulation. Cohesin performs these functions as a DNA tether and potentially a DNA-based motor. At least one of its DNA binding activities involves entrapment of DNA within a lumen formed by its four subunits. Cohesin's DNA binding activity can be reconstituted in vitro by incubating cohesin with DNA, ATP and cohesin loader. We show that wild-type cohesin incubated with ADP aluminum fluoride (cohesinADP/AlFx) can form stable cohesin-DNA complexes. We speculate that a single nucleotide state of cohesin, likely cohesinADP-Pi, is capable of initially dissociating one interface between cohesin subunits to allow DNA entry into the cohesin lumen and subsequently interacting with the bound DNA to stabilize DNA entrapment. We also show that cohesinADP/AlFx binding to DNA is enhanced by the cohesin loader, suggesting a function for the loader other than stimulating cohesin's ATPase activity. Finally, we show that the loader remains stably bound to cohesinADP/AlFx after DNA entrapment, potentially revealing a function for the loader in

34 tethering the second DNA substrate. These results provide important clues on how SMC complexes like cohesin can function as both DNA tethers and motors.

81 Investigating mitochondrial VDAC dynamics and viral repression during yeast sporulation. S.O.Y. Chau, J. Gao, F. Chowdhury, M. Meneghini Molecular Genetics, University of Toronto, Toronto, CA. We previously showed that sporulation is a bona fide context of programmed cell death (PCD) in budding yeast, during which the remnant parental cell undergoes programed autolysis through vacuolar rupture. Provocatively, yeast PCD is accompanied by the fragmentation of genomic DNA from uncellularized meiotic products in a manner dependent on NUC1, the homolog of mitochondrial endoG. Indeed, a hallmark of apoptosis is fragmentation of genomic DNA through endoG and other nucleases. The role of this widespread phenomenon remains unclear however as perturbation of the responsible nucleases has no known consequence for PCD execution. This is also the case with yeast meiotic PCD as deletion of NUC1 has no consequence for mother cell autolysis. These observations raise the question: are there any adaptive roles for the activation of DNA fragmentation pathways that accompany PCD and what evolutionary pressures may have driven this? We have now discovered that programmed evacuation of Nuc1 from mitochondria during sporulation protects spores from detrimental consequences caused by hyper-accumulation of intracellular double stranded RNA (dsRNA) viruses that chronically infect yeast. We have also discovered that Nuc1 evacuation requires the voltage dependent anion channel (VDAC) Por1 and its paralog Por2, which appears to be meiotic specific. Our ongoing cell biological characterizations of Por1/2 suggest that Por2 distinguishes a separate population of mitochondria and these mitochondria collaborate for antiviral activity through Nuc1 release. The Por1/2-Nuc1 pathway targets the L-A virus for repression during sporulation. L-A encodes a rudimentary viral particle that upholds its propagation while its satellite “Killer” virus encodes a secreted toxin and requires the L-A viral particles for its own replication. Interestingly, in strains infected with the Killer virus, the Por1/2-Nuc1 pathway collaborates with the “superkiller” SKI complex to prevent a lethal accumulation of Killer toxin within the spores. To confirm that the lethality we observed was Killer dependent, we generated strains infected with L-A but not Killer. Fascinatingly, resulting meiotic progeny lacking both SKI complex and NUC1 function displayed strong respiratory defects that were caused by L-A itself, revealing that unrestrained L-A infection causes mitochondrial defects. Our ongoing experiments address this mutually inhibitory relationship between dsRNA viruses and mitochondria.

82 Inheritance and Biogenesis in Saccharomyces cerevisiae. A.K. Chemel, M. Chan San Francisco State University, San Francisco, CA. Saccharomyces cerevisiae, commonly known as budding yeast, forms a single bud and divides asymmetrically during its cell cycle. Previous research has shown vacuoles are crucial for cell cycle progression and suggests that the mammalian lysosome (the equivalent of the yeast vacuole) may also play a crucial role in mammals. Inheritance is a key pathway for ensuring proper distribution of organelles from the mother yeast to its bud and understanding this highly regulated process may shed light onto cancer and tumor prevention. Although inheritance is a highly regulated process, it is unclear in what pattern inheritance occurs. Biogenesis is measured using genetically modified yeast with GFP bound to VPH1, a protein abundant in vacuole membrane. Inheritance is measured using FM-4-64, a red dye that’s trafficked to the vacuole. The cells are followed through a full stage of cell division and are documented with 3-D images using a spinning disk confocal microscope. I hypothesized that inheritance occurs in a steady progressive manner over time, however data is suggesting that inheritance may occur in a discrete pattern.

83 Cdc28 and Fus3 synergistically regulate mating pheromone signaling dynamics via a shared multi-site phosphorylation region on the scaffold protein Ste5. A. Colman-Lerner1,2, M. Repetto1,2, M. Winters3, A. Bush1,2, W. Reiter4, D. Hollenstein4, G. Ammerer4, P. Pryciak3 1) Department of Physiology, Molecular, and Cell Biology University of Buenos Aires, Buenos Aires, Argentina; 2) IFIBYNE, University of Buenos Aires - National Scientific and Technical Research Council (CONICET), Buenos Aires, Argentina; 3) Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA; 4) Department for Biochemistry, Max F. Perutz Laboratories, University of Vienna, Vienna, Austria. Normal cell behavior requires the ability to detect and respond to extracellular signals. To study how cells integrate these signals with the internal cell state, we used as a model system the response to mating pheromone in the budding yeast S. cerevisiae, which has an antagonistic relationship with the cell cycle. The pheromone response pathway MAPK (Fus3) arrests the cell cycle by activating the CDK inhibitor Far1. Conversely, when cells pass START, the CDK (Cln2-Cdc28) blocks pheromone response by phosphorylating the scaffold protein Ste5 near a plasma membrane binding (PM) domain, which prevents its association with the plasma membrane. Here, using quantitative time-lapse microscopy, we examined Ste5 membrane recruitment dynamics at different cell cycle stages. Surprisingly, in S-phase, where Ste5 recruitment should be blocked, pheromone stimulation caused an initial recruitment followed by a steep drop-off. This delayed inhibition revealed a requirement for both CDK activity and negative feedback from the pathway MAPK Fus3. Mutagenesis, mass spectrometry, and electrophoretic analyses suggested that the CDK and MAPK modify shared sites flanking the PM domain, and that these sites are most extensively phosphorylated when both kinases are active and able to bind their docking sites on Ste5. Our results suggest that the synergy between these kinases depends on a priming role of Fus3, in which it phosphorylates threonine residues. Then, these pThr residues become docking sites for the Cks1 subunit of CDK, which then proceeds to complete the phosphorylation of the remaining sites. Functionally, one consequence of the joint action by two kinases (as opposed to solo control by CDK alone) is to broaden the time-window in the cell cycle during which the CDK can effectively 35 block the mating response. Another is to make this inhibition more sensitive to other inputs or cell states that can regulate Fus3 in a way that reduces or enhances CDK control of mating. In conclusion, our findings indicate that the Ste5 scaffold protein behaves as a regulatory node whereby distinct kinases can target a shared set of phosphorylation sites to jointly control the affinity of a membrane interaction domain. The net impact on the signaling pathway can range from a mild modulation to severe quenching, depending on the regulatory inputs. These properties highlight how multiple phosphorylation sites, with different probabilities of being phosphorylated, plus cooperative interactions between kinases, can expand the options for kinase-mediated regulation.

84 Parallel and opposing regulators of the highly disordered transcriptional activator, Gln3, and their roles in the control of nitrogen catabolite repression (NCR) sensitive gene expression. Terrance G Cooper, Jennifer Tate, Rajendra Rai Microbiology, Immunology & Biochemistry, University of Tennessee Health Science Center, Memphis, TN. Yeast thrive in the luxurious nitrogen environment of rotting fruit and vegetables. However, a rain storm can swiftly wash them into the hostile, nitrogen poor environment of the surrounding soil. Survival is all about how they manage these transitions from good to poor nitrogen environments. The objective of this management is to selectively utilize the most abundant and easily metabolized nitrogen sources, relying on poor nitrogen sources only when nothing better is available. At the same time intracellular nitrogen homeostasis must be maintained. Selective utilization and homeostasis are achieved through (Nitrogen Catabolite Repression-) NCR-sensitive control of the major nitrogen catabolic transcription activators Gln3 and Gat1. In luxurious nitrogen conditions these GATAA-binding activators are sequestered in the cytoplasm and as a result, NCR-sensitive transcription is minimal. As the nitrogen environment deteriorates, Gln3 and Gat1 relocate to the nucleus and dramatically increase transcription of the genes required to import and catabolize a variety of poor nitrogen sources scavenged from the environment. Autophagy, which is also controlled by NCR-sensitive transcription, dramatically increases as well. The TorC1 kinase complex was originally thought to be the principle contributor to NCR-sensitive Gln3 regulation. However, Gln3 responds to 5 distinct nitrogen conditions each of which requires a unique set of regulators. Using residue substitutions throughout the highly disordered Gln3 protein, we show TorC1 control only partially accounts for NCR-sensitive Gln3 intracellular localization. Gcn2 kinase-mediated General Amino Acid Control (GAAC) is equally critical with the Gcn2 and TorC1 kinases functioning independently and in opposition to one another. Nnk1 kinase is also required for nuclear Gln3 import. The Gcn2 and Nnk1 kinases likely function upstream of the negative Gln3 regulator, Ure2, whereas 14-3-3 proteins Bmh1/2, not previously associated with Gln3 regulation, likely function downstream of Ure2. Nuclear Gln3 import is also more complex than previously recognized, consisting of two major and one minor Nuclear Localization Sequences. During these studies, we identified a small Gln3 region [the Ure2 Relief Sequence (URS)] whose integrity is only needed when Ure2 is present. Finally, a third level of Gln3 regulation is imposed within the nucleus. In high glutamine, or presence of a glutamine analogue, Gln3 exits from the nucleus in the absence of a requirement to bind to its GATAA targets within NCR-sensitive promoters. In contrast, as glutamine levels decrease, GATAA binding becomes requisite for Gln3 to exit from the nucleus. It is only through the concerted actions of this full array of regulatory components that NCR can effectively manage intracellular homeostasis in the face of unreliable environments. Supported by NIH grant GM-35642-27.

85 Cdc42 pathways control social cooperation in fungal microbes. P.J. Cullen1, J. Chow1, H.M. Dionne1, A. Prabhakar1, A. Mehrotra1, J. Somboonthum1, B. Gonzalez1, M. Edgerton2 1) Department of Biology, SUNY Buffalo, Buffalo, NY; 2) Department of Oral Biology, SUNY Buffalo, Buffalo, NY. In microbes, cooperation among individuals can lead to the assembly of multicellular structures by mechanisms that remain unclear. Budding yeast and other fungal species undergo a nutrient foraging response called filamentous growth, which requires interactions among cells. By examining filamentous growth from the perspective of microbial social cooperation, we identify a response where cells collectively produced macroscopic structures called invasive aggregates. Signaling pathways that regulate filamentous growth, including a mucin (Msb2)-dependent MAP kinase pathway that is controlled by the polarity GTPase Cdc42, were required for aggregate invasive growth. Cdc42-dependent pathways regulated aggregate formation by the expression of products known to promote social cooperation in microbes (e.g. metabolic enzymes and adhesion molecules). Cdc42 pathways also regulated changes in cell shape and polarity to promote the collective assembly of groups of filaments into higher-order structures. We hypothesized that the role of Cdc42 pathways in regulating social interactions in microbes might underlie their ubiquitous roles in controlling developmental morphogenetic responses in higher eukaryotes. In line with this possibility, Cdc42 pathways were required for the production of multicellular traits by directed selection experiments in the laboratory.

86 Establishing a yeast model system to study Transthyretin aggregation. E.E. Davis, A.L. Manogaran Marquette University, Milwaukee, WI. Transthyretin amyloidosis (ATTR) is an age related terminal disease associated with the misfolding and aggregation of the Transthyretin protein. The Transthyretin aggregates assemble into structures called amyloid, which are extremely stable, and resistant to protease and detergent treatments. This amyloid accumulates within the heart and peripheral nervous system leading to cardiomyopathy and neuropathy, and ultimately death. The most common form of ATTR arises from spontaneous misfolding of the wildtype Transthyretin allele, leading to late onset of the disease, however mutant alleles can lead to earlier disease onset. ATTR has been difficult to study in humans due to the timing of onset, high variability in presenting phenotypes, and low diagnosis rates. Furthermore, mouse models are limited since expression of wildtype or mutant human 36

Transthyretin alleles does not lead to extensive aggregation. Only upon knock down of a heat shock transcription factor, which leads to global changes in proteostasis, would aggregation be observed. Therefore, mouse models are difficult to use when assessing how subtle changes in the proteostasis machinery affect Transthyretin aggregation. A simple in vivo system is then required in order to understand the cellular mechanisms that impact the aggregation of Transthyretin. Here, we describe a Saccharomyces cerevisiae model in which Transthyretin forms two distinct types of aggregates. Expression of either wildtype or mutant alleles of Transthyretin, fused to GFP, resulted in the presence of fluorescent inclusions. Using biochemical assays, Transthyretin was found to be present in populations of multiple sizes with varying properties. One population of aggregates were extremely large and appeared to disassemble upon treatment with detergent. A second lower molecular weight population formed stable aggregates that were SDS-insensitive. These data suggest Transthyretin is capable of forming amyloid-like aggregates within yeast, allowing for further study into how proteostasis networks may impact Transthyretin aggregation and disassembly within an in vivo model.

87 Novel mechanisms of Hsf1 activity revealed by deep mutational scanning. M.W. Dorrity1,2, E.M. Morton1, S. Fields1,3,4, C. Queitsch1 1) Department of Genome Sciences, University of Washington, Seattle, WA; 2) Department of Biology, University of Washington, Seattle, WA; 3) Howard Hughes Medical Institute, University of Washington, Seattle, WA ; 4) Department of Medicine, University of Washington, Seattle, WA. The activity of Heat shock factor 1 (Hsf1) in regulating the heat shock response is conserved among all eukaryotes, and can play a role in such biological processes as longevity, climate adaptation, and oncogenic transformation Upon sensing an increase in temperature, Hsf1 trimerizes and binds DNA to activate heat shock genes. Here, we use high-throughput fitness measurements of >500,000 variants in the trimerization domain of the Saccharomyces cerevisiae Hsf1 to investigate mechanisms of temperature-specific Hsf1 function. We find that cell division rate under heat-shock can be modulated by mutations at particular helical positions in the coiled-coil trimerization domain, and find similar phenotypes are conferred in S. cerevisiae by natural trimerization domain variants from other species. Furthermore, exceptional Hsf1 trimerization variants increase the fitness of yeast cells under heat-shock conditions beyond that of wild-type Hsf1. Hsf1 trimerization variants have altered DNA-binding patterns in vivo, and altered transcriptional programs, suggesting that trimerization modulates target specificity. We suggest that Hsf1 variants with altered trimerization can modulate cell division in response to changing temperatures.

88 Pan2-Pan3 deadenylase complex is involved in the cell growth on non-fermentable carbon source. Long Duy Duong, Kenji Irie Department of Molecular Cell Biology, Graduate School of Comprehensive Human Sciences, and Faculty of Medicine, University of Tsukuba, Tsukuba, Japan. The cytoplasmic deadenylases play crucial roles in regulation of mRNA translation and stability. In the yeast S. cerevisiae, there are two major deadenylases, Ccr4-Not and Pan2-Pan3 complexes. It has been believed that Pan2-Pan3 complex initially trims the mRNA poly(A) tail and then Ccr4-Not complex mainly shortens the tail. Previously, we have shown that the poly(A) tail of LRG1 mRNA is lengthened in ccr4∆ mutant in the stationary phase, which can be shortened by deletion of Pbp1, and this shortening is caused by Pan2-Pan3 deadenylase complex (Duy et al., PLoS One. 2017). However, it is remained unclear how Pan2-Pan3 activity is regulated in the later growth phases. In this study, by using genetic approach, we found that ccr4∆ pan2∆ and ccr4∆ pan3∆ mutants show growth defects in the media containing glycerol and lactate as non-fermentable carbon sources. The ccr4∆ pan2∆ and ccr4∆ pan3∆ mutants grow similarly to the ccr4∆ mutant in glucose-based media, suggesting that Pan2-Pan3 complex has important roles in the growth on non-fermentable carbon sources. By using the multicopy suppressor screening approach, we have found that over-expression of REX2 or STM1 suppresses the growth defect of ccr4∆ pan2∆ mutant in glycerol-based media. REX2 encodes for the 3'-5' exonuclease, and STM1 encodes a protein required for optimal translation under nutrient stress. Unexpectedly, we found that deletion of STM1 also suppresses the growth defect of ccr4∆ pan2∆ mutant on glycerol-lactate media, indicating that Stm1 has an inhibitory effect on cell growth, which may be involved in translational regulation. Furthermore, we found that Pan3 is phosphorylated in the glycerol-based media, suggesting that Pan2-Pan3 activity may be regulated via this phosphorylation. Taken together, our results demonstrated that Pan2-Pan3 complex has a role in the cell growth on non-fermentable carbon source.

89 Defining harmful yeast proteins by measuring overexpression limits. Y. Eguchi1, K. Makanae1, Y. Hori2, T. Hasunuma2, H. Moriya1 1) Research Core for Interdisciplinary Sciences, Okayama University, Japan; 2) Graduate School of Science, Technology and Innovation, Kobe University, Japan. Protein expression levels within the cell are coordinated to maximize cellular functions. Ultimate overexpression of a protein could disturb the coordination and lead to cause growth defects, and this phenomenon is known as the protein burden. According to the concept of the protein burden, nonharmful proteins can be overexpressed up to the limit of protein synthesis capacity of a cell. In contrast, harmful proteins should not be overexpressed up to the limit due to their detrimental effects. However, the expression limit that causes the protein burden is still unclear, and thus nonharmful and harmful proteins remain poorly defined. To estimate the protein burden limit and to distinguish nonharmful or harmful proteins upon overexpression, we systematically measured overexpression limits of glycolytic proteins in Saccharomyces cerevisiae. We applied the genetic Tug- Of-War (gTOW) method by which we can measure the limit of a target gene/protein overexpression. An artificial gene in which each protein was expressed from the TDH3 promoter was cloned into the gTOW plasmid, and the protein expression level in 37 the cell and its copy number limit were measured. We simultaneously analyzed enzymes that had mutations in their catalytic centers. We estimated that the limits of some glycolytic proteins were up to 15% of total cellular protein. These limits were unrelated to proteins’ catalytic activities. Some proteins had low expression limits, explained by their localization and metabolic perturbations. Even if a gene transcription was induced by strong promoter, an extreme high optimized codon usage was needed for an expressing protein up to the protein burden limit level. Therefore, we established a framework to distinguish nonharmful or harmful proteins upon overexpression. We further developed a method named “Tug-Of-War within a Fusion protein” (TOW-Fu) to evaluate if a target protein can be expressed up to the expression limit by using dihydrofolate reductase fusion protein dependent on methotrexate resistance. TOW-Fu permits the measurement expression limit of a target protein as quantified by a growth rate in a cell expressing the target protein. We analyzed the expression limits of 94 proteins encoded on Ch. I in S. cerevisiae by using TOW-Fu and estimated that only minor portion of yeast protein could be expressed up to the protein burden limit level.

90 Mapping Functional Domains of the Cell Fusion Protein Prm1p. Lucille Moholt-Siebert, Maya Stevens, Niya Paul, Samantha Tran, Aine Piedad, Alex Engel Dept. of Biology, Mills College, Oakland, CA. Cell fusion is a membrane fusion event required for gametic fusion and the development of syncytial tissues. The identities and mechanisms of proteins that mediate cell fusion are not as well defined as for the proteins that mediate intracellular membrane fusion. We utilized the mating process of haploid yeast gametes to investigate the molecular basis for cell fusion in Saccharomyces cerevisiae. The pheromone-induced gene PRM1 is required in one cell of a mating pair for efficient cell membrane fusion. prm1 x prm1 mating pairs fail to fuse apposed cell membranes and can undergo contact dependent cell lysis. PRM1 encodes a multipass membrane protein that is conserved among yeasts; however, how Prm1p functions to promote membrane fusion and which domains of Prm1p contribute to that function are unknown. We tested if the expression of fungal orthologs of PRM1 could rescue the mating defect of prm1 mutants. Using a fluorescence-based cytoplasmic mixing assay to quantify cell fusion efficiency, we found that expression of N. crassa PRM1 did not rescue the mating defect of prm1 mutants. Furthermore, localization of GFP-tagged N. crassa Prm1p showed that this heterologously expressed protein is retained in the ER and intracellular compartments of shmooing cells, in contrast to the typical shmoo tip localization of endogenous Prm1p. We plan to make various chimeric Prm1p proteins to investigate which Prm1p domains are required for shmoo tip localization and for membrane fusion promoting activity. These orthologous Prm1p proteins may also help in identifying Prm1p-interacting proteins that participate in the membrane fusion step of yeast cell fusion.

91 Role of the C-terminal region of yeast monocarboxylate transporter Jen1 in its glucose-induced and ubiquitination-dependent inactivation by α-arrestin Rod1. S. Fujita, K. Gomi, T. Shintani Tohoku University, Graduate School of Agricultural Science, Sendai, Miyagi, JP. In response to rapidly changing external environment and certain stresses, cells displace and rearrange their plasma membrane (PM) transporters for maintaining intracellular homeostasis. Signal-dependent ubiquitin modification of PM transporters triggers its selective endocytic internalization and sorting to the vacuole/lysosome for degradation. In yeast, an E3 ubiquitin ligase Rsp5 is responsible for ubiquitination of the PM transporters, which requires a specific α-arrestin to be selectively targeted by Rsp5. JEN1 encodes a monocarboxylate (pyruvate and lactate) transporter, which undergoes glucose- induced endocytic degradation. Although the α-arrestin Rod1 may act as an Rsp5 adaptor during the glucose inactivation of Jen1, it is unclear how Rod1 recognizes Jen1. Here, we identify the Jen1 region important for its glucose-induced endocytosis. Jen1 is predicted to have 12 transmembrane domains and its N- and C-termini are faced to the cytoplasm. Deletion and mutational analyses revealed that the N- and C-terminal cytosolic regionswere required for glucose-dependent Jen1 degradation. Particularly, the C-terminal 20-amino-acid region (referred as Jen1C) plays a major role in glucose-elicited interaction between Jen1 and Rod1. We further evaluated whetherthis region is sufficient to promote glucose-induced and Rod1-dependent internalization of Jen1.To address this, we first transplanted the Jen1 C-tail to high-affinity methionine permease Mup1, whose endocytic degradation is elicited by a high concentration of methionine in an Art1 α-arrestin- dependent manner. After glucose addition, Mup1−Jen1C−GFP was internalized from the PM, whereas Mup1−GFP was retained in the PM. In the rod1∆ cells, glucose-induced internalization of Mup1−Jen1C−GFP was also impaired. These results suggest that glucose-induced Mup1−Jen1C degradation occurs through recognition of the Jen1 C-tail by Rod1. Moreover, we observed the mutation of two lysine residues localized in Jen1 C-tail blocked glucose-induced ubiquitination and endocytosisof Mup1−Jen1C. These mutations also decreased glucose-induced internalization of Jen1. These results indicated that the Jen1 C-tail is sufficientfor recognition and ubiquitination by Rod1-Rsp5 complex. Therefore, we concluded that the Jen1 C-tail acts as the degron for Jen1 in response to glucose stimuli.

92 Facultative sex driven by protein-based inheritance: Investigating the cellular consequences of a novel prion element, [STE+]. Raymond Futia1, Daniel Jarosz2 1) Biology, Stanford University, Stanford, CA; 2) Chemical and Systems Biology, Stanford University, Stanford, CA. Prions are proteins that can adopt multiple conformations, at least one of which is self-templating. Ensuing changes in protein activity are heritable over long biological timescales, in a manner that does not follow Mendel’s rules. Aggregation of PrP, the first prion protein discovered, is associated with devastating neurodegenerative disease. Yet other prions can promote adaptive traits in organisms ranging from yeast to mice. A screen of all S. cerevisiae open reading-frames identified proteins with prion-like properties including, Sli15, a component of the yeast chromosome passenger complex (CPC). The CPC 38 is a master regulator of mitosis, most notably monitoring the spindle assembly checkpoint and the onset of anaphase. Interestingly, the prion state is associated with an insensitivity to mating pheromone and a consequent inability to mate, a phenotype with no obvious relationship to the canonical functions of the CPC. We therefore refer to the prion state as [STE+]. Acquisition of [STE+] fuels transcriptomic changes in metabolic and mating pathway components, providing a logical basis for the observed phenotype. In addition, this transcriptomic effect displays a spatial bias, with downregulated genes clustering in the centromere-proximal half of each chromosome arm. Assessment of CPC activity suggests that decreased activity in [STE+] cells – particularly the CPC’s phosphorylation of histones – may result in such a chromatin signature and that this signature may represent the mechanism by which [STE+] phenotypes manifest. This novel sterile behavior associated with a CPC component, represents a molecular relationship between the decision to divide or mate – potentially one that can be environmentally regulated through Sli15’s prion behavior.

93 Identification of human genes enhancing the toxicity of α-synuclein. I. Haider, S. Chen, E. Hayden, S. Ju, Q. Zhong Biological Sciences, Wright State University, Dayton, OH. Parkinson's disease (PD) is an incurable and debilitating neurodegenerative disorder that affects 1-2% of the population above age 65. Following the identification of causal genes in familial forms of PD, the molecular basis of the disease is emerging. With 90% of disease cases being sporadic, our understanding of PD remains vastly incomplete. A pathological hallmark of PD is the formation of Lewy body, primarily composed of the abnormal aggregation of α-synuclein. Both missense mutations and increased expression level of α-synuclein are genetically linked to PD. Overexpression of α-synuclein leads to protein aggregation and induces cell death in many cellular and genetic model systems, including yeast. Although Lewy body is observed in more than 90% of PD patients, the protein sequence or expression level of α-synuclein is not necessarily altered. We, therefore, hypothesize that there are other genetic factors that could increase the propensity of α- synuclein to misfold and enhance its toxicity. To test this hypothesis, we carried out a proof-of-principle genetic screen to overexpress 585 human genes in a yeast model expressing α-synuclein at a level that does not lead to aggregation or toxicity. This small-scale screen yielded 17 enhancers that significantly increase the toxicity of α-synuclein. Some of the enhancer proteins directly induce the aggregation of α-synuclein or change its membrane localization. One of the enhancer protein has been previously associated with other neurodegenerative diseases. Ongoing research focuses on expanding the search of genetic enhancers of α-synuclein toxicity among 14,000 human genes. Yeast models expressing α-synuclein have been subjected to several genome-wide screens to identify modifier genes. Fruitful as such research has proven to be, most studies start with models already exhibit cellular defects. Such screens would not be effective to uncover genetic buffering mechanisms that initiate α-synuclein proteinopathy. Moreover, not all human genes necessarily have functionally conserved homologs in yeast. Non-conserved human genes should not be left out in search for genetic modifiers. The ability to uncover modifiers that induce the toxicity of α-synuclein at low expression level and to directly test all human genes would expand our understanding of PD from a global perspective.

94 Genetic screen to identify human modifiers of ALS protein FUS toxicity. E. Hayden, A. Chumley, S. Chen, Q. Zhong, S. Ju Biological Sciences , Wright State University, Dayton, OH. ALS, also known as Lou Gehrig’s disease, is a particularly devastating neurodegenerative disease with no cure. Patients with ALS suffer from rapid degeneration of motor neurons in the brain and spinal cord, leading to progressive muscle weakness and death usually within five years. There are over 20 genes associated with ALS including a number of RNA binding proteins such as TDP-43 and FUS. ALS linked mutations in FUS largely cluster in the nuclear localization signal and cause mislocalization of the protein from the nucleus into cytoplasmic stress granules. FUS protein is extremely toxic to yeast and forms cytoplasmic aggregates which colocalize with p-bodies and stress granules. We and others have taken advantage of this model to study the toxicity of FUS and search for genetic modifiers of FUS mediated toxicity. A previous genetic screen in our lab using a yeast gene library uncovered 5 yeast genes which rescue FUS toxicity. Importantly, the human homologue to one of the yeast genes was tested in a neuronal cell and animal model of ALS where it also rescued toxicity. With our success in translating findings from yeast to neuronal and animal models we have performed a genetic screen on the FUS yeast model using a library of human genes. This is a novel library which will allow us to directly identify human modifiers of toxicity. To introduce the plasmid library into yeast a unique approach using yeast mating was used. We have identified numerous genes which rescue FUS mediated toxicity. Ongoing experiments aim to test whether the aggregation, localization and expression level of FUS is changed by these human genes. Additionally, we are exploring the possibility that the modifiers directly interact with FUS protein in yeast.

95 Regulation of mating in the methylotrophic yeast Komagataella phaffii. L. Heistinger1, J. Moser2,3, N.E. Tatto1,2, M. Valli1,2, B. Gasser1,2, D. Mattanovich1,2 1) Department of Biotechnology, BOKU - University of Natural Resouces and Life Sciences, Vienna, AT; 2) Austrian Centre of Industrial Biotechnology (ACIB), Vienna, AT; 3) School of Bioengineering, University of Applied Sciences FH-Campus, Vienna, AT. The methylotrophic yeast Komagataella phaffii (Pichia pastoris) is an industrially relevant protein production host with a preferentially haploid lifestyle that is able to form diploid cells by mating. Wild type strains of K. phaffii are homothallic and cells can undergo mating-type switching by inversion of the chromosomal region encoding the mating type (MAT) genes (Hanson et al., 2014). As a result, cultures consist of a mixture of cells of both mating types, which, up to now, hindered the investigation of mating-type-specific gene expression and phenotypes. We generated stable heterothallic K. phaffii strains of 39 opposite mating type by targeted deletion of a genomic region required for mating-type switching (Heistinger et al., 2018). These strains with defined mating types were then used for characterization of mating gene regulation and mating-type- specific gene expression.

Unlike Saccharomyces cerevisiae, K. phaffii encodes four functional MAT genes which are only transcribed under nitrogen limitation conditions. We could show that mating-type regulation in K. phaffii is similar to other pre-whole-genome duplication yeasts. Characterization of deletion mutants showed that in haploid cells, MATa2 and MATα1, respectively, are essential for the activation of mating-type-specific genes, while in diploid cells the expression of MATa1 and MATα2 is required for spore formation. Analysis of RNA sequencing data of cells under mating conditions also allowed the identification of the so far unknown K. phaffii a-factor pheromone genes. Using gene deletion strains and synthetic mating factor peptides we could show that both of the two identified MFA genes code for a variant of the a-factor pheromone and are required for efficient mating of K. phaffii a-type cells. Furthermore, the mating behavior of α-factor deletion strains and the response of K. phaffii to synthetic α-factor pheromone were investigated.

Hanson S. J. et al., Proc Nat Acad Sci USA 2014, 111: E4851-8. Heistinger L. et al., Mol Cell Biol 2018, 38: e00398-17.

96 Effects of Rfa2 N-terminal Phosphorylation on Adaptation-deficient Yeast. Cristian Hernandez, Trevor Baumgartner, Stuart Haring North Dakota State University, Fargo, ND. Determining the role of the Replication Factor A2 (Rfa2) N-terminus on cell cycle regulation and DNA damage checkpoint progression is crucial for studying how cells prevent genetic defects. Cells must be able to efficiently and accurately repair DNA damage in a temporally-controlled manner. If cells are unable to do so, they are at a very high risk of developing permanent DNA mutations, which are directly correlated with cancer and other cellular diseases. To prevent mutations, it is especially important for cells to be able to address DNA damage before they replicate their DNA or segregate their chromosomes by mitosis. Before the phases of the cell cycle that involve DNA metabolism (i.e., DNA synthesis and mitosis), checkpoints are elicited to provide the necessary time to do one last check to ensure that the genome is intact. The N- terminus of the single-stranded DNA-binding protein Rfa2 plays a crucial role in the response to DNA damage, especially in regulating checkpoint function. Checkpoints are utilized by all eukaryotic cells and require precise coordination of many proteins. Using yeast as a model eukaryotic organism, gene deletions of checkpoint machinery required for checkpoint exit (adaptation) were generated to assess the ability of cells to progress through cell cycle checkpoints. We show here how Rfa2 modification affects adaptation when these genes are deleted, to determine the pathway through which Rfa2 mediates checkpoint exit and the return to cell growth.

97 Dissecting the genetic architecture of aneuploidy tolerance in wild yeast strains. J. Hose, K. Clowers, A. Gasch Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI. Aneuploidy, the state of having an abnormal copy number of one or more chromosomes, produces severe consequences in many organisms, especially during development. Yet many cancerous cells carry extra chromosomes with little detrimental effect. Saccharomyces cerevisiae has been reported to exhibit severe stress when carrying extra chromosomes, displaying a transcriptional stress response, proteotoxic stress and proliferative defects. Many of these studies utilized aneuploid strains of the common W303 laboratory strain background. However, we previously reported that aneuploidy is relatively common in wild yeast strains isolated from unique environmental niches. These wild aneuploid strains do not exhibit the proliferative defects reported in previous studies of the W303 strain. To better understand the impact of aneuploidy in wild yeast strains, we are undertaking a genetic analysis to dissect the patterns of aneuploidy tolerance in wild and lab yeast. We mated wild oak-soil strain YPS1009 that is naturally aneuploidy for chromosome XII (Chr 12) to W303 with an extra copy of Chr 12 and characterized aneuploid segregants. We scored aneuploidy sensitivity in each segregant based on growth rate/colony size and propensity to lose the extra Chr 12 after two days of serial passaging. Segregants with big colony size that maintained Chr 12 aneuploidy were scored as tolerant of aneuploidy, while segregants with small colony size that reverted to euploidy were classified as intolerant of aneuploidy. Genetic analysis reveals that auxotrophies in W303 contribute to aneuploidy sensitivity, in addition to two other loci that underlie aneuploidy sensitivity. Genetic mapping to identify the responsible alleles and genes is underway.

98 CDC15 and SPS1 act together to coordinate timely meiotic cytokinesis. Scott Paulissen1,2, Christian Slubowski1,3, Cindy Hunt1, Joe Roesner1,4, Linda Huang1 1) Department of Biology, Univeresity of Massachusetts Boston, Boston, MA 02125; 2) NIH, Bethesda, MD 20892; 3) Department of Surgery, BIDMC, Boston, MA 02115; 4) Merck, Boston, MA 02115. Under starvation conditions, diploid yeast cells undergo sporulation to produce four haploid spores. During this process, four prospore membranes will grow to surround each of the haploid nuclei. Meiotic cytokinesis occurs through the closure of the prospore membrane around the newly formed haploid nucleus. Once the prospore membrane closes, it serves as the template for spore wall deposition and will ultimately become the plasma membrane for the new spore. Our previous work has shown that SPS1, which encodes a STE20-kinase of the GCKIII subclass, is required for timely closure of the prospore membrane. We have now found that the hippo-like kinase encoded by CDC15 acts similarly to regulate prospore membrane 40 closure. We find that Cdc15 is in a complex with Sps1, and CDC15 is required for Sps1 phosphorylation. As Cdc15 is a member of the mitotic exit network, we investigated the role of downstream pathway members DBF2, DBF20, MOB1, and CDC14 in meiotic cytokinesis. We find that SPS1 and CDC15 are required for sustained Cdc14 nucleolar release during meiosis. We also find that SPS1 and CDC15 are not required for the known role of DBF2, DBF20, and MOB1 in spore number control. Taken together, our results suggest that different portions of the mitotic exit network have been recruited to serve distinct functions during meiosis.

99 Rts1/B56 regulatory subunit of PP2A is required for Cyclin-dependent kinase activation and anaphase onset. Sam Kajjo, Erin Kennedy, Adam Rudner University Of Ottawa, Ottawa, CA. Cyclin-dependent kinase (Cdk1) drives multiple cell cycle transitions. For example, during mitosis Cdk1 activity is required for centrosome duplication, mitotic spindle assembly, changes in the dynamics of microtubules, chromosome condensation and attachment to the spindle, and to trigger anaphase onset. Two models have been proposed to explain how Cdk1 can accomplish its many roles: 1) Cdk1 binds to nine different cyclins (the Cln and Clb proteins) which are required for its activity and have been shown to provide some specificity to Cdk1. 2) Stepwise increases in the activity of Cdk1/cyclin complexes has been proposed to trigger distinct events in the cell cycle. The best example of a stepwise change in Cdk1 activity is its regulation by Wee1 and Cdc25. Swe1 (the budding yeast Wee1) phosphorylates tyrosine 19 (Y19) on Cdk1 and inhibits its activity, while Mih1 (the budding yeast Cdc25) dephosphorylates this residue and activates Cdk1. This inhibition of Cdk1 plays a conserved role in eukaryotes as the target of G2 size control mechanisms and of the DNA replication and damage checkpoints. In budding yeast Swe1-dependent checkpoints arrest cells at metaphase rather than in G2, suggesting that Swe1/Mih1 regulation of Cdk1 plays an important role in triggering anaphase onset. Our recent work showed that Ptp1 and PP2A- Rts1/B56 work redundantly with Mih1 to regulate Cdk1-Y19 dephosphorylation and activation during mitosis, and these three phosphatases may allow for precise changes in Cdk1 activity. PP2A-Rts1/B56 is believed to be solely a serine/threonine phosphatase, and we are currently testing if purified PP2A-Rts1/B56 has activity in vitro against tyrosine phosphorylated Cdk1. This novel activity for PP2A may require post-translational modifications or sub-stoichiometric binding partners. Complementing these in vitro studies we are testing if deletion of all three phosphatases, in mih1∆ rt1∆ ptp1∆ cells, arrests cells at metaphase.

100 Using genetic buffering relationships identified in fission yeast to reveal susceptibilities in cells lacking hamartin or tuberin function. Ashyad Rayhan, Adam Faller, Ryan Chevalier, Alannah Mattice, Alexander Timoshenko, Jim Karagiannis Department of Biology, The University of Western Ontario, London, Ontario, CA. Tuberous sclerosis complex is an autosomal dominant disorder characterized by benign tumors arising from the abnormal activation of mTOR signaling in cells lacking TSC1 (hamartin) or TSC2 (tuberin) activity. To expand the genetic framework surrounding this group of growth regulators, we utilized the model eukaryote Schizosaccharomyces pombe to uncover and characterize genes that buffer the phenotypic effects of mutations in the orthologous tsc1 or tsc2 loci. Our study identified two genes: fft3 (encoding a DNA helicase) and ypa1 (encoding a peptidyle-prolyl cis/trans isomerase). While the deletion of fft3 or ypa1 has little effect in wild-type fission yeast cells, their loss in tsc1Δ or tsc2Δ backgrounds results in severe growth inhibition. These data suggest that the inhibition of Ypa1p or Fft3p might represent an “Achilles’ heel” of cells defective in hamartin/tuberin function. Furthermore, we demonstrate that the interaction between tsc1/tsc2 and ypa1 can be rescued through treatment with the mTOR inhibitor, torin-1, and that ypa1Δ cells are resistant to the glycolytic inhibitor, 2- deoxyglucose. This identifies ypa1 as a novel upstream regulator of mTOR and suggests that the effects of ypa1 loss, together with mTOR activation, combine to result in a cellular maladaptation in energy metabolism that is profoundly inhibitory to growth. Lastly, to determine if the identified genetic interactions are specific to S. pombe, or are conserved in more developmentally complex eukaryotes, we used siRNA to knockdown the expression of the mouse ypa1 and fft3 orthologs (Ppp2r4 and Smarcad1, respectively) in Tsc2+/+TP53-/- and Tsc2-/-TP53-/- mouse embryonic fibroblasts (MEFs). No significant decrease in viability was observed upon knockdown of Smarcad1 in either Tsc2+/+TP53-/- or Tsc2-/-TP53-/- MEFs. In contrast, knockdown of Ppp2r4 resulted in a 26% decrease (p=0.0099) in the number of viable Tsc2-/-TP53-/- MEFs in culture, while having no significant effect (p=0.2074) on Tsc2+/+TP53-/- MEFs. Thus, similar to the negative genetic interaction observed between fission yeast tsc2Δ and ypa1Δ mutants, MEFs deficient in tuberin activity display reduced viability in conjunction with Ppp2r4 knockdown.

101 Mitotic substrates of SUMO proteases: Building a better SUMO Trap. O. Kerscher, R Yin, C Harvey Biology, College of William & Mary, Williamsburg, VA. SUMO proteases of the SENP/Ulp family are master regulators of both sumoylation and desumoylation and regulate SUMO homeostasis in eukaryotic cells. Moreover, they play important roles in mitosis. For example, in budding yeast, loss of Ulp1- mediated desumoylation leads to cell-cycle progression defects and cell death. This observation suggests that Ulp1 plays a key role in the sumoylation dynamics of important cell-cycle regulatory proteins. Several nuclear and cytosolic proteins involved in DNA replication and mitosis have been identified as Ulp1 desumoylation substrates. However, the specific Ulp1 targets required for cell cycle progression have yet to be identified. Our previous studies revealed that Ulp1 requires specific structural features of its catalytic domain for substrate targeting. We also identified a substrate-trapping fragment of Ulp1 (UTAG) that allows us to identify Ulp1-specific desumoylation substrates. Here we compare and contrast determinants of 41

SUMO targeting, binding, and processing in Ulp1 SUMO protease domains derived from S. cerevisiae and K. marxianus, a thermotolerant yeast strain (Peek et al., 2018). Our data suggests that a SUMO-trapping protein derived from this thermotolerant yeast strain may have distinct advantages to study the transient interactions with SUMO and mitotic Ulp1 substrates, especially under stress conditions.

102 The analysis on termination mechanism of autophagy. Shintaro Kira, Masafumi Noguchi, Takeshi Noda Center for Frontier Oral Science, Department of Dentistry, Osaka University, Suita, Osaka, JP. The yeast Saccharomyces cerevisiae cells degrade their own components by autophagy to supply nutritional sources for their survival under nitrogen starvation condition. On the other hand, we showed that autophagy is eventually terminated after 10- 12 hr nitrogen starvation. By screening of knock-out library and the DAmP knock-down library, we identified an uncharacterized gene involved in the autophagy termination and named it Tag1. Tag1 is a single transmembrane protein and localized in the vacuolar membrane. TORC1 protein kinase regulates autophagy through multiple phosphorylation of Atg13; among them, Atg13 S379 phosphorylation is crucial for autophagy regulation. Atg13 S379 was dephosphorylated in short period of nitrogen starvation, and rephosphorylated in 24 hr starvation. Such S379 rephosphorylation was independent of TORC1 activity. In ∆tag1 cell, however, the S379 rephosphorylation was significantly decreased in 24 hr starvation. In wild-type cell, autophagosome formation site PAS was formed in response to nitrogen starvation, however, it was disassembled after 24 hr starvation. Instead, PAS was kept assembled in ∆tag1 cell in 24 hr starvation. As formation of PAS was regulated by the Atg13 S379 phosphorylation, Tag1 terminates autophagy through Atg13 partial rephosphorylation and subsequent PAS disassembly.

103 Genetic requirements for translocon-associated quality control. Avery M. Kirschbaum, Christopher J. Indovina , Sarah M. Engle, Sheldon G. Watts, Samantha M. Turk, Danielle L. Overton, Cade J. Orchard, Eric M. Rubenstein Department of Biology, Ball State University, Muncie, IN. Translocation into the endoplasmic reticulum (ER) is essential for modification and function of many proteins. Proteins are transported into the ER through a channel called the translocon. Occasionally, proteins moving through the translocon aberrantly or persistently occupy the channel. Although functional ER translocation has been intensely studied, translocon- associated quality control mechanisms have not been fully elucidated. Impairment of translocon quality control correlates with compromised cellular health. Additionally, the human protein apolipoprotein B (apoB), the major protein component of low-density lipoproteins (i.e. bad cholesterol), can become stalled in the translocon if its lipid binding partners are not present in the ER. In both yeast and mammals, homologs of the ubiquitin ligase Hrd1 ubiquitylate translocon-clogging proteins, thereby targeting them for degradation. Hrd1-mediated degradation of translocon-associated proteins occurs without several cofactors required for other Hrd1-mediated degradation pathways, suggesting a unique mechanism of substrate recognition and destruction. We hypothesize that Hrd1 functions with a distinct group of cofactors to recognize and degrade translocon- clogging proteins. A yeast growth-based screen was performed to identify genes required for degradation of a model Hrd1 substrate that aberrantly associates with the translocon. A variety of genes were identified, some of which have roles in other protein degradation pathways. We are genetically and biochemically validating roles for these identified genes, and others, in translocon-associated protein degradation. Our results suggest that a complex system of mechanisms collaborate to clear aberrantly engaged translocons. A better understanding of the mechanisms that clear clogged translocons could contribute to new treatment options for high cholesterol.

104 Cln3-Cdk1 promotes the G1/S transition by phosphorylating RNA Pol II C-terminal domain repeats on S5 at SBF- regulated promoters. M. Kõivomägi, J. Turner, M. Swaffer, J. Skotheim Department of Biology, Stanford University, Stanford, CA. Proper progression through the cell cycle is orchestrated by cyclin-dependent kinases (Cdks) and their activating subunits, cyclins. In budding yeast, the G1 cyclin Cln3 is an upstream activator of G1/S progression. Cln3 forms a complex with Cdk1 and has been thought to partially inactivate the transcriptional inhibitor Whi5. Inactivation of Whi5 relieves inhibition of the transcription factor SBF, whose target genes include the downstream G1 cyclins Cln1 and Cln2. Cln1/2-Cdk1 complexes complete the inactivation of Whi5 via a positive feedback loop that drives the G1/S transition. In the present study, we have investigated the kinase activity of Cln3-Cdk1 and compared it with other budding yeast cyclin-Cdk1 complexes. In sharp contrast to Cln2-Cdk1-Cks1 complexes, Cln3-Cdk1 shows very weak kinase activity towards Whi5 in vitro and no detectable kinase activity in vivo. This result fits with our recent finding in Schmoller et al (2015) that cell growth drives G1/S progression primarily by diluting the cell cycle inhibitor Whi5, but it reopens the question of how Cln3-Cdk1 promotes G1/S progression. To address the question of Cln3 drives the G1/S transition, we undertook a search for new Cln3 targets using a candidate- based in vitro screen and phosphoproteomic methods. We found that the Rpb1 C-terminal unstructured region, which contains multiple repeats of the sequence YSPTSPS, is a specific substrate for Cln3-Cdk1. Using in vitro kinase assays with different mutant versions of the CTD repeats, we discovered that Cln3-Cdk1 specifically phosphorylates the Ser5 residue in each repeat. We hypothesize that Cln3-Cdk1 may play a role similar to that of the known Ser5 CTD kinase Ccl1-Kin28, which is responsible for basal transcriptional initiation. However, Cln3 only functions at specific promoters. To test this hypothesis, we applied the anchor-away technique, a system that allows the rapamycin-dependent induction of protein-protein binding. Remarkably, directing the Ccl1 or Kin28 subunits to SBF via its Swi6 subunit partially rescues the large cell-size phenotype 42 of cln3∆ cells and participates in rescuing viability of cln1∆cln2∆cln3∆ cells. This suggests that Cln3-Cdk1 may indeed function similarly to the Ccl1-Kin28 complex at SBF-regulated promoters. Further supporting this model, we find Cln3 at SBF-regulated promoters via ChIP-seq analysis. Together, these results suggest that Cln3-Cdk1 regulates G1/S progression via phosphorylation of Rbp1 CTD repeats at SBF regulated promoters. In summary, Cln3-Cdk1 does not phosphorylate Whi5 as previously expected, but rather phosphorylated the RNA Pol II CTD at SBF-regulated promoters to trigger the G1/S transition. Our work redraws the model of the G1/S regulatory network in budding yeast and expands our knowledge of the molecular mechanisms through which Cdk can trigger cell cycle progression.

105 Role of chaperone-mediated protein quality control in stress-induced relocalization of histone deacetylases. A. Kumar1,2, V. Mathew1, P. Stirling1,2 1) Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, CA; 2) Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, CA. Cells experiencing environmental stress must coordinate the repair of cellular damage with the transcriptional and proteome remodelling associated with stress recovery. The coordination of transcription changes with protein quality control (PQC) is not well understood. Here, we focus on two histone deacetylases, Rpd3 and Hos2, both of which are important for transcription dynamics and are sequestered to nuclear PQC compartments upon methyl methanesulfonate (MMS) treatment. We determined the molecular chaperones that are required for Rpd3/Hos2 deposition in PQC structures. As with other nuclear PQC substrates, BTN2 deletion results in only reduction of nuclear foci, while HSP42 deletion leads to complete removal of both nuclear and cytoplasmic foci. We are currently exploring additional modifiers, and the effects of TORC1 and ATM/ATR signaling on Rpd3/Hos2 relocalization. Moreover, we are exploring the impact of PQC sequestration on Rpd3 assembly with its partner proteins. Building on this preliminary results, our goal is to help link chromatin remodeling complexes and stress recovery in the nucleus and thus help take a step forward in understanding how cells coordinate transcriptional responses to stress with remodeling of the nuclear proteome.

106 Multiplicity of LAMMER kinase-dependent G1/S progression in fission yeast. S.J. Kwon, S Shin, S.H. Jang, H.M. Park Microbiology & Molecular Biology, Chungnam national university, Daejeon, KR. LAMMER kinases have various cellular functions in eukaryotes. In Schizosaccharomyces pombe, LAMMER kinase, Lkh1, is crucial in regulating cell cycle, especially G1/S progression. It targets two cell cycle related proteins: CDK (cyclin dependent kinase) inhibitor, Rum1 and a negative regulator of MBF (G1/S transcription factors), Yox1. Lkh1 is reported to phosphorylate Thr110 residue of Rum1 and up-regulate its activity. To identify how Lkh1 positively regulates Rum1 activity, further experiments were performed as follows; Pull-down assay was performed to test whether Lkh1-dependent phosphorylation on Rum1 facilitates the interaction with Cdc2-cyclin complex. Effect of Lkh1-dependent phosphorylation on Rum1 stability was measured by cycloheximide chase assay. Finally, cellular localization of Rum1 in wild type and lkh1 deletion mutant was observed with fluorescence microscopy. These experiments showed that the interaction with Cdc2, stability, and cellular localization of Rum1 were not affected by Lkh1-dependent phosphorylation. Cdk inhibitors inhibit productive binding with ATP and active conformation of Cdc2-cyclin complex. Therefore, it was proposed that the phosphorylation on Thr110 by LAMMER kinase may have an effect on not interaction with Cdc2-cyclin complex, but conformation of it. We also found that Lkh1 phosphorylates Thr40, 41 residues of Yox1. Microarray analysis with the lkh1 deletion mutant revealed four cell cycle genes up-regulated, of which expression are known to be modulated by the G1/S specific MBF. Effects of Lkh1-dependent phosphorylation on in vivo functions such as cell morphology and intracellular localization of Yox1 were investigated. No dramatic changes in the ratio of cell length to width in Yox1 mutants were observed. When cellular localization of phospho- mutation forms of Yox1 was observed with fluorescence microscopy, Yox1T40,41A was less frequently located at nucleus than Yox1WT. The results presented here indicate multiple functions of LAMMER kinase in G1/S progression of S. pombe cell cycle: activation of Cdc2-inhibitor Rum1 and a MBF-repressor, Yox1.

107 Yeast models of PMM2 deficiency, a Congenital Disorder of Glycosylation. J.P. Lao, E.O. Perlstein Perlara PBC, South San Francisco, CA. Perlara PBC is a public benefit corporation committed to discovering small molecule therapeutics for rare genetic diseases using genetic models such as yeast, worms, and flies in high-throughput drug discovery screens. Our parallel, multi-model whole animal screening approach leverages shared evolutionary pathways to accelerate the discovery of potential cures. We have partnered with Maggie’s Cure to identify therapeutics for the most common form of congenital disorder of glycosylation (PMM2-CDG). PMM2-CDG is an inherited metabolic disorder that impairs the production of glycoprotein, and affects many parts of the body leading to hypotonia, developmental delay, lethargy, and multiple organ failures.

PMM2 encodes an enzyme, phosphomannomutase 2, that catalyzes the conversion of mannose-6-phosphate to mannose-1- phosphate. This is then converted into GDP-mannose and is an important intermediate in the glycosylation pathway. The Saccharomyces cerevisiae ortholog SEC53 is 55% identical to PMM2. We generated yeast models for a high-throughput drug discovery screen that corresponds to PMM2 disease-causing alleles: F119L, R141H, and V231M. These correspond to F126L, R148H, and V238M in SEC53. We expressed these variants under four different promoters, TEF1 > ACT1 > SEC53 > REV1, to alter the amount of protein the cell produces over a 40-fold range. We determined that the R148H allele is lethal 43 under any expression level, which is consistent with its reported lack of enzymatic activity. Under the endogenous SEC53 promoter, F126L and V238M alleles show compromised growth as measured by absorbance at 600 nm. F126L shows a more severe growth defect than V238M. Their growth defects correspond to their enzymatic activity of 25% and 38% respectively. Doubling the expression of F126L by driving it with the ACT1 promoter improves growth, while two-fold expression of V238M fully restores growth. We also generated heterozygous diploids to mimic compound heterozygous patient alleles: F126L- R148H, V238M-R148H. We found that these strains show similar growth defects as the F126L and V238M haploids. We have completed screening a 2,650-compound Microsource Spectrum library of FDA approved drugs, bioactive tool compounds, and natural products to identify small molecules that rescue the growth defects of these strains.

108 Post-translational Modifications of Lysines Within Replication Factor A (RFA) Direct both Checkpoint Entry and Exit. W. Larson, S. Haring North Dakota State University, Fargo, ND. Replication Factor A (RFA) is a complex that binds to single-stranded DNA (ssDNA) to maintain the fidelity of genomic information during cellular replication and in the event of DNA damage. When genotoxic stress occurs, RFA binds to ssDNA and recruits proteins necessary to carry out the DNA damage response. The repair of damaged DNA requires the establishment of a checkpoint to ensure that cellular division is delayed until DNA lesions have been repaired. Checkpoint exit can occur after the damage has been repaired (checkpoint recovery) or in the presence of persistent DNA damage after a prolonged delay (checkpoint adaptation). One type of post-translational modification (phosphorylation) appears to play a major role in checkpoint adaptation. The contribution of other post-translational modifications that occur on RFA in response to DNA damage have yet to be determined.

To study the importance of how post-translational modifications of RFA regulate its function, one or more subunits of the RFA complex were mutated, such that all lysines within the subunit(s) were replaced with arginines. This lack of lysine residues prevents post-translational modifications such as sumoylation, ubiquitination, acetylation, and methylation from occurring within the mutated subunit. We show that “lysine-less” Rfa2 influences the ability of yeast to exit a checkpoint and resume the cell cycle, in response to irreparable DNA damage. Adaptation-deficient cells were induced to undergo checkpoint adaptation more readily when transformed with a vector containing the lysine-less Rfa2 mutant gene. Interestingly, cells containing the lysine-less Rfa2 gene also demonstrated more extensive Rfa2 phosphorylation and greater incidence of adaptation compared to wild-type cells, supporting that post-translational modification of lysines within Rfa2 may negatively regulate its phosphorylation, which in turn, helps prevent checkpoint exit. Conversely, lysine-less Rfa1 mutant cells display an inability to fully activate a checkpoint, indicating that modification of lysines on this subunit may be important for positive regulation of checkpoint activity. The role of lysine modifications on Rfa1 and Rfa2 appear to both coordinately enforce checkpoint entry (activation), and we propose later phosphorylation of Rfa2 and checkpoint exit requires reversal of lysine modifications.

Support: This work was supported by a National Science Foundation grant (NSF-CAREER-1253723) awarded to SJH.

Corresponding Author’s Email: [email protected]

109 Distinct roles for COMPASS subunits Swd1 and Swd3 during meiosis. Brandon M. Trainor2, Kerri A. Ciccaglione2, Miranda Czymek1, Michael J. Law1 1) Biology Program, Stockton University, Galloway, NJ; 2) GSBS, Rowan University, Stratford, NJ. Differentiation requires cells to integrate intrinsic and extrinsic information into a program of morphogenesis. When deprived of fermentable carbon and nitrogen, diploid cells of the budding yeast Saccharomyces cerevisiae will execute meiosis. Meiosis is characterized by one round of DNA replication followed by two rounds of nuclear division to yield four genetically distinct haploid gametes. After DNA replication, synapsis, and homologous recombination, cells commit to completing meiosis. After commitment, de novo membrane synthesis occurs resulting in a protective coating for the haploid gametes. Mistakes in pre- or post-commitment processes can result in checkpoint arrest or cell death. Post-translational histone modifications are involved in all aspects of chromatin biology. Histone H3K4 methylation (H3K4 me) represents one of the most well-studied of the histone modifications. Since lysines can be methylated up to three times, both the H3K4 me level and location can dictate chromatin behavior. The Set1-containing COMPASS complex is responsible for catalyzing all H3K4 me in yeast. Biochemical analyses have revealed that specific COMPASS subunits are required to catalyze precise H3K4 me levels. For example, Swd1 and Swd3 form a heterodimeric complex that is essential for H3K4 me both in vitro and in vivo. In addition, Swd1 and Swd3 are required for Set1 stability and COMPASS complex integrity, suggesting that these two subunits are functionally inseparable. While much is known about Swd1 and Swd3 in H3K4 me catalysis, less is understood regarding their role in important biological processes including meiosis. Here, we report distinct functions for Swd1 and Swd3 during yeast meiosis. While SET1 and SWD1 are required for meiotic commitment, SWD3 is critical for late meiosis and spore morphogenesis. Western blots indicate that mutations in SWD1 and SWD3 destabilize Set1 and abolish H3K4 me during meiosis, agreeing with their mitotic roles. This suggests that the meiotic phenotypes of swd1Δ and swd3Δ yeast mutants are independent of both H3K4 me catalysis and Set1. Interestingly, we have found that Set1 and Swd1 are important for efficient homologous recombination and spindle assembly, suggesting that COMPASS coordinates these events prior to commitment. These data are consistent with a model in which Swd1 and Swd3

44 may function as independent COMPASS subcomplexes that are required for progression through meiotic commitment and spore morphogenesis.

110 Cortical dynein pulling mechanism is regulated by differentially targeted attachment molecule Num1. W.-L. Lee, S. Omer Department of Biological Sciences, Dartmouth College, Hanover, NH. Cortical dynein generates pulling forces via microtubule (MT) end capture/shrinkage and lateral MT sliding mechanisms. In Saccharomyces cerevisiae, the dynein attachment molecule Num1 interacts with endoplasmic reticulum (ER) and mitochondria to facilitate spindle positioning across the mother-bud neck, but direct evidence for how these cortical contacts regulate dynein-dependent pulling forces is lacking. Using live cell imaging technique, we show that loss of Scs2/Scs22, ER tethering proteins, resulted in defective Num1 distribution and loss of dynein-dependent MT sliding, the hallmark of dynein function. Cells lacking Scs2/Scs22 performed spindle positioning via MT end capture/shrinkage mechanism, requiring dynein anchorage to an ER- and mitochondria-independent population of Num1, dynein motor activity, and CAP-Gly domain of dynactin Nip100/p150Glued subunit, but not the MT plus end depolymerase Kip3 or Kar3. Additionally, a CAAX-targeted Num1 rescued loss of lateral patches and MT sliding in the absence of Scs2/Scs22. These results reveal distinct populations of Num1 and underline the importance of their spatial distribution as a critical factor for regulating dynein pulling force.

111 Towards targeted LAMB3 PTC readthrough. M. Loffler, Anna Adamec Department of Biosciences, University of Salzburg, Salzburg, Salzburg, AT. In a premature termination codon (PTC) condition, cellular feedback signaling cascades cause repetitive shut-down of translational and transcriptional elongation. The requirement for coupling both processes can be observed as a necessary adaption during damage and repair. Continuous production of dysfunctional proteins requires energy, resources, labeling and final decay. Instead of constantly recycling misleading mRNA transcripts and misfolded protein products through chains of processing and decay, cells use advanced tools to circumvent and adapt to the PTC situation. In 2017, we established both, a genome-edited patient specific keratinocyte LAMB3 (R635X) PTC readthrough model system (1), and a Saccharomyces cerevisiae luc2-human gene-PTC-hRluc readthrough evaluation system (2). Comparisonsof patient and model system mass spectrometry readouts were used to identify patient specific insufficiencies/requirements and potential therapeutic targets in human keratinocytes. In the keratinocyte LAMB3 PTC regulative, essential amino acid and vitamin shortage, loss of salvage pathways and energy demanding cellular workarounds enforce the burden of extracellular matrix (ECM) degradation and repair. Preliminary results from the yeast evaluation system indicate that basal PTC readthrough of luc2-human gene-PTC-hRluc fusion proteins under the bi-directional GAL1,10 promoter can be achieved under non-induced conditions. Therefore, we propose that this system can be used for fast-track pre-evaluation of supplement based and transcript specific, basal PTC readthrough responses in yeast. Such pre-screened human PTC mRNA transcript variant conditions can be applied to targeted PTC readthrough in human keratinocytes, where exon-in/exclusion in mRNA PTC conditions causing higher demands of essential resources.

1. E18_02 (P_147200_30, Land Salzburg, University of Salzburg) 2. E17_03 (P_147200_30, Land Salzburg, University of Salzburg)

112 Proteasome activity is influenced by the HECT_2 Protein Ipa1 in budding yeast. A. Lutz1, S. Schladebeck1, C. Renicke1, R. Spadaccini2, H.-U. Mösch1, C. Taxis1 1) Department of Genetics, Faculty of Biology, Philipps-Universität Marburg, Germany; 2) Department of Science and Technology, Universita’ degli studi del Sannio, Benevento, Italy. The ubiquitin–proteasome system (UPS) controls cellular functions by maintenance of a functional proteome and degradation of key regulatory proteins. Central to the UPS is the proteasome that adjusts the abundance of numerous proteins, thereby safeguarding their activity or initiating regulatory events. Here, we demonstrate that the essential Saccharomyces cerevisiae protein Yjr141w/Ipa1 (Important for cleavage and PolyAdenylation) belongs to the HECT_2 (homologous to E6-AP carboxyl terminus_2) family. We found that five cysteine residues within the HECT_2 family signature and the C-terminus are essential for Ipa1 activity. Furthermore, Ipa1 interacts with several ubiquitin-conjugating enzymes in vivo and localizes to the cytosol and nucleus. Importantly, Ipa1 has an impact on proteasome activity, which is indicated by the activation of the Rpn4 regulon as well as by decreased turnover of destabilized proteasome substrates in an IPA1 mutant. These changes in proteasome activity might be connected to reduced maturation or modification of proteasomal core particle proteins. Our results highlight the influence of Ipa1 on the UPS. The conservation within the HECT_2 family and the connection of the human HECT_2 family member to an age-related degeneration disease might suggest that HECT_2 family members share a conserved function linked to proteasome activity.

113 Regulation of mitochondrial DNA levels by endosomal transport and UDP-glucose homeostasis. Y. Ma1,2, A. Rosebrock3, A. Caudy1,2 1) Department of Molecular Genetics, University of Toronto, Toronto, ON, CA; 2) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, CA; 3) Department of Pathology, Stony Brook School of Medicine, State University of New York, USA. Mitochondrial DNA (mtDNA) is essential for respiratory growth of all eukaryotic cells. Abnormalities of mtDNA have been 45 observed in many human diseases and during aging. We propose that insight into mtDNA regulation will allow a deeper understanding of disease pathology and provide targets for intervention. We performed a whole-genome screen measuring mtDNA level in each individual mutant from two whole genome haploid Saccharomyces cerevisiae deletion collections. We compared mtDNA levels in the S288C background to those in a newly derived version of the deletion collection where we used a novel counterselection approach to introduce a wild-type HAP1 allele. S288C strains carry a Ty1 element insertion in the transcription factor Hap1 that creates a hypomorphic HAP1 allele resulting in the reduced respiratory function of S288C derived strains. We measured the mtDNA content by TaqMan quantitative PCR for each strain of the two deletion collections, including an additional 700 wild-type control strains. Surprisingly, the S288C wild-type strain contains more mtDNA than the wild-type strain with the ancestral HAP1 gene, suggesting that cells carrying the S288C allele respond to the respiratory defect by increasing mtDNA. By examining the levels of mtDNA in each deletion mutant, we identified several potential regulators of mtDNA. Among them, the most interesting hit is the ESCRT complex that is required for intracellular transportation of many proteins and lipids. We propose that the ESCRT complex is involved in a novel mitochondrial turnover process independent of the canonical autophagy pathway and that blocking this turnover leads to accumulation of mtDNA. Another group of candidate mtDNA regulators is involved in UDP-Glucose metabolism which may affect the glycosylation of amino acid transporters. We will report our progress characterizing the effects of these mutations using microscopy and other approaches.

114 Destabilization of eIF3a leads to the formation of stress granules in Saccharomyces cerevisiae. I. Malcova, L. Senohrabkova, J. Hasek Laboratory of Cell Reproduction, Institute of Microbiology of the Czech Academy of Sciences, Prague 4, CZ. Eukaryotic cells response to environmental stresses by sequestering proteins, mRNAs and their complexes into various non- membraneous cytoplasmic inclusions. When the stress is robust enough to arrest translation, stress granules (SGs) are formed, however, their composition varies with the applied stress. In our previous studies, we have described the formation of stress granules in yeast cells induced by robust heat shock 46°C. These accumulations harbor components of the translation machinery, mRNAs, and proteins affecting mRNA dynamics. Here we present formation of SGs upon moderate heat shock of 42°C due to a structural destabilization of the initiation translation factor 3a (eIF3a) and subsequent translational arrest. The wild-type eIF3a stays diffused in the cytoplasm and the translation is not arrested but slowed down only at this temperature. 42°C-SGs formed by the eIF3a mutant contain translational initiation, elongation and termination factors as well as heat shock proteins e.g. Hsp104, Hsp42, Hsp26. Eno2mRNA and mRNA degrading proteins Dcp2 and Xrn1 were also found colocalizing with 42°C-SGs. Neither small nor large ribosomal subunits do accumulate at 42°C. Although the protein composition is similar, the 42°C-SGs are less numerous and more robust than SGs formed at 46°C. Dissolution of 42°C-SGs is much faster than those formed upon the robust heat stress and does not depend on the main yeast disaggregase Hsp104. We identified 42°C-SGs in the vicinity of endoplasmic reticulum and mitochondria rather than in the cytosol. Our results demonstrate that affecting the function and structure of an essential translation component such as the eIF3 complex leads to sequestration of proteins and mRNAs even upon less severe stresses. This work was supported by the grant from the Czech Science Foundation CSF16-05497S

115 Toxicity and infectivity: insights from de novo prion formation. A.L. Manogaran, J. Sharma, B. Wisniewski, E. Legan, D. Lyke, J. Dorweiler Dept of Biological Sciences, Marquette University, Milwaukee, WI. Prion and other neurodegenerative diseases are associated with misfolded protein assemblies called amyloid. Prions were originally set apart from other amyloid diseases in that they were infectious. However, recent studies have suggested that amyloids associated with these other diseases have prion-like infectivity. Research has begun to uncover common mechanisms underlying transmission of amyloids, yet the mechanisms underlying amyloid formation and infectivity remains unclear. Here, we take advantage of the yeast prion called [PSI+], which is the prion form of the Sup35 protein, to uncover the early steps of amyloid formation and infectivity in vivo. While [PSI+] formation is quite rare, the prion can be induced by overexpression of the prion domain of the Sup35 protein. When fused to GFP, overexpression leads to the formation of fluorescent cytoplasmic structures. Using 4D live cell imaging, we observed that fluorescent structures appear through four different pathways to yield [PSI+] cells. In most of the cases, small foci appear within the cytoplasmic diffuse fluorescence. These foci are transiently mobile and then become a stationary aggregate. Once stationary, the majority of the fusion protein appears to be sequestered to the aggregate near the cellular cortex. Using mathematical modeling and analysis of actin mutants, our data suggests that the aggregate movement to the cortex is non-random and possibly requires intact actin networks. We also found that lysates from cells containing newly formed aggregates were able to convert [psi−] cells to [PSI+] cells, suggesting that newly formed prion aggregates are infectious. To begin to understand the mechanisms that underlie formation, we looked at genetic mutants in which prion formation is altered. Our previous work shows that toxicity is associated with cells containing newly formed aggregates in strains lacking the VPS5 gene. Our recent studies show that the infectivity of vps5Δ lysates is similar to wildtype. However, the physical appearance of the intracellular aggregates is different. Instead of large immobile aggregates, vps5Δ strains have an additional population of small mobile foci. We speculate that changes in the cellular milieu in vps5Δ strains may reduce the cell’s ability to efficiently recruit and sequester newly formed prion particles into central inclusion sites, resulting in toxicity.

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116 Identifying Selective Growth Inhibitors of Cells with High Mutation Burdens. J. Mares, R. Zabinsky, M. Zeman, R. She, T. Silvers, D. Jarosz Chemical and Systems Biology, Stanford University, Menlo Park, CA. Many cancers are surprisingly robust to high mutation burdens despite the fact that most mutations are deleterious. To identify small molecule inhibitors of this mutation-buffering property, we developed a model system in the yeast Saccharomyces cerevisiae. We propagated defined, independent mutator lineages to create clonal cell populations that harbor thousands of distinct mutations. We then performed a high throughput small molecule screen on our model system to identify selective inhibitors of growth in cells with high mutation burdens. We identified small molecules, including several FDA approved drugs, that inhibit growth of multiple lineages in a mutation burden dependent manner. Top hits implicate autophagy and calcium signaling as mechanisms for buffering the deleterious effects of recently acquired mutations. Further experiments using forward genetics approaches identified the targets of our other hits and further elucidate the mechanisms cells employ to buffer the deleterious effects of new mutations. Our findings suggest new pharmacological approaches that might be used to target cancers with high mutation burdens.

117 Systematic Mapping of Conditional Genetic Interactions in Saccharomyces cerevisiae. V. Messier1, J. Nelson2, M. Costanzo1, B. VanderSluis2, M. Rahman Ahm2, B. San Luis1, E. Shuteriqi1, M. Usaj1, S. Sharifpoor1, Y. Chen1, S. Dharwada1, B. Andrews1, C. Myers2, C. Boone1 1) University of Toronto, Donnelly Centre, Toronto, Canada; 2) University of Minnesota, Minneapolis, MN, USA. Genetic interactions (GIs) involving loss-of-function alleles have been systematically analyzed in budding yeast under standard growth conditions, creating a reference genetic network. We modified Synthetic Genetic Array (SGA) analysis to investigate the effect of 15 different stress conditions known to broadly affect yeast physiology on the GI network. We selected 30 query mutant strains that show broad GIs across many bioprocesses and crossed them against a 1,200 mutant strain diagnostic array by SGA, constructing 36,000 double mutants, which were then screened for condition-specific GIs. We identified a genetic network encompassing 13.7% (73, 843) of conditionally tested double mutants. The large majority of GIs are observed in standard media with similar or significantly enhanced fitness defects on stress conditions, respectively covering 8.1% (43,760) and 1.6% (9,174) of all tested interactions. We revealed condition-specific novel GIs, representing 0.8% (4,174) of probed double mutants, while standard media fitness defect could be ‘lost’ for 0.9% (5,015) of tested GIs upon specific stress treatment. These novel and ‘lost’ interactions were enriched for genes targeted by the specific stress-inducing conditions, suggesting their importance for cellular adaptation. Several groups of genes showed a condition-specific increase in GI profile similarity. For example, we observed that 117 genes involved in mRNA decay had GI profiles that were more similar when cells were grown in the presence of MMS, HU, benomyl and tunicamycin, and also showed novel GIs with Processing body (P-body) genes. Using high-content microscopy we explored the effect of these genes on selected mRNA decay pathway proteins (Lsm1p, Lsm8p, Pat1p and Dcp1p) in stress-induced cells and identified groups of mutants that shared specific mRNA decay pathway phenotypes. These mutants, enriched for nucleopore, karyopherin and heterogeneous nuclear ribonucleoproteins (hnRNPs) genes, perturb Lsm1p, Pat1p and Dcp1p localization to P-bodies that are involved in mRNA translation repression and degradation, while mislocalizing nuclear splicing factor Lsm8p into cytoplasmic foci. Collectively, the observed subcellular changes help us explain these stress-specific mutant strain fitness defects. Adopting an unbiased approach to study conditional GI network identified highly informative and novel functional links that we exploited to mechanistically associate gene mutations to their phenotypes.

118 Investigating the Novel Impacts of a Mutant NUD1 on the Mitotic Exit Network. Victoria Mingione, Michael Vannini , Courtney Sniffen , Anupama Seshan Department of Biology, Emmanuel College, Boston, MA. The Mitotic Exit Network (MEN) in S. cerevisiae is a protein signaling cascade that effects the M phase to G1 transition during the cell cycle. Homologs of the MEN in mammalian cells also govern cell cycle transitions. For example, the MEN protein Nud1, a spindle pole body (SPB) component, is homologous to Centriolin, a centrosomal protein that plays a crucial role in cell cycle progression. However, there are gaps in knowledge regarding the mechanisms of Centriolin activity, and defects in its function have been implicated in cancer. Our group has identified a novel hyperactive allele of NUD1. In this study, we evaluated the ability of cells containing the hyperactive Nud1 protein to bypass cell-cycle regulatory checkpoints. We also analyzed the stability of this mutant Nud1 protein and its effects on the activity and regulation of other MEN components. We found that the hyperactive Nud1 mutant promotes a robust bypass of the spindle positioning checkpoint (SPoC) in anaphase cells, and a partial bypass of the spindle assembly checkpoint (SAC) in metaphase cells. We also found that the mutant Nud1 bypasses a temperature-sensitive mutation in the MEN GTPase TEM1 and does not function by recruitment of Tem1 to SPBs. However, the mutant Nud1 does require the activity of the kinases Cdc15 and Mob1-Dbf2. Using a cycloheximide assay, we confirmed that the mutant Nud1 protein does not exhibit increased stability. Instead, using co-immunoprecipitation analysis and live-cell microscopy, we found that the mechanism of the mutant NUD1 allele is likely the early recruitment of Dbf2 to the mother SPB during metaphase. These data are consistent with the hypothesis that this Nud1 mutant has an additional phospho-docking site for Mob1-Dbf2, which in turn causes hyperactive MEN activation. In support of this hypothesis, the mutant Nud1 allele can partially suppress mutations in other key Nud1 phospho-binding sites. Research aimed at characterizing this NUD1 mutant, which shares homology with the cytokinesis domain of Centriolin, may provide clues toward elucidating how a hyperactive Centriolin protein could impact microtubule organization, chromosome segregation and cytokinesis in mammalian cells.

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119 Characterization of the catalytically-inactive phosphatase Oca1 as a pseudoenzyme that enhances the activity of the phosphatase Siw14. V.A. Morrissette1, E.A. Steidle1, H. Wang2, R.J. Rolfes1 1) Biology, Georgetown University, Washington, DC; 2) Inositol Signaling Group, NIEHS, Research Triangle, NC. Inositol pyrophosphates are high-energy signaling molecules found ubiquitously across eukaryotes and are involved in diverse pathways such as DNA repair, yeast virulence, human immune response, glycolysis, energy homeostasis, and the general stress response, although mechanisms are not fully understood. We recently characterized Siw14 from Saccharomyces cerevisiae as a novel inositol pyrophosphate phosphatase that cleaves the 5-position β-phosphate from

InsP7 to form InsP6. We are interested in understanding the mechanisms that regulate the activity of Siw14. Oca1 and Oca2 are paralogs of Siw14 and were examined for their enzymatic activity using generic and specific substrates. Using purified, recombinant GST-fusion proteins, Oca1 and Oca2 were each found to be catalytically inactive in vitro. To measure in vivo activity, we deleted siw14Δ, oca1Δ and oca2Δ from yeast and measured levels of endogenous [3H]-labeled inositol pyrophosphates. There was a 6.5-fold increase in InsP7 in the siw14Δ mutant and less than a 2-fold change in the oca1Δ and oca2Δ mutants. We expressed the OCA1, OCA2 and SIW14 genes in HEK293 cells; we saw a 90% reduction in the endogenous labeled InsP7 pools with SIW14 expression but no changes to the pools when OCA1 and OCA2 were expressed. Oca1 and Oca2 were found to carry substitutions in critical active site residues, consistent with their lack of catalytic activity. These data suggest there might be nonenzymatic role for Oca1 and Oca2; pseudoenzymes affect the subcellular localization, substrate binding, or enzymatic activity of a homologous, catalytically-active enzyme. To test the hypothesis that Oca1 and Oca2 are pseudoenzymes, we purified recombinant GST-tagged protein and mixed each one with purified Siw14, and assayed phosphatase activity. The Siw14 phosphatase activity increased approximately 2.5-fold with Oca1 using generic and specific substrates. However, there was no change in the activity of Siw14 when Oca2 was added to the reaction. On-going experiments are addressing other possible mechanisms of action for Oca2.

120 The 5PP-IP5 phosphatase Siw14 regulates levels of inositol pyrophosphates and confers stress resistance through Msn2/4. V.A. Morrissette, E.A. Steidle, R.J. Rolfes Biology, Georgetown University, Washington, DC. Cells and organisms integrate information about nutrient conditions and environmental stresses to inform growth and survival. Our lab recently described the novel inositol pyrophosphate phosphatase Siw14 that cleaves the 5-position β- phosphate from IP7 to form IP6. The siw14Δ mutant accumulates inositol pyrophosphates (PP-InsPs) IP7 and IP8 at levels 6.5- fold and 1.6-fold above WT levels, respectively. The siw14D mutant of Saccharomyces cerevisiae is resistant to a number of cell stressors including nutrient deprivation, heat, oxidative, and osmotic stresses. We examined the changes in global gene expression in WT and siw14Δ mutant cells, and found increased expression of stress-response genes in the mutant under non-stress conditions. This finding of partial induction of the stress-response, in a mutant that accumulates PP-InsPs, is consistent with the previously reported inability to induce a normal environmental stress response in a mutant that cannot synthesize PP-InsPs. These observations suggest that the high levels of PP-InsPs found in the siw14D mutant generate a cellular response similar to that of WT cells responding to stress. We considered two mechanisms by which PP-InsPs could affect the general stress response. First, the activity of the histone deacetylase Rpd3L is known to require PP-InsPs for activity; thus, modulation of PP-InsP levels could alter gene expression through Rpd3L. Alternatively, transcriptional activity by the general stress response transcription factors Msn2 and Msn4 could be altered in the siw14Δ mutant. We used epistasis to examine the stress response phenotype when the stress-resistant siw14Δ mutant was combined with the sensitive rpd3ibp single or the msn2Δ msn4Δ double mutants. Unexpectedly, we found that the double siw14Δ rpd3ibp mutant was as resistant to oxidative stress as the siw14Δ mutant, indicating that siw14Δ is epistatic to rpd3ibp; this finding was unexpected as rpd3ibp cannot mount a normal stress response. We found that the siw14Δ msn2Δ msn4Δ triple mutant had the same sensitivity to stress as the msn2Δ msn4Δ double mutant, indicating that msn2Δ msn4Δ are epistatic to siw14Δ. Thus, the effects of the loss of Siw14, likely the accumulation of PP-InsPs, are acting upstream of Msn2/4.

121 The yeast kinase Ksp1 regulates cellular stress response. N. Mutlu1, D. Sheidy1, P. Andrews2, A. Kumar1 1) Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI ; 2) Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI. Pseudoyphal growth is a cellular stress response where yeast cells form elongated multicellular filaments, similar to processes of filamentous development required for virulence in pathogenic yeast such as C. albicans. We have identified KSP1 as a gene required for wild-type yeast pseudohyphal growth, likely through its association with the Target of Rapamycin Complex (TORC1). KSP1 was first identified as a high-copy suppressor of a mutation in the nucleotide exchange factor SRM1. It negatively regulates autophagy via TORC1, and its localization changes during pseudohyphal growth. We have found that Ksp1 kinase activity is required for pseudohyphal growth in S. cerevisiae. The signaling network of Ksp1, however, has not been studied directly on a proteome-wide scale in a filamentous background. To address this, we undertook an analysis of Ksp1 signaling through quantitative phosphoproteomics, identifying proteins differentially phosphorylated in a catalytically inactive kinase-defective Ksp1 mutant under conditions of nitrogen and glucose stress. Analysis of the proteins differentially phosphorylated upon loss of Ksp1 kinase activity identifies a statistically significant set of proteins associated with mRNA-protein (mRNP) granules. mRNPs, encompassing P-bodies and stress granules, present an additional form of stress response thought to regulate mRNA translation. We find that Ksp1 localizes to mRNP granules in a filamentous background, in agreement with recent studies of mRNPs in a non-filamentous yeast strain. Moreover, the kinase activity of Ksp1 is required for wild-type localization of several mRNP component proteins/regulators upon growth to a high cell density, 48 including Pbp1 and the P21-activated kinase ortholog Ste20. We further find that Ksp1 kinase activity regulates the localization of TORC1 and that Ksp1 colocalizes with TORC1 under stress. A catalytic residue in the kinase domain of Ksp1 is important for this colocalization. TORC1 is known to be a key regulator for translation under stress and preliminary yeast two hybrid data identify a putative interaction between Ksp1 and the cap binding translation initiation factor eIF4E. Collectively, these results suggest a function for Ksp1 in coordinating TORC1-signaling, translation and the regulation of mRNP dynamics, likely through phosphorylation of key effectors. Ongoing studies are aimed at identifying the mechanism through which this signaling is coordinated.

122 Novel role for Skn7p and the Calcium Signaling Pathway in Endoplasmic Reticulum Stress survival. A.E. Olivares, E.A. Dominguez-Martin, S. Gonzalez-Behn-Eschenburg, L. Kawasaki, R. Coria Departamento de Genetica Molecular, Instituto de Fisiologia Celular, UNAM, Ciudad de Mexico, Coyoacan, MX. SKN7 is a gene coding for a very peculiar, and understudied, transcription factor (TF). SKN7 has a two-component system receiver domain as well as a Heat Shock TF-homology region. Initially described as an effector for the Saccharomyces cerevisiae’s phosphorelay system, Skn7 has been also considered as one of two master transcription factors of the Oxidative Stress Response in yeast. In this work, we describe yet another, independent role for Skn7 in the cell’s physiology. We found that Skn7 is required for the survival to Endoplasmic Reticulum (ER) stress; we demonstrated that its function in this process did not require the receiver aspartate residue of the phosphorelay system, and neither was related to its role in oxidative stress. Instead SKN7 interacted genetically with another transcription factor, i.e, the main transcriptional regulator of the Calcium Signaling Pathway: CRZ1. This genetic interaction was studied further, and we found that many of the genes involved in the Calcium Signaling Pathway were as well needed for ER stress survival. We also found that Crz1p translocates from the cytosol to the cell’s nucleus (a landmark of its activation) in response to ER stress, and that Skn7p and Crz1p hold a physical interaction in the nucleus. Finally, we studied the expression of FKS2, a previously reported gene for the calcium pathway, in response to ER stress, in both wild type and skn7 and crz1 mutant cells. We found that ER stress induces a strong expression of FSK2 in the wild type cells but not in the mutant cells, indicating that both Skn7 and Crz1 are required to induce FSK2 expression under ER stress. These results imply a novel function for Skn7 and give clear evidence for the participation of the Calcium Signaling Pathway in the survival to ER stress. This work was supported by grants: CONACyT CB-254078 and DGAPA, UNAM, PAPIIT IN210616 to RC. AO is a Master student of the Biochemical Science Program, UNAM

123 Selective autophagy and remodeling of the endoplasmic reticulum during budding yeast meiosis. G.M. Otto, G.A. Brar Molecular and Cell Biology, UC Berkeley, Berkeley, CA. The endoplasmic reticulum (ER) is a membrane-bound organelle whose diverse, essential functions rely on the maintenance of a complex membrane architecture. During acute stress or cell differentiation, the ER must incorporate new protein constituents while degrading parts of the organelle that are no longer required, often undergoing dramatic changes in morphology in the process. Despite the clear link between ER structure and function, how changes to these features are coordinated during times of cellular change is poorly understood. The ER is dramatically remodeled during meiosis in budding yeast, as the cortical ER detaches from the plasma membrane and collapses around the dividing nuclei before partitioning into maturing spores. Here, we show that the ER is degraded by selective autophagy during meiosis, coincident with ER detachment from the plasma membrane. Selective ER autophagy is regulated by developmentally timed expression of the autophagy receptor Atg40. Mutants that fail to detatch the ER from the cell cortex during meiosis show reduced ER autophagy, suggesting a link between the two processes. Preliminary data suggest that the unfolded protein response, historically defined as a stress response pathway but with conserved roles in development, may link ER collapse to the induction of selective ER autophagy.

124 The yeast claudin Dcv1 contributes to the polarization of mating functions. C. Pai, M. Sukumar, D. Stone Biological Sciences, University of Illinois at Chicago, Chicago, IL. Chemotaxis (directed cell movement) and chemotropism (directed cell growth) are processes that are essential to a wide range of biological phenomena. Both chemotactic and chemotropic cells exhibit a remarkable ability to interpret the shallow chemical gradients and sense direction. Establishment and maintenance of front versus back cell polarities are crucial for cell migration and polarized cell growth. The mating response of the budding yeast S. cerevisiae is the best-studied chemotropic model to date. Two haploid mating types, MATa and MATα, can sense the pheromone secreted by the opposite type, polarize their growth toward the closest mating partner, and fuse to form diploid zygotes. During the mating response, the asymmetric distribution of specific proteins and plasma membrane (PM) lipids is vital for the pheromone signaling and polarized mating functions. The pheromone receptor (Ste2) and other proteins required for mating polarize to the chemotropic growth site and center at the mating projection (shmoo). PM lipids such as phosphatidylserine (PS) and ergosterol polarize to the mating projection, whereas phosphatidylinositol-4,5-bisphosphates (PIP2) distributes to the back of the cell. However, how the polarity of these proteins and PM lipids is established and maintained is unclear. In a directed genetic screen, we found that deletion of the yeast claudin homolog, DCV1, caused depolarization of the receptor. In mating mixtures, the receptor crescents in dcv1∆ cells were not centered toward the mating partners. Additionally, dcv1∆ cells exhibited an aberrant distribution of PM lipids in shmooing cells. In WT shmooing cells, Dcv1-RFP and Ste2-GFP are inversely localized: Dcv1 concentrated at the back of the cell and the receptors polarized to the mating projection. These results are consistent with the conclusion that in higher eukaryotes, claudins promote PM lipid segregation and act as a PM barrier. We 49 propose that Dcv1 provides a PM barrier during the mating response. This barrier promotes and maintains the polarization of mating-specific proteins and PM lipids that are critical for polarized mating functions. Consistent with our hypothesis, time- lapse imaging of the erg6∆ cells in mating mixtures showed significant defects in receptor polarization and orientation. However, unlike WT and dcv1∆ cells, erg6∆ cells did not bud at the center of the fusion zone, suggesting that ergosterol is required to properly localize polarity proteins that mark the correct bud site.

125 The shared role of the Rsr1 GTPase and Gic1/Gic2 in Cdc42 polarization. Pil Jung Kang1, Kristi Miller2, Julia Guegueniat1, Laure Beven1, Hay-Oak Park1,2 1) Department of Molecular Genetics, Ohio State Univ, Columbus, OH; 2) Molecular Cellular Developmental Biology Program, Ohio State Univ, Columbus, OH. The Cdc42 GTPase plays a central role in polarity development in species ranging from yeast to human. In budding yeast, Cdc42 is necessary for both selection of a proper bud site as well as polarity establishment. While multiple events including the polarized organization of actin and septin cytoskeletons and targeted secretion are necessary for bud emergence, how these events are coordinated with cell cycle progression remain unclear. We previously discovered biphasic polarization of Cdc42 during G1 in haploid cells: Cdc42 is activated sequentially by two GDP-GTP exchange factors – first by Bud3 and subsequently by Cdc24. Here we report that the Rsr1 GTPase shares a partially redundant role with Gic1 and Gic2, two closely related Cdc42 effectors, in Cdc42 polarization during the first period of G1. We find that cells depleted for Rsr1 and both Gic proteins are defective in recruitment of new septin ‘clouds’, suggesting that biphasic Cdc42 polarization in the G1 phase is likely coupled to stepwise assembly of the septin ring for bud emergence. Interestingly, overexpression of CDC42 bypasses requirement of Rsr1 and both Gic proteins for septin recruitment. While the role of Rsr1 in Cdc42 polarization depends on spatial cues, Gic proteins promote Cdc42 polarization by reducing its mobility at the incipient bud site. We propose that the first phase of Cdc42 polarization is mediated by positive feedback loops that function in parallel – one involving Rsr1 via local activation of Cdc42 in response to spatial cues and another involving Gic1 or Gic2 via reduction of diffusion of active Cdc42.

126 Septin-associated proteins Aim44 and Nis1 traffic between the bud neck and the nucleus in the yeast Saccharomyces cerevisiae. A. M. Perez, J. Thorner Molecular and Cell Biology, University of California, Berkeley, Berekely, CA. In budding yeast, septin-based structures reproducibly adopt different stereotypical states of organization in a highly reproducible and cell cycle-dependent manner. These septin-based structures serve central roles in coordinating events required for proper cell morphogenesis and timely execution of cytokinesis. Considerable evidence supports the view that a primary function of these septin arrays is to serve as a scaffold for the highly localized recruitment of a host of other proteins and enzymes that carry out necessary cell cycle-specific processes. For instance, the inhibition of Cdc42-dependent bud site re-establishment at the division plane is mediated by a multi-protein complex that localizes at the bud neck. This complex consists, in part, of the Aim44, Nba1 and Nis1 proteins. We are currently investigating the molecular mechanisms that direct these proteins to the septin cytoskeleton in order to better understand the role of septins in the spatiotemporal regulation of the cell cycle. Using live cell imaging, we have observed that Aim44 co-localizes with septins from G1 phase through cytokinesis, whereas Nis1 and Nba1only arrive at the bud-neck during G2/M and continue to mark the cytokinetic site after cell septation. We have also found that expression levels play a critical role in targeting Aim44 and Nis1 to the bud-neck. When overproduced, both proteins shift their subcellular distribution predominantly to the nucleus (Aim44 localizes at the nuclear envelope and the plasma membrane, whereas Nis1 accumulates within the nucleus), indicating that these proteins normally undergo nucleocytoplasmic shuttling. Of the 14 yeast karyopherins, Kap121/Pse1 and Kap123/Yrb4 are the primary importins for Aim44, whereas Kap123/Yrb4 and Kap111/Mtr10 appear to be the major importins for Nis1. Moreover, in the absence of the exportin Kap124/Xpo1/Crm1, Nis1 expressed at its endogenous level fails to be exported from the nucleus. Likewise, in the absence of Nba1, endogenously-expressed Nis1 also accumulates in the nucleus. Consistent with their mutual interaction, overexpressed Aim44 or Nis1 compete for the pool of Nba1 and displace it from the bud neck. Collectively, our results indicate that a previously unappreciated level at which localization of septin-associated proteins is controlled is via regulation of their nucleocytoplasmic shuttling, which places constraints on their availability for complex formation with other partners at the bud neck.

127 DNA alkylating drug methyl methanesulfonate causes a cell division arrest program distinct from a G1 or G2/M checkpoint or from DNA damage adaptation, that resembles an endocycle. H. Phenix1,2, M. Tepliakova2, C. Nwosu2, D. Jedrysiak1,2, M. Downey2,4, T. Perkins1,3,4, M. Kærn1,2,5 1) Dept. of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, CA; 2) Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, ON, CA; 3) Ottawa Hospital Research Institute, Ottawa, ON, CA; 4) Dept. of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, CA ; 5) Dept. of Physics, University of Ottawa,. DNA damage adaptation is a phenomenon in which prolonged or irreparable DNA damage leads to an override and deactivation of the DNA damage checkpoint (DDC) pathway. To investigate whether adaptation could explain why a marker of DDC pathway activity, RNR3 transcription, is repressed by high but not low doses of the DNA alkylating agent methyl methanesulfonate (MMS), we examined Rad53p electrophoretic mobility and cell division cycle progression as a function of MMS dose. We find that Rad53p mobility is consistent across dose, but not cell division state. Low dose exposure synchronizes cells in S or G2, whereas high dose exposure synchronizes cells in G1. We hypothesize that high dose exposure may allow cells to override the G2 checkpoint by a mechanism distinct from adaptation. This could explain why about 25% of 50 cells exposed to high dose exhibit characteristics of cells that have re-entered G1 from S or G2 phase, and thus simultaneously posses both a bud and canonical markers of G1 cells.

128 Posttranslational modifications modulate α-synuclein cytotoxicity and degradation fate in a yeast model of Parkinson´s Disease. B. Popova1,2, A. Kleinknecht1,2, D. Lazaro2,3, O. Valerius1,2, T. Outeiro2,3, G. Braus1,2 1) Institute of Microbiology and Genetics, Department of Molecular Microbiology and Genetics, University of Goettingen, Goettingen, Germany; 2) Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Goettingen, Germany; 3) University of Goettingen Medical School, Department of NeuroDegeneration and Restorative Research, Goettingen, Germany. Parkinson’s disease (PD) is characterized by loss of dopaminergic neurons and presence of α-synuclein (αSyn) protein inclusions. The accumulation of αSyn is critical for the development of the disease. Human αSyn is subject to a variety of posttranslational modifications including ubiquitination, SUMOylation, phosphorylation, acetylation, nitration, and glycation. However, little is known about the interplay between these modifications and the consequences for αSyn toxicity and aggregation. Dysregulation of posttranslational modifications has been associated with many disease conditions and was linked to the pathogenesis of the disease. The yeast Saccharomyces cerevisiae represents established system for modelling human diseases. Expression of human αSyn in yeast resembles the pathology of the disease resulting in cytotoxicity and aggregate formation. We characterize the interplay of phosphorylation and nitration of αSyn and its impact on the ratio of clearance by autophagy versus the ubiquitin-proteasome system. αSyn is abundantly phosphorylated at serine S129 and possesses four tyrosines (Y39, Y125, Y133, and Y136) that can be posttranslationally modified by nitration or phosphorylation. We demonstrated that a-synuclein is nitrated in vivo leading to the formation of stable αSyn dimers originating from covalent cross-linking of two tyrosine residues. Tyrosine nitration can contribute to αSyn toxicity or can be part of a cellular salvage pathway when di-tyrosine-crosslinked dimers are formed. The Y133 residue, which can be either phosphorylated or nitrated, determines whether S129 is protectively phosphorylated and αSyn inclusions are cleared. This complex interplay with S129 phosphorylation demonstrates a dual role for C-terminal tyrosine residues that control αSyn protein turnover.

129 CDK targets regulating the coordination of nutrients and polarized cell growth. E.B. Powell, D.P. Toczyski Biochemistry & Biophysics, UCSF, San Francisco, CA. As cells divide, they must coordinate the cell cycle with growth. These events are regulated by cyclin-dependent kinases (CDKs). As yeast cells commit to entering the cell cycle, they must remodel their membranes and cell walls to allow polarized growth. Chitin, an important structural component of the cell wall, is deposited in a polarized manner and modulated in response to stress, a process controlled by the intracellular trafficking of cell wall biosynthesis enzymes. Here we establish that Isr1, a largely uncharacterized kinase with homology to Raf, is tightly regulated to maintain cell wall homeostasis. In conjunction with transcriptional regulation, Isr1 is phosphorylated by the CDK Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex. This largely confines Isr1 protein levels to the time of bud emergence. Stabilization of Isr1 compromises chitin deposition, as evidenced by calcufluor-white resistance. Thus, control of Isr1 protein levels may provide the cell with an additional layer of regulation to allow for dynamic remodeling of the cell wall in response to environmental and cellular inputs. We will present these data and ongoing studies examining the Isr1 substrates responsible for this phenotype and other CDK substrates that feed into this pathway.

130 Role for a Cdc42p-dependent MAP Kinase pathway in regulating bud morphogenesis in yeast. A. Prabhakar, J. Chow, P.J. Cullen Department of Biological Sciences at SUNY-Buffalo, Buffalo, NY. Cell polarity is a multifaceted process governed by the concerted action of evolutionarily conserved regulatory proteins. In yeast, the Rho GTPase Cdc42p controls the establishment of polarity and polarized growth to produce a daughter cell or bud. Cdc42p also regulates signal transduction pathways. In response to nutrient limitation, Cdc42p regulates the filamentous growth Mitogen Activated Protein Kinase (fMAPK) pathway, which controls a morphogenetic response that generates a new cell type. We show here that the fMAPK pathway regulates aspects of bud morphogenesis. Activation of the fMAPK pathway suppressed the growth/polarity defects of the cdc24-4 mutant, which has problems establishing a new growth site. Suppression was dependent on Gic2p, a direct effector of Cdc42p and transcriptional target of the fMAPK pathway. Suppression also required the formin Bni1p, which nucleates actin filament assembly at sites of polarized growth. We also show that activation of the fMAPK pathway induced multiple growth projections in wild-type cells. One of the plasma- membrane sensors of the fMAPK pathway, Sho1p, impacted aspects of Cdc42p-dependent polarity outside its role in regulating the fMAPK pathway. Another transcriptional target of the fMAPK pathway, Rax2p, functioned with Gps2p and Nba1p to exclude Cdc42p-fMAPK signaling from dormant growth sites, which may help focus Cdc42p activity to sites of bud emergence. Collectively, the study demonstrates a role for a differentiation-type MAPK pathway in regulating aspects of bud morphogenesis.

131 A new yeast peroxin, Pex36, a functional homologue of mammalian PEX16, functions in the ER-to-peroxisome trafficking of peroxisomal membrane proteins. JC Farré1, K Carolino1, OV Stasyk2, OG Stasyk2,3, Z Hodzic1, G Agrawal1, A Till4, M Proietto1, J Cregg5, AA Sibirny2,6, S Subramani1 1) University of California San Diego, UCSD, La Jolla, CA, USA; 2) National Academy of Sciences of Ukraine, Lviv, Ukraine; 3) Ivan Franko National University of Lviv, Lviv, Ukraine; 4) University of Bonn Medical Faculty, Bonn, Germany; 5) Keck Graduate Institute of Applied Life Science, Clairemont, CA, USA; 6) University of 51

Rzeszow, Rzeszow, Ukraine. BACKGROUND: Peroxisomes are ubiquitous, single membrane-bound organelles present in almost all eukaryotic cells. They participate in a wide variety of metabolic processes, many of which are related to the metabolism of lipids and reactive oxygen species. Peroxisomal membrane proteins (PMPs) traffic to peroxisomes by two mechanisms: 1) direct insertion from the cytosol into the peroxisomal membrane and 2) indirect trafficking to peroxisomes via the endoplasmic reticulum (ER). In mammals and yeast, several PMPs traffic via the ER in a Pex3- and Pex19-dependent manner. In Komagataella phaffii (Pichia pastoris) the indirect trafficking of Pex2, but not that of Pex11 or Pex17, depends on Pex3, but all PMPs tested for indirect trafficking require Pex19. In mammals, the indirect trafficking of PMPs also requires PEX16, a protein that is absent in most yeast species. Other than the requirement of these factors, the machinery and mechanisms that regulate the indirect trafficking of PMPs to the peroxisomes via the ER are still poorly understood.

RESULTS: In this study, we isolated a new gene in K. phaffii, which encodes a new PMP. Using cellular and molecular approaches (confocal and electronic microscopy, growth curve assays, yeast two-hybrid, western blot, complementation of mutant strains with homologous proteins) we elucidated the role and the function of this new protein that we named Pex36. Pex36 is essential for cell growth in conditions that require peroxisomes for the metabolism of certain carbon sources like methanol. A growth defect in cells lacking Pex36 can be rescued by the expression of human PEX16, S. cerevisiae Pex34, or by overexpression of the endogenous K. phaffii Pex25. Conversely, the expression of Pex36 restores the normal phenotype in human ΔPex16 fibroblasts. Pex36 is non-essential for peroxisome proliferation, but in the absence of the functionally redundant protein Pex25, it becomes essential and less than 20% of ΔPex36 cells show import-incompetent peroxisome-like structure. In the absence of both proteins, peroxisome biogenesis and the intra-ER sorting of Pex2 and Pex11C are seriously impaired, likely by affecting Pex3 and Pex19 function.

132 The mRNA-binding protein Cth2 post-transcriptionally regulates gene expression in response to iron deficiency. A. M. Romero1, L. Ramos-Alonso1, A. Perea1, T. Jordá1, C. Ros-Carrero1, P. Miró1, M. T. Martínez-Pastor2, S. Puig1 1) Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain; 2) Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, Valencia, Spain. Iron is an indispensable micronutrient for all eukaryotic organisms due to its participation as a redox cofactor in many metabolic pathways including respiration and the biosynthesis of DNA, proteins and lipids. Iron imbalance leads to the most frequent human nutritional deficiency on the globe. In response to iron scarcity, the conserved Saccharomyces cerevisiae mRNA-binding protein Cth2 coordinates a global remodeling of the cellular metabolism by promoting the degradation of multiple mRNAs encoding highly iron-consuming proteins and facilitating iron utilization in essential processes. Here, we reveal a sophisticated mechanism for the phosphorylation and degradation of Cth2 protein that is necessary for the adaptation to iron deficiency. By using protein-protein interaction experiments, we study the imprinting of Cth2 mRNA targets during the process of mRNA decay. Finally, we show initial data on how Cth2 regulates lipid biosynthesis. Collectively, these results highlight the relevance of Cth2 protein on the adaptation to iron imbalance.

133 Characterization of the Sks1 target residue S837 and the phosphorylated residues in Linker 2 of the yeast multidrug transporter Pdr5. H. Rahman1, S. Patrick Joly1, J. Carneglia1, M. Robertello1, M. Lausten2, J. Golin1 1) Biology, The Catholic University of America, Washington, DC; 2) Haverford College, Haverford, PA. Identification of interacting proteins is critical to an understanding of how ABC transporters function. We identified an intergenic suppressor Sks1, a serine-threonine kinase, in the background of a loss of function mutation (E1013A) of yeast multidrug transporter Pdr5. Resistance is restored when Sks1 overexpressed in E1013A and significantly increased when overexpressed in the wildtype strain. However, deletion of Sks1 in the WT background does not have any effect. Of significance was the analysis of the Sks1 target residue S837 located in the linker connecting the drug and ATP-binding domains. To our surprise, we found that S837A and S837D mutants show increased drug resistance and drug transport relative to the wild-type strain. In addition to S837A, we investigated the role of the rest of five additional phosphoserine residues in linker 2 region by constructing alanine substitutes. Our data indicate that all of these mutants showed significant drug resistance to the Pdr5 drug substrates cycloheximide and cerulenin. Furthermore, rhodamine 6G transport is enhanced. These observations suggest that phosphorylation may modulate Pdr5 function. The ATPase activity of single and double mutants is not different from WT Pdr5. Understanding how these alanine mutants' increase resistance without further increasing ATPase activity will be critical to evaluating the role of phosphoserine residues in the linker 2 region of Pdr5.

134 Deletion of deubiquitinase UBP6 exacerbates disease severity in the yeast model of Niemann-Pick type C disease. Tamayanthi Rajakumar1, Katsumi Higaki 2, Stephen Sturley3, Andrew Munkacsi1 1) School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand; 2) Division of Functional Genomics, Tottori University, Yonago, Japan; 3) Department of Biology, Barnard College at Columbia University, New York, New York, USA. Niemann-Pick type C (NP-C) disease is a fatal, pediatric neurodegenerative disease due to the lysosomal accumulation of 52 cholesterol and sphingolipids. Currently, there is no effective therapy to treat NP-C disease. We hypothesize genetic epistasis regulates NP-C disease severity and is thus a powerful means to identify novel therapeutic strategies. Using the yeast model of NP-C disease (ncr1∆), a genome-wide conditional synthetic lethality screen was conducted using myriocin to mimic the disruption of sphingolipid metabolism in human patients. We identified 46 genes in 10 evolutionarily conserved processes are required to tolerate disrupted sphingolipid metabolism in NCR1-deficient cells. Network analysis of these 46 genes identified epistatic mechanisms that specifically respond to sphingolipid metabolism. The central hub within this interactome network was UBP6, a deubiquitinating enzyme that regulates proteasomal degradation via ubiquitin homeostasis. Experimental evidence supported this network analysis in which the ubiquitin-dependent intracellular trafficking pathways were defective only in ncr1∆ubp6∆ cells. Sphingolipid-specific growth defects in ncr1∆ubp6∆ were rescued by ubiquitin ligase genes. As NP-C disease is a lysosomal storage disease, additional pathways mediating transport to and from the vacuole (the yeast equivalent of the mammalian lysosome) are currently being characterized. Since we show that the deletion of UBP6 exacerbates lethality of the yeast model of NP-C disease, we predict that activation of the UBP6 ortholog (Usp14) will reverse NP-C disease severity in human patient cells.

135 TOR Complex 2 is down-regulated by MAPK phosphorylation. F. M. Roelants1, K. L. Leskoske1, A. Emmerstorfer- Augustin1, C. M. Augustin2, J. M. Hill1, J. Thorner1 1) Department of Molecular and Cell Biology, University of California, Berkeley, CA; 2) Department of Mechanical Engineering, University of California, Berkeley, CA. Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 senses the status of the cell envelope is not fully understood. Here we report that the TORC2 subunit Avo2 is a direct target of the cell wall integrity (CWI) MAPK Slt2. Activation of Slt2 either by overexpression of a constitutively-active allele of Pkc1 or by auxin-induced degradation of Sln1 resulted in hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its downstream effector protein kinase Ypk1. Cells expressing a phosphomimetic allele of Avo2, or in which Avo2 is absent, exhibit increased sensitivity to myriocin and to acetic acid stress, two phenotypes indicative of diminished Ypk1 function. Additionally, phosphomimetic Avo2 displayed decreased association with the primary catalytic subunit (Tor2) of TORC2. Collectively, these findings show that Avo2 is necessary for full TORC2 activity and that Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Our results demonstrate for the first time that TORC2 function is regulated by phosphorylation.

136 NASA’s BioSentinel mission: an adaptable platform for life science studies in multiple space environments. Sergio Santa Maria, Sofia Tieze, Lauren Liddell, Sharmila Bhattacharya Space Biosciences, NASA Ames Research Center, Moffett Field, CA. Ionizing radiation presents a major challenge to human exploration and long-term residence in space. The deep-space radiation environment is omnidirectional, of low flux, and includes highly energetic particles that can generate a series of DNA lesions, including DNA double strand breaks (DSBs). While progress identifying and characterizing biological radiation effects using Earth-based facilities has been significant, no source duplicates the unique space radiation environment.

NASA’s BioSentinel mission will conduct the first study of biological response to space radiation outside Low Earth Orbit (LEO) in almost 50 years. BioSentinel is a biosensor-based nanosatellite planned to launch in late 2019 as a secondary payload on NASA’s first Exploration Mission (EM-1), from which it will be deployed on a lunar fly-by trajectory and into a heliocentric orbit. Our biosensor uses S. cerevisiae to measure the DNA damage response to ambient space radiation, which will be compared to information provided by an onboard radiation dosimeter and to data obtained in LEO and on Earth. BioSentinel will carry at least two yeast strains, including: (1) a wild type strain as a control for yeast health and unrepairable damage, and (2) a DNA repair defective strain that cannot repair DNA double strand breaks and serves as a radiation sensitive control. Desiccated cells will be carried within microfluidic cards, and each card will be activated by medium addition at different time points during the mission. Cell growth and metabolic activity will be tracked continuously via optical density. One reserve set will be activated only if a solar particle event (SPE) occurs during the mission.

BioSentinel will thus address strategic knowledge gaps related to the biological effects of space radiation and will provide an adaptable platform to perform human-relevant measurements in multiple space environments, including the International Space Station (ISS), on and around other planetary bodies, and other exploration platforms.

Supported by NASA’s Advanced Exploration Systems.

137 Single Cell Analysis of Vacuolar pH and Size Using Confocal Microscopy. Roberto Carlos Segura, Mark Chan Biology, San Francisco State University, San Francisco, CA. In order for ion storage, protein degradation and protein folding to occur properly, vacuolar pH must lie within a specific range. Due to the breadth of the vacuole’s function, pH regulation has direct implications for cellular age. It could provide insight into human diseases such as lysosomal storage diseases and Alzheimer’s disease. Current methodologies—including flow cytometry and fluorimetry—are unable to quantify other measurements of interest in individual cells that could affect vacuolar pH, or they provide qualitative information on pH. Our goal is to develop a microscopy-based method to provide 53 quantitative pH measurements of individual vacuoles in single cells, using Saccharomyces cerevisiae as a model system. To observe vacuolar pH, confocal microscopy was used to collect single-cell data using the pH-sensitive vacuolar dye, BCECF. Based on the ratiometric properties of the dye, a calibration between fluorescence and pH was determined and used to determine vacuolar pH. Through the development of this method, one would be able to measure vacuolar pH to a comparable degree as flow cytometry and fluorimetry, with the added benefits of other measurements. After developing this microscopy-based method, future experiments could be done to characterize the relationship between vacuolar pH and other structural features within the cell.

138 Activation of autophagy promotes yeast prion curing. Francesca Shilati, Joanna Obaoye, Mitch Oddo, Anita Manogaran Department of Biological Sciences, Marquette University, Milwaukee, WI. Protein misfolding can lead to the formation of stable protein aggregates states called amyloids. Amyloids are associated with many human diseases, such as Alzheimer’s disease, Parkinson’s disease, and transthyretin amyloidosis. The [PSI+] prion, which is the misfolded, aggregated form of the yeast translational release factor, Sup35p, forms amyloid in yeast. Studies focused on [PSI+] can help us understand how quality control mechanisms manage amyloids within the cell. [PSI+] is propagated through a mechanism that utilizes the chaperone system. Hsp104p, along with other co-chaperones, shear [PSI+] aggregates into smaller pieces that can be transferred to daughter cells for continued propagation. Inactivation of Hsp104p, through low levels of guanidine hydrochloride (GuHCl), prevents shearing and leads to growth of the prion aggregate over time. These large aggregates are retained in the mother cell, leaving the daughter cell free of the prion. Complete loss of the [PSI+] prion in the presence of GuHCl, takes many generations, indicating that this is a slow process. While it is well established that the chaperone system is important for prions, it is unclear how other quality control mechanisms contribute to prion propagation. A previous study observed that dimethyl sulfoxide (DMSO) cures the [PSI+] prion. We found that low concentrations of DMSO had no effect on prion stability, yet increased concentrations up to 5% DMSO lead to complete curing of a [PSI+] variant. Compared to GuHCl, DMSO action on [PSI+] is rapid, curing the entire population of cells within four generations. In DMSO treated cells, Sup35p shifted from an aggregated state to a soluble state, suggesting that the aggregate may be disassembled into monomeric Sup35 protein. However, it is unclear what cellular mechanisms are activated by DMSO to cure prions. Recent studies have suggested that DMSO induces autophagy in mammalian cells. To determine whether there is a link between DMSO, autophagy, and prion curing, we screened several autophagy mutants for their ability to prevent DMSO mediated curing. We found that while DMSO treated wildtype cells showed 100% curing, bulk autophagy mutants showed approximately 8% [PSI+] curing by DMSO, indicating that DMSO effects are significantly reduced in autophagy mutants. Taken together, our data suggest that [PSI+] curing may be mediated by activation of autophagy.

139 Functional analysis of Ykt6 in autophagic process. S. Urano, N. Kikuchi, K. Gomi, T. Shintani Graduate Sch Agricultura, Tohoku Univ, Sendai, Miyagi, JP. Autophagy is a cellular process by which cellular components are sorted to the lysosome/vacuole for degradation. In response to nutrient depletion, autophagosomes are formed de novo. Despite advances in understanding functions of Atg proteins, it remains unclear what is a source of autophagosome membrane and how membrane sources are integrated to forming autophagosomes. Recently, it has been reported that soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) are involved in autophagic process. Ykt6 is one of SNAREs and known to function in multiple vesicular transport pathways including ER-to-Golgi trafficking, intra-Golgi trafficking, and trafficking to the vacuole. In this study, we analyzed autophagy in several ykt6 mutants of Saccharomyces cerevisiae. In ykt6-11 and ykt6-12 mutant strains, autophagosome formation was not completed under non-permissive temperature. On the other hand, a fusion of autophagosome to the vacuole was inhibited in ykt6-13 mutant at any temperature. Because vacuolar localization of Vam3, a SNARE protein required for the autophagosome-vacuole fusion, is defective in the ykt6-13 mutant, it was plausible that the defect might be indirect. Therefore, we attempted to isolate ykt6 mutants defective only in autophagic process. The resulting mutant ykt6-104 exhibited the defect in the fusion between autophagosome and the vacuole at 37ºC although the mutant grew normally at 37ºC and Vam3 was properly localized at the vacuolar membrane at 37ºC. This suggests that Ykt6 is directly involved in the autophagosome-vacuole fusion. Although Ykt6 also plays a role in autophagosome formation, it is still unclear whether Ykt6 is directly involved in this process.

140 Effects of the cell cycle on vacuole size in S. cerevisiae. J. Sims, M. Chan Biology Department, San Francisco State University, San Francisco, CA. Lysosomes are organelles vital for degrading subcellular proteins no longer needed by the body. Malfunctions in degradative ability may lead to lysosomal storage disorders such as Tay-Sachs Disease.1 We study vacuoles in S. cerevisiae yeast as a homologous model for lysosomes in humans. We ask whether changes in vacuole size and degradative function are dependent on the cell cycle. We hypothesize that the progression of the cell cycle influences changes in vacuolar size. Furthermore, we hypothesize that the progression of the cell cycle also governs the vacuole’s degradative ability. If we find a link between the changes in vacuolar morphology, cell cycle progression, and degradative function, our results would mean that we could potentially predict the efficiency of degradative function based on morphology. The future implication of this project could allow improved degradative function through manipulation of the cell cycle.

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141 Yeast prion [PSI+] is under surveillance by nonsense-mediated mRNA decay factors. M. Son, R. Wickner LBG, NIDDK/NIH, Bethesda, MD. The [PSI+] prion, a self-propagating amyloid of the Sup35 protein with a folded in-register parallel β-sheet architecture, produces detrimental effects on yeast. For limiting its pathogenic effects, yeast should have evolved systems to repress prion generation before its arising or to block propagation after it arises. In a genetic survey for anti-prion factors, using the yeast knockout deletion collection, UPF1/NAM7 and UPF3/NMD3, core components of the nonsense-mediated mRNA decay (NMD) apparatus, were frequently detected and shown to be anti-prion factors. In absence of these Upf proteins including Upf2p, also a crucial component for NMD, [PSI+] arises more frequently and further nearly all of the [PSI+] prion variants arising in the mutants are eliminated by restoring normal levels of these proteins. The curing ability of Upf proteins is not correlated with previously reported functions such as helicase, ATP-binding, and RNA-binding activities, but closely related with their binding with Sup35p and formation of the Upf1p-Upf2p-Upf3p complex (Upf complex). In addition, purified Upf1p at sub- stochiometric concentrations inhibits Sup35p amyloid formation in vitro. The anti-prion function of Upf proteins may be attributed to the monomeric Upf proteins and the Upf complex competing with Sup35p amyloid fibers for available Sup35p monomers or to association of the Upf complex with amyloid filaments blocking addition of new monomers. Our results indicate that maintenance of normal protein-protein interactions can prevent prion generation and propagation, and even reverse the process.

142 Sorting out the JUNQ: the spatial nature of protein quality control. E. Sontag1, J-H. Chen2, G. McDermott2, D. Gestaut1, C. Larabell2,3, J. Frydman1 1) Biology, Stanford University, Stanford, CA; 2) University of California San Francisco, San Francisco, CA ; 3) Lawrence Berkeley National Laboratory, Berkeley, CA. A healthy proteome is essential for survival; therefore, cells have evolved elaborate protein quality control (PQC) systems to maintain protein homeostasis, or proteostasis. Work from our lab and others has demonstrated that many PQC pathways rely on the spatial sequestration of misfolded proteins into specific PQC compartments. Since defects in PQC are linked to human diseases such as Alzheimer’s, Parkinson’s, cancer and even aging, there is a critical need to better understand the complex PQC machinery as well as the mechanisms of spatial sequestration, inclusion formation and clearance. Misfolded proteins are actively sequestered into a number of distinct PQC compartments that are conserved from yeast to mammals, but have been best described in the yeast system. The juxtanuclear quality-control compartment (JUNQ), which forms when the proteasome is impaired, contains soluble misfolded proteins that can be refolded or cleared. A recent study proposed that the JUNQ actually resides inside the nucleus and should be renamed to INQ. To better understand how nuclear and cytoplasmic misfolded proteins are sorted to different PQC compartments, we performed Structured Illumination Microscopy and cryo soft X-ray tomography of reporter proteins subject to PQC. Using these tools, we characterize the formation of nuclear and cytoplasmic PQC compartments and their impact on cell viability. Our results provide insights into the formation and functions of the compartments critical to understanding the etiology of various misfolded protein diseases and for developing novel and potent therapeutic strategies.

143 Translation kinetics and co-translational proteostasis in health and disease. K.C. Stein, J. Frydman Department of Biology, Stanford University, Stanford, California. Maintaining proteostasis by generating functional and properly folded proteins is crucial to every cellular process. Disruption of protein quality control can lead to protein misfolding and aggregation, which are key hallmarks of aging and many human diseases. Understanding how cells synthesize and maintain a functional proteome is critical for elucidating the mechanisms underlying aging and age-related diseases. At the heart of synthesizing a healthy proteome is the ribosome, centered at the interface of translation and protein quality control. The ribosome intricately coordinates mRNA translation with a network of ribosome-associated machinery, including molecular chaperones, to fold newly synthesized proteins or degrade aberrant translation products. This coupling of mRNA translation and nascent protein folding relies on precise regulation of translation kinetics. Increasing evidence indicates that the kinetics of translation elongation are an important determinant of protein fate: faster elongation can increase protein abundance, whereas slower elongation (ribosome stalling) can facilitate protein folding. Defects in translation kinetics and co-translational machinery can impair protein biogenesis and enhance the formation of protein aggregates. Yet, how translation kinetics and protein quality control machinery are coordinated to regulate co-translational proteostasis remains poorly defined.

We sought to determine the principles of translation elongation that dictate chaperone-mediated co-translational protein folding, and how aging might disrupt translation kinetics and the folding of nascent polypeptides. Using selective ribosome profiling with yeast, we have identified distinct mechanisms that influence the Hsp70 SSB and the essential chaperonin TRiC/CCT to associate with ribosome-nascent chain complexes. In addition, we have found that aging disrupts translation kinetics and overwhelms nascent polypeptide quality control to lead to the aggregation of newly synthesized proteins. By understanding how translation kinetics are regulated and dictate co-translational proteostasis, our work provides mechanistic insight into the principles that define both the maintenance and collapse of proteostasis, and the role that co-translational events have in the progression of aging and age-related protein misfolding disorders.

144 Genetic dissection of the signaling pathway required for the cell wall integrity checkpoint. Y. Sukegawa, T. Negishi, Y. Kikuchi, K. Ishii, M. Imanari, F. Ghanegolmohammadi, S. Nogami, Y. Ohya Department of Integrated Biosciences, 55

Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan. The cell cycle of budding yeast is tightly controlled by cell cycle checkpoint. At the cell cycle checkpoint, when an abnormality occurs in the cell cycle, it ensures cell survival by stopping the cell cycle progression until the abnormality is corrected. In budding yeast, there is a cell wall integrity checkpoint that monitors cell wall abnormalities as well as checkpoints such as DNA damage, DNA replication, actin skeleton, and spindle formation. The cell wall of budding yeast is an essential structure to survive the harsh environment. When cell wall synthesis is inhibited in Saccharomyces cerevisiae, cell cycle arrests in G2 phase before spindle formation due to cell wall integrity checkpoint. It has been confirmed that the dynactin complex (Arp1, Jnm1, and Nip100) regulates the expression of cyclin Clb2 via the forkhead transcription factor Fkh2 in this cell wall integrity checkpoint mechanism. In this study, we isolated new factors involved in this checkpoint, by genetic screening of deletion mutants. In addition to the previously identified dynactin complex, the Las17 complex, in particular the components Bzz1 and Vrp1, also plays a role in this checkpoint. In addition, the cell cycle arrest is achieved by transcriptional downregulation of the mitotic cyclin Clb2 by phosphorylation and degradation of the S-phase transcription factor Hcm1 and transcriptional regulation of the transcription factor Fkh2. The HOG MAPK pathway and the CWI MAPK pathway were also found to be essential for the checkpoint function. By further analysis, the hierarchical relationship of these factors was clarified. From the above results, the cell wall synthesis checkpoint mechanism functions in the order of the HOG MAPK pathway, the dynactin complex / Bzz1 / Vrp1, and the CWI MAPK pathway. CWI MAPK phosphorylates and induces degradation of Hcm1. As a result, transcriptional regulation of Fkh2 is induced, and expression of Clb2 is suppressed.

145 Genetic interaction mapping in quiescent yeast cells identifies novel functional relationships with core signaling kinases. S. Sun1,2, N. Brandt1,2, Y. Qin3, D. Miller1,2, M. Costanzo44, C. Boone4, D. Gresham1,2 1) Center for Genomics and System Biology, New York University, New York, USA; 2) Department of Biology, New York University, New York, USA; 3) Department of Biology, New York University, Shanghai, China; 4) Donnelly Center for Cellular and Biomolecular Research, University of Toronto, Canada. Most eukaryotic cells spend the majority of their lifetime in a quiescent state defined as the temporary and reversible exit from proliferation. In humans, dysregulation of quiescence is associated with age-related diseases and cancers. Conversely, quiescent tumor cells, or eukaryotic microbial pathogens, are frequently refractory to drug treatments that target active processes in cell proliferation. In unicellular organisms, cell exit the mitotic cycle and enter quiescence (or G0) in response to diverse essential nutritional starvations, e.g glucose, nitrogen, phosphorous. Cell growth and quiescence in yeast is controlled by evolutionarily conserved signaling pathways including the target of rapamycin complex 1 (TORC1), AMPK and Ras/Protein kinase A (PKA) pathways. How these pathways sense diverse signals to coordinately mediate initiation and maintenance of quiescence is poorly understood. To understand how signaling pathways integrate environmental information to initiate quiescence we performed genome-wide genetic interaction mapping in quiescent conditions. We constructed haploid prototrophic double deletion libraries comprising ~4,900 non-essential gene deletion strains to identify interactions with the core kinases TOR1, RIM15 and PHO85 using synthetic genetic array (SGA). We identified quiescent specific genetic interaction in different pro-quiescence conditions (carbon, nitrogen, and phosphorous starvation) using pooled assays and barseq. We identified multiple conditional genetic interactions that vary depending on the pro-quiescent signals. We found that genes involved in ER Associated Degradation (ERAD) show a coherent positive interaction profile with RIM15 under nitrogen starvation, suggesting that RIM15 regulation of ERAD activity in response to nitrogen starvation is essential for quiescence. Our results point to a rich spectrum of condition-specific genetic interactions that underlie cellular viability across a diversity of conditions.

146 Phenomic screen reveals that the lysine acetyltransferase NuA4 has an unexpected role in regulating glycogen synthesis and mitochondrial morphology in Saccharomyces cerevisiae. Elizabeth Walden1,2, Roger Fong1,2, Trang Pham1,2, Sylvain Huard1,2, Hana Knill1,2, Mary-Ellen Harper1,2, Kristin Baetz1,2 1) Biochemistry Molecular Biology and Immunology, University of Ottawa, Ottawa, Ontario, CA; 2) Ottawa Institute of Systems Biology, University of Ottawa, Ottawa, Ontario, CA. We have recently determined that the Saccharomyces cerevisiae lysine acetyltransferase complex NuA4 is required for the activity and localization of Acetyl-CoA carboxylase. This motivated us to ask whether NuA4 was impacting the cellular localization of other metabolic proteins. By combining synthetic genetic analysis with high-throughput microscopy we assessed the impact of the deletion of the NuA4 structural subunit EAF1 on the cellular localization of 381 GFP-tagged metabolic proteins. We determined that the deletion of EAF1 impacted the localization or signal of 23 GFP-tagged metabolic proteins, including enzymes involved in glycogen metabolism and mitochondrial function. In the absence of EAF1 there is an increase in the protein levels of glycogen synthase enzymes Gsy1 and Gsy2 along with glycogen debranching enzyme Gdb1, which results in increased glycogen reserves. We determined that the increase in glycogen biosynthesis proteins is partially dependent on NuA4-dependent regulation of the stress response transcription factors Msn2 and Msn4. In addition, over half of the NuA4-dependent changes in protein localization were associated with mitochondrial proteins. Instead of linear mitochondrial structures largely found around the cell periphery, cells containing eaf1Δ or mutants of the catalytic domain of NuA4, esa1-ts, have hyper-elongated mitochondria that are chaotically distributed. Compared to wild type cells, in eaf1Δ cells, the mitochondrial volume is increased approximately 3 fold and this is associated with an increase in mitochondrial function as measured by extracellular flux analysis and a decrease in sensitivity to a mitochondrial inhibitor. Despite aberrant structure, the mitochondria of eaf1Δ cells are still able respond to environmental stress, suggesting the hyper-elongation 56 mitochondrial phenotype is not due a defect in mitochondrial fission or fusion. Though we are still presently dissecting the molecular mechanisms by which NuA4 regulates the mitochondria dynamics, we do know this is not through the regulation of known targets of NuA4 implicated in metabolism such as SNF1/AMPK1. Overall our study has uncovered an important role for NuA4 in remodeling cellular metabolism.

147 Make Copper Great Again – Exploring the Unique Mode of Toxicity by Hybrid Carboxymethyl Cellulose Copper Nanoparticles. M.J. Winans1, N. Nurmalasari2, S. Smith2, J.E. Gallagher1 1) Biology, West Virginia University, Morgantown, WV; 2) Physics, South Dakota School of Mines, Rapid City, South Dakota. Copper has been used since antiquity as a broad spectrum antimicrobial agent to prevent water-born diseases, protect agricultural food securities, and treat medical ailments. Novel carboxymethyl cellulose (CMC) nanoparticles engineered with copper (c-CuNPs) enhance the toxicity of copper to S. cerevisiae via a unique mode of action when compared to soluble, copper sulfate. Reactive oxygen species (ROS), such as the hydroxyl radicals are generated through Fenton reactions with transition metals in aqueous solutions. C-CuNPs are a promising emerging nanotechnology composed from timber industry byproduct. Imbedded into poly vinyl alcohol plastics, these nanoparticles used in medical devices and food packaging may ultimately be beneficent to society.

In this study we found the antioxidants N-acetylcysteine (NAC), a glutathione (GSH) precursor, could not rescue growth inhibition due to c-CuNP toxicity, but GSH rescued both copper forms by serial dilutions and spotting. Utilizing flow cytometry and confocal imaging, copper nanoparticle were found dispersed close to the cell wall. Loss of Alpha arrestins Aly1 and Aly2, which regulate endocytosis at the plasma membrane by recruitment of ubiquitin ligase Rsp5, rescued yeast from c-CuNPs, but not CuSO4. Ongoing experiments are focused on deciphering the role of the vacuole in c-CuNP toxicity, ROS production, and how metabolism is affected. We hypothesize that endocytosis transports c-CuNPS to the acidic vacuole where c-CuNPs impart cellular damage. This value-added product is poised for studies on biocompatibility and nosocomial infection prevention studies, further validating its application today. The exact cellular mechanism, of carboxymethyl cellulose is unknown and further research is warranted in their role in ROS production, biocompatibility, and ability to provide microbial safety in industry.

148 Forming Protein Complex Pages at the Saccharomyces Genome Database. E. D. Wong1, G. A. Binkley1, S. R. Engel1, K. Karra1, B. H. Meldal2, S. Orchard2, M. S. Skrzypek1, S. Weng1, T. K. Sheppard1, J. M. Cherry1, The SGD Project 1) Department of Genetics, Stanford University, Palo Alto, CA; 2) European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire. Proteins do not always function in isolation, but often interact with other proteins or nucleic acids to form stable macromolecular complexes that play roles in important cellular processes and pathways. One of the goals of Saccharomyces Genome Database (SGD; www.yeastgenome.org) is to provide a complete picture of budding yeast biological processes. To this end, we collaborated with the Molecular Interactions team that produces the IntAct database at EMBL-EBI to manually curate the complete yeast complexome. These data, from a total of 585 complexes, were initially available in YeastMine (https://yeastmine.yeastgenome.org/yeastmine/begin.do), our data warehouse, as well as the Complex Portal (https://www.ebi.ac.uk/complexportal/home).

We have now incorporated macromolecular complex data into our main SGD database and designed pages to make these data easily available to researchers. The pages contain referenced summaries focused on the composition and function of individual complexes. In addition, detailed information about how subunits are associated in the complex; their stoichiometry and the physical structure are displayed when available. Finally, we have network diagrams displaying shared members and GO annotations between complexes. Macromolecular complexes will continue to be updated and curated as more data become available.

149 Cooperative membrane binding with Rng10 targets the F-BAR protein Rga7 to the fission yeast division site. Y. Liu1, N.A. McDonald2, S. Naegele1, K.L. Gould2, J.Q. Wu1 1) The Ohio State University, Columbus, OH; 2) Vanderbilt University, Nashville, TN. F-BAR family proteins bind to phospholipids of the plasma membrane and play important roles in various cellular processes such as endocytosis, cell motility, and cytokinesis. However, the mechanisms and the regulations of their cellular localization are not well understood. Previously we identified a novel coiled-coil protein, Rng10, in Schizosaccharomyces pombe that is required for the proper cellular localization of the F-BAR protein Rga7 to the division site and the cell tips. Here we investigate the mechanism of Rga7 and Rng10 interaction and how this affects Rga7 targeting on the plasma membrane. We find that both the Rga7 F-BAR domain and the Rng10 C-terminus (amino acids 951-1038) can bind membranes independently in vitro.

Rga7 prefers PI(4,5)P2 phospholipids, whereas Rng10(951-1038) binds various phospholipids with no obvious selectivity. In accord with Rga7’s dependence on Rng10 for localization, we find that the Rga7 F-BAR domain directly interacts with

Rng10(751-950) with a Kd of 0.3 µM. Rng10(751-950) localizes to the division site mainly at the contractile ring instead of throughout the division plane due to the loss of plasma-membrane binding and this localization depends on Rga7. Similarly, Rga7 localization is also restricted to the ring in cells expressing only Rng10(751-950). In membrane co-binding experiments, Rng10 and Rga7 bind to each other and membranes non-exclusively. Moreover, Rng10 dramatically increases Rga7’s affinity 57 for membranes at physiological concentrations. Collectively, our study shows that membrane binding capacity is not sufficient to localize the F-BAR protein Rga7, which instead requires an additional interaction with Rng10 for stable association with the plasma membrane.

150 Remembering the Stressful Past: Elucidating Mutation-Independent Mechanisms of Drug Resistance. J. Xie, D. Garcia, C. Gill, D. Jarosz Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA. By poisoning rapidly dividing cancer cells, chemotherapy has been an effective means of extending the lifespans of many cancer patients. However, the prevalence of drug resistance in tumors – and collateral damage to healthy tissues – have been major road blocks to improving the efficacy of this treatment modality. Thus, understanding the mechanisms of drug resistance could shift the balance in this life and death struggle for survival in favor of healthy cells. Although much research has focused on identifying mutations that confer cancer drug resistance, epigenetic heterogeneity could be a hidden force promoting drug resistance. Previous studies have shown that both chromatin- and protein-based epigenetic states can be induced in response to stress. These heritable ‘molecular memories’ can confer a fitness advantage during future exposures to stress. We tested >300 isogenic yeast isolates against a panel of 54 stresses, including multiple chemotherapeutics. Many of these conditions induced adaptive epigenetic states at a very high frequency, orders of magnitude more frequent than adaptive mutations. In addition, they exhibited a non-Mendelian pattern of inheritance characteristic of yeast prions. One such epigenetic state was robustly induced by transient exposure to carmustine, an alkylating agent commonly used to treat recurrent glioblastoma. To identify the genes involved in the adaptive response to carmustine, we screened the non-essential yeast knockout collection and the DAmP collection of essential genes. We found that the molecular memory of carmustine is mediated by a complex network of genes involved in DNA replication, chromatin modification and metabolism. Further characterization of this epigenetic-based mechanism of drug resistance will have important implications for the design and delivery of chemotherapeutics and our fundamental understanding of adaptation.

151 Evolutionarily conserved pathways prevent mislocalization of CENP-A for chromosome stability in yeast and human cells. Munira Basrai1, Lars Boeckmann1, Sultan Ciftci-Yilmaz1, Jessica Eisenstat1, Prashant Mishra1, Kentaro Ohkuni1, Austin Rossi1, Roshan Shrestha1, Tianyi Zhang1, Mahfuzur Rahman6, Michael Costanzo2, Anastasia Baryshnikova7, Chad Myers6, Peter Kaiser4, Dan Foltz5, Richard Baker3, Charlie Boone2 1) Dept Genetics, NCI/NIH, Bethesda, MD; 2) University of Toronto; 3) Univ. of Massachusetts Med. School ; 4) Univ. of California, Irvine ; 5) Northwestern Univ. Feinberg School of Medicine; 6) Univ. of Minnesota ; 7) Princeton Univ. Faithful chromosome segregation prevents chromosomal instability (CIN), a hallmark of aggressive tumors and other diseases. The evolutionarily conserved centromeric histone H3 variant CENP-A (Cse4 in budding yeast, Cid in flies) is essential for chromosome segregation. Overexpression of Cse4 or Cid causes its ectopic mislocalization to chromosome arms and promotes CIN in yeast and flies respectively. Overexpression and mislocalization of CENP-A have been reported in numerous cancers and is correlated with poor prognosis. To define pathways that prevent mislocalization of Cse4 we performed genome-wide synthetic dosage lethality (SDL) screens to identify mutants that exhibit growth sensitivity when Cse4 is overexpressed. Among the top hits are genes that encode the evolutionarily conserved ubiquitin ligase (SCF), replication dependent kinases (DDK) and the replication-independent histone chaperone (HIR) complexes. We determined that SCF- Met30, SCF-Cdc4, DDK and HIR proteins regulate proteolysis of Cse4 to prevent its mislocalization for chromosome stability. For studies with human cells we used a HeLa cell line stably overexpressing CENP-A and provide the first evidence to show that CIN results from mislocalization of overexpressed CENP-A in human cells. Depletion of the human homologs of HIR and SCF-Met30 show increased mislocalization of CENP-A and a CIN phenotype similar to our findings in yeast cells. In summary, we have identified evolutionarily conserved pathways that regulate proteolysis and localization of Cse4/CENP-A for genome stability in yeast and human cells. The results from the screen will offer insights into how CENP-A mislocalization contributes to aneuploidy in cancers and identify potential therapeutic targets for CENP-A overexpressing cancers.

152 A diverse and dynamic SUMO-modified proteome during meiosis. Nikhil Bhagwat1,2, Shannon Owens1, Jay Boinapalli1, Alex Ditzel1, Jeffrey Johnson3, Nevan Krogan3, Neil Hunter1,2 1) Microbiology and Molecular Genetics, University of California, Davis, CA; 2) Howard Hughes Medical Institute, Chevy Chase, MD 20815; 3) Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA. Post-translational modification of proteins by conjugation of Small Ubiquitin-like MOdifier (SUMO) to the ε-amino group of target lysine serves to alter the stability, interactions, subcellular localization and molecular function of the substrates. In meiosis, SUMO plays key roles in promoting homologous recombination required for appropriate segregation of chromosomes and crossover formation, and the assembly of synaptonemal complexes (SCs), the proteinaceous zipper-like structures that connect homologous chromosomes. Proteome-wide identification of SUMOylation target sites is precluded by the low abundance, labile nature and poor mass signature produced on mass spectrometry (MS) by the modified peptides. These technical challenges have hindered research on SUMO targets. We have developed a pipeline for mapping SUMOylation sites during budding yeast meiosis. By employing a modified SUMO we are able to enrich and detect peptides with SUMO remnants using MS. Additionally, using strains that sharpen the meiotic synchrony and purification of SUMOylated proteins under denaturing conditions we were able to generate an unprecedented picture of SUMOylation dynamics throughout meiosis. We identified over 1900 modified lysines in over 600 targets. Using label-free quantification (LFQ) allows us to plot the SUMOylation levels of important proteins at key milestones, which provides insight into the 58 physiological roles of their SUMOylation. Finally, mutating SUMOylation sites on a key meiotic double-stranded break formation protein Mer2 reveals an unexpected role of SUMO in the initiation of homologous recombination.

153 Investigations on possible roles for DNA sequences and specific chromatin environments in promoting dissociation of the FACT complex from the 3’ ends of genes following transcription. S. Byrd, B. Hoyt, D. Habenicht, H. Prowse, S. Ozersky, G. Harris, J. Harper, A. Crocker, C. Turkal, A. Duina Hendrix College, Conway, AR. To promote transcription elongation, the Facilitates Chromatin Transactions (FACT) complex, composed of the Spt16 and Pob3 proteins in yeast, works in parallel with RNA polymerase II by facilitating the disassembling and assembling of nucleosomes while moving toward the 3' end of genes. In previous studies from our laboratory using the Saccharomyces cerevisiae model system, we identified a region on the side of the nucleosome, which we refer to as the ISGI (Influences Spt16- Gene Interactions) region, that is required for proper dissociation of FACT from the 3' ends of transcribed genes. To further investigate the mechanisms that control FACT dissociation from genes, we are carrying out studies to determine possible roles for DNA sequences and specific chromatin environments in this process. To determine if DNA sequences play a role in FACT dissociation from genes, we have generated fourteen internal deletions within the 3’ end of the constitutively expressed gene PMA1 and are currently carrying out chromatin immunoprecipitation (ChIP) assays to determine if any of these deletions impair Spt16 dissociation from the 3’ end of PMA1. As a way to assess if specific chromatin environments are required for proper Spt16 dissociation from DNA, we are carrying out two mass spec-based experiments to define the chromatin environments (i.e., collection of all proteins and histone modifications) at 3’ ends of genes in wild-type and ISGI-mutant cells – difference in chromatin environments between these cells may point to key proteins or histone modifications that regulate FACT dissociation from genes. Collectively, these studies will provide a more complete understanding of the factors that promote Spt16 dissociation from genes following transcription.

154 Interactions between histone variant H2A.Z and linker histone H1 in Saccharomyces cerevisiae meiosis. L. Chigweshe, A. J. MacQueen, S. G. Holmes Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT. Meiosis in Saccharomyces cerevisiae is associated with unique chromosome choreography and chromatin reorganization. In vegetative cells, the histone variant H2A.Z, encoded in budding yeast by the HTZ1 gene, has a significant role in gene expression, DNA double strand break repair and maintenance of euchromatin-heterochromatin boundaries, however, its function in meiosis and sporulation remains undefined. We show that htz1Δ homozygous diploid strains have wild type sporulation efficiency but exhibit reduced spore viability. Interestingly, our preliminary results indicate that these strains exhibit interval-specific increases in recombination frequency. We have also examined linker histone H1’s role in meiosis. In agreement with published data, we found that cells lacking H1 have wild type levels of sporulation efficiency and spore viability, and exhibit no changes in recombination map distance. However, loss of H1 can suppress some of the decrease in spore viability seen in htz1Δ strains. Surprisingly, strains lacking both H2A.Z and H1 exhibit a marked increase in the frequency of meiotic gene conversion events. We are currently assessing whether H2A.Z affects synaptonemal complex assembly by analyzing meiotic spread chromosomes for presence and proper localization of synaptonemal complex specific proteins Zip1, Ecm11/Gmc2 and Red1. Overall our results demonstrate that H2A.Z and H1 interact to ensure a proper chromatin context for the execution of meiosis in budding yeast.

155 Activation of Rev3-dependent error-prone mismatch repair in Exo1-deficient Saccharomyces cerevisiae strain. B. Dahal, F. Kadyrov, L. Kadyrova, V. Gujar Biochemistry and Molecular Biology, Southern Illinois University Carbondale, Carbondale, IL. Eukaryotic DNA mismatch repair (MMR) is an important intracellular mechanism that suppresses spontaneous mutations caused by mispaired bases resulting from DNA damage or replication errors. These mutations if not repaired are the basis of numerous diseases including cancer. Thus, inactivation of genes involved in MMR increases the risk of cancer initiation and development. One of the proteins actively involved in MMR is Exonuclease-1 (Exo1), a 5′ →3′ exo n u clease w h ich p articip ates in excision of mismatch in daughter strand after mismatch recognition. Studies have showed that MMR can function properly in absence of Exo1 via Polymerase-δ dependent strand displacement (Exo1-independent MMR). However, recent studies have showed that Exo1-independent MMR is error-prone in yeast and inactivation of Exo1 predisposes mice to cancer development. As knowledge about Exo1-independent MMR mechanism is limited, we tried to study Exo1-independent MMR in heterochromatin and euchromatin using Saccharomyces cerevisiae as a key model organism. In this study, we employed URA3 genetic reporter in two different heterochromatic loci (HMR and subtelomeric region) and a euchromatic locus to investigate if Exo1-independent MMR is error-prone in overall chromatin environment. Prior studies have showed that mutation rate is higher in exo1∆ strain compared to that of wild-type strain in both heterochromatic and euchromatic loci. Our study revealed that deletion of REV3 (catalytic subunit of Pol-ζ polymerase) in exo1∆ strain resulted in decrease of mutation rate (similar to wild-type rate) suggesting that exo1∆ mutations are Rev3-dependent. Detailed mutation rates and spectrum as well as statistical and comparative analyses suggested that absence of Exo1 during MMR leads to activation of Rev3-dependent translesion bypass during which mutational intermediates are formed leading to error-prone MMR in both chromatin environments.

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156 Assembly, Characterization and Application of the Yeast Synthetic Chromosome XII. Junbiao Dai1,2, Zhouqing Luo1,2, Lihui Wang2, Yicong Lin2, Weimin Zhang2, Guanghou Zhao2, Yizhi Cai1,3 1) Center for Synthetic Genomics, Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Shenzhen, CN; 2) Center for Synthetic and Systems Biology, School of Life Sciences, Tsinghua University; 3) Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, M1 7DN, Manchester, UK. New technologies to synthesize DNA provides a great opportunity to completely redesign the entire genome of an organism. Together with several other groups worldwide, we teamed up to re-synthesize a designer eukaryotic genome, Sc2.0. In my lab, a 976,067-base pair linear chromosome, synXII, was designed and assembled using a two-step method, producing a functional chromosome. The ribosomal gene cluster (rDNA) on synXII was retained during the assembly process and subsequently replaced by a modified rDNA unit used to regenerate rDNA at three distinct chromosomal locations. The signature sequences within rDNA, commonly used as the molecule barcode of a species, were swapped to generate a Saccharomyces strain that would be identified as Saccharomyces bayanus. Furthermore, we designed a reporter of SCRaMbLEd cells using efficient selection (ReSCuES) to isolate SCRaMbLEd strains based on the loxP-mediated switch of two auxotrophic markers, which not only allow us to rapidly identify strains of interest from a SCRaMbLEd synthetic yeast population, but also provides methods to dissect the underlying mechanisms of resistance.

157 Evaluation of the contributions of RNA Pol II and other proteins in promoting the dissociation of the FACT complex from the 3' ends of genes at the end of the transcription process. M. Edwards, J. Campbell, S. Ozersky, A. Duina Hendrix College, Conway, AR. In eukaryotic cells, histone chaperones can manipulate chromatin environments to facilitate a variety of processes that occur on DNA, including gene transcription. FACT (FAcilitates Chromatin Transactions) is a highly conserved histone chaperone complex that plays important roles during transcription initiation and elongation by interacting directly with nucleosomes. Previous research from our laboratory has identified a region on the nucleosome – which we refer to as the ISGI (Influences Spt16-Gene Interactions) region – that is required for proper dissociation of FACT from the 3’ ends of genes at the end of the transcription process. We have recently initiated a series of studies to further investigate the mechanisms that regulate FACT dissociation from genes. Here, we describe experiments to determine if specific proteins promote FACT dissociation from the chromatin template following transcription. In one set of studies, we are assessing if RNA Pol II has a role in this process by testing whether factors that facilitate Pol II termination also play a role in promoting dissociation of Spt16 from genes. To date, our results suggest that Pol II dissociation promotes Spt16 dissociation. In other studies, an SGA (Synthetic Gene Array)-based screen using and ISGI mutant as the query strain has identified proteins that may contribute to proper Spt16-gene dissociation. One of the proteins identified in the screen is Rtt103, a factor involved in Pol II dissociation, and we are currently performing ChIP assays to determine if Rtt103 is required for Spt16 dissociation from genes. We expect that continuation of these studies will lead to new insights into roles of specific proteins in promoting dissociation of Spt16 from genes following gene transcription.

158 Mutational and functional analysis of the Saccharomyces cerevisiae Pif1 DNA helicase. C. Geronimo1, S. Singh2, R. Galletto2, V. Zakian1 1) Department of Molecular Biology, Princeton University, Princeton, NJ; 2) Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, Saint Louis, MO. Pif1 family DNA helicases are conserved from bacteria to humans and perform diverse functions that promote genome integrity. Saccharomyces cerevisiae Pif1 (ScPif1) is the founding and best-studied member of this family. ScPif1 is multi- functional by both genetic and biochemical assays and plays multiple, diverse roles in maintaining the nuclear and mitochondrial genomes. Given the diversity of its biological functions, we conducted a mutational analysis of ScPif1 to determine the importance of specific residues, motifs, and domains for effects on distinct in vivo functions. Pif1 can be separated into three regions structurally: N-terminus, helicase domain, and C-terminus. The Pif1 helicase core domain is highly conserved, while the N- and C-termini vary in both length and sequence among different family members. In addition to the seven canonical helicase motifs, the helicase domain contains a 23 amino acid region, called the Pif1 signature motif (SM), that is unique to this family of helicases, but whose in vivo function(s) had not been determined. Therefore, we conducted a study to determine the functional importance of the SM in Pif1 family helicases, using budding yeast ScPif1 as the model. Using genetic assays, deletions and substitutions within the SM were defective in maintaining mitochondrial DNA, inhibition of telomerase, and maturation of Okazaki fragments. Key SM mutants were also tested for ATPase activity and substrate binding. Overall, our study demonstrated that the SM is essential for all tested in vivo functions and is critical in vitro for ATPase activity, but not substrate binding. In addition to our study of the SM, we are extending our analysis to other regulatory regions and domains whose function in vivo are unknown. Analysis of the amino terminus identified a small region that is essential for one of ScPif1’s nuclear functions, and loss of the carboxyl terminus disrupts nuclear functions without compromising mitochondrial function. We are also testing mutations that others have shown impair specific in vitro activities, such as G4 binding or DNA polymerase delta stimulation, with the goal of determining which of these activities are needed for individual in vivo functions.

159 Cohesin binding to DNA requires residues in the interface between Smc3p and Mcd1p. V.A. Guacci, F.A. Chatterjee, B. Robison, D. Koshland Molecular & Cell Biology, UC-Berkeley, Berkeley, CA. Sister chromatids are held together by an evolutionarily conserved “cohesin complex” composed of 4 subunits, Mcd1 (Scc1), 60

Smc3, Smc1 and Scc3. Budding yeast cohesin binds DNA in late G1 then tethers sisters chromatids from their formation in S phase through metaphase. Prior to replication, cohesin’s binding to DNA is unstable. During S phase, Eco1p (Ctf7p) mediated acetylation of Smc3p stabilizes cohesin-DNA binding and enables sister chromatid cohesion. The mechanism of how cohesin binds DNA and mediates cohesion remains controversial. Mcd1p, Smc3p and Smc1p form a trimeric ring and evidence indicates that DNA is entrapped within this ring. The interface between Mcd1p (N-terminal helices) and Smc3p (N and C- terminal helices) are thought to be crucial for DNA binding as mutants in these helices are inviable. One model posits that this interface opens to allow DNA entry, whereas another model posits the interface is a gate that opens to allow DNA exit unless closed by Smc3p acetylation. We examined the role of the Smc3p and Mcd1p interface by charactering interface smc3 or mcd1 mutants in either an Smc3p or Mcd1p degron background, respectively. As expected, interface mutants are inviable in the degron background. Surprisingly, Western Blot analysis reveals that Mcd1p is absent. We previously showed that Mcd1p is degraded unless both Smc3p and Smc1p are present. Thus, the interface mutants impair cohesin trimer formation and or stability, making it impossible to ascertain whether the interface is regulated to control DNA entry or exit. To overcome the degradation of Mcd1p in interface mutants, we utilized an Smc3-Mcd1 fusion. A wild-type Smc3-Mcd1p fusion was previously shown to support viability. We show that the wild-type fusion protein binds DNA, and binding requires both Scc3p cohesin subunit and cohesin loader, indicating it functions similar to normal cohesin. We find that the mutant Smc3-Mcd1 fusion protein is also present in cells. However, the mutant fusion cannot support viability. Moreover, the mutant fusion protein fails to bind DNA yet still binds Scc3p and the cohesin loader. Our results demonstrate that opening the interface between Smc3 and Mcd1p is not required for cohesin DNA loading as the fusion binds DNA. The fact that fusion mutants fail to bind DNA indicates the interface has a function distinct from any proposed role in keeping the interface closed. We propose that the interface between Smc3p and Mcd1p regulates cohesin conformation to promote DNA binding, possibly by regulating other cohesin sub-unit interfaces.

160 Meiotic Crossing Over Requires Sumoylation of the MutS Homolog Msh4 . Wei He, Nikhil Bhagwat, Neil Hunter UC Davis. Crossing over is essential for the faithful disjunction of homologous chromosomes during the first meiotic division. Regulatory processes that control crossing over remain poorly characterized. Our study shows that the pro-crossover function of the conserved meiosis-specific recombination factor, MutS gamma (a complex of Msh4 and Msh5), requires its sumoylation, which is essential for the crossover activity of MutS gamma.

161 Mechanisms of myotonic dystrophy type 2-causing CCTG DNA repeat instability. JE Narvanto, D Papp, JC Kim Biological Sciences, California State University San Marcos, San Marcos, CA. Myotonic dystrophy type 2 (DM2) is a genetic disorder characterized by progressive muscle wasting and weakness that is caused by expanded CCTG tetranucleotide DNA repeats in an intron of the ZNF9 gene. Individuals can have upwards of 11,000 CCTG repeats at the disease-causing locus. Unlike CTG trinucleotide repeats, which are responsible for DM1 and have been extensively studied, much less is known about the molecular mechanisms of CCTG repeat expansions and contractions. However, like CTG repeats, CCTG repeats have been shown to form secondary structures in vitro, which may contribute to their instability. We established a budding yeast experimental system to analyze CCTG repeats in vivo. (CCTG)100 was cloned into the intron from the ACT1 gene, and inserted into the URA3 reporter gene. The entire DNA cassette was integrated into a chromosomal location (Chr III) near ARS306, an efficient and early-firing replication origin. Under non-selective conditions, we observed 1-5% frequency of expansions and contractions. The expansions were small-scale (

162 CDC7 modulates silencing at HMRae** via H4 K16 acetylation and CAF-1 in Saccharomyces cerevisiae. Tiffany Young1, Yi Cui2, Joseph Irudayaraj3, Ann Kirchmaier1 1) Dept. Biochemistry, Purdue University, West Lafayette, IN; 2) Dept. Ag. & Biological Engineering, Purdue University, West Lafayette, IN; 3) Dept. Bioengineering Univ. IL Urbana-Champaign, Urbana, IL. CAF-1 is an evolutionarily conserved H3/H4 histone chaperone that plays a key role in replication-coupled chromatin assembly and is targeted to the replication fork via interaction with PCNA, which, if disrupted, leads to epigenetic defects. In Sacchromyces cerevisiae, when the silent mating-type locus HMR contains point mutations within the E silencer at Rap1p and Abf1p binding sites, Sir protein association and silencing is lost. Mutation of CDC7, encoding a S phase-specific kinase, or of subunits of the H4 K16-specific acetyltransferase complex SAS-I, however, restore silencing to this crippled HMR, HMRae**. Here, we report that loss Cac1p, the largest subunit of CAF-1, also restores Sir-dependent silencing at HMRae**, and silencing in both cac1Δ and cdc7 mutants is suppressed by overexpression of SAS2. We demonstrate Cdc7p and Cac1p interact in vivo in S phase, but not G1, consistent with observed cell cycle-dependent phosphorylation of Cac1p. Moreover, chromatin becomes hypoacetylated at H4 K16 in both cdc7 and cac1Δ mutants, and silencing at HMRae** is restored in cells expressing cac1p mutants lacking Cdc7p phosphorylation sites. Combined, these and additional results support a model in which Cdc7p regulates replication–coupled histone modification via a CAC1-dependent mechanism involving H4 K16ac deposition, and thereby silencing, but other CAF-1-dependent chromatin assembly activities are retained in the absence of phosphorylation at Cdc7p consensus sites.

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163 The effects of Tbf1 and Reb1 on Telomere function. E Pasquier, A Krallis, R Wellinger Université de Sherbrooke, Sherbrooke, Quebec, CA. Subtelomeric regions contain protein binding sites that may vary between chromosomal ends. Since proteins bound to the subtelomeres as well as those associated to the telomeric repeats can govern the behaviour of telomeres, they account for some of the variability in the properties between all telomeres. Tbf1 and Reb1 are two essential yeast proteins that function as transcription factors on promoters of a multitude of genes. On gene promoters, both have roles in nucleosome exclusion, and Reb1 plays an important role in transcription termination for DNA polymerase I. They are also the most prevalent and ubiquitous proteins found on subtelomeres, although little is known about their function in these regions. The aim of this project is to better understand the contributions and effects of Tbf1 and Reb1 to telomere behavior. Previous results suggested that some properties of Tbf1 and Reb1 include effects on telomere length maintenance and anti- silencing (Arnéric and Ligner, EMBO reports, 2007; Berthiau et al., EMBO J, 2006; Fourel et al. EMBO J. 1999). These data were obtained using artificially constructed chromosome ends. In our study, we will assess the contributions of Tbf1 and Reb1 by mutating their binding sites in wild type subtelomeric areas. This will allow us to evaluate changes in telomere length maintenance and the propagation of silencing that nucleates in telomeric repeats in the absence of these proteins. Furthermore, the relevance of Tbf1 and Reb1 as transcription factors at the subtelomeres will be studied by measuring changes in TERRA expression in their absence. By determining the relevance of Tbf1 and reb1 to telomere function, this study aims to uncover the roles of subtelomeres at chromosomal ends, providing another tool to understand mechanisms essential for genome stability. I will be presenting our working model as well as preliminary results obtained.

164 The many roles of Elg1 in the maintenance of genome stability and chromatin states. Soumitra Sau, Keren Shemesh, Batia Liefshitz, Shay Bramson, Martin Kupiec School of Molecular Cell Biology and Biotechnology, Tel Aviv Univ, Tel Aviv, Israel. During DNA replication and repair, DNA polymerases are tethered to the processivity factor PCNA, a homotrimeric ring that encircles DNA. Timely unloading of PCNA from the replication fork (each Okazaki fragment carries one such ring) is essential for genome integrity. The yeast Elg1 interacts with the Rfc2-5 subunits of the Replication Factor C (RFC) complex to form an RFC-like complex (RLC). The Elg1-RLC is the principal unloader of chromatin-bound PCNA, and thus plays a central role in DNA replication, DNA recombination and genomic stability maintenance. Mutations in Elg1 lead to hyper-recombination, chromosome loss, longer telomeres, hyper-transposition, problems in sister chromatid cohesion and chromatin silencing, and sensitivity to DNA damage agents. Thus, Elg1 affects a number of central processes in the cell. In addition, Elg1 is a target of the DNA damage Response (DDR): when cells are subjected to DNA damage, it becomes phosphorylated at its Serine 112 in a Mec1 (ATR)-dependent fashion. Using a chromatin-fractionation assay, we show that phosphor-dead elg1 mutants are defective in PCNA unloading upon DNA damage, but are proficient for PCNA unloading during normal S-phase replication. Our results show however that, paradoxically, Elg1 plays a role in eliciting the DDR, in addition of being its target. Another important phenotype of Δelg1 mutants is a higher rate of de-silencing of the HML and HMR mating type cassettes. We will discuss possible mechanisms by which Elg1 function links chromatin maintenance and epigenetic memory to DNA replication and DNA repair.

165 The Mec1(ATR) checkpoint facilitates the elongation of short telomeres by upregulating RNR activity. Andre maicher1, Inbal Gazy1, Andrei Chabes2, Martin Kupiec1 1) School of Molecular Cell Biology and Biotechnology, Tel Aviv Univ, Tel Aviv, IL; 2) 2Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87, Umeå, Sweden. Ribonucleotide reductase (RNR) provides the precursors for the generation of dNTPs, which are required for DNA synthesis and repair. Here, we investigated the function of the major RNR subunits Rnr1 and Rnr3 in telomere elongation in budding yeast. We show that Rnr1 is essential for the sustained elongation of short telomeres by telomerase. In the absence of Rnr1, cells harbor very short, but functional, telomeres, which cannot become elongated by increased telomerase activity or by tethering of telomerase to telomeres. Rnr3 activity cannot replace Rnr1 in this telomeric function. Our results suggest that telomerase has a "basal activity" mode that is sufficient to compensate for the "end-replication-problem" and does not require the presence of Rnr1 and a different "sustained activity" mode necessary for the elongation of short telomeres, which requires an upregulation of dNTP levels and dGTP ratios specifically through Rnr1 function. By analyzing telomere length and dNTP levels in different mutants showing changes in RNR complex composition and activity, we provide evidence that the Mec1(ATR) checkpoint protein promotes telomere elongation by increasing both dNTP levels and dGTP ratios through Rnr1 upregulation in a mechanism that cannot be replaced by its homolog Rnr3.

166 Synapsis and recombination unite at Zip1’s N terminal tip. K. Voelkel-Meiman1, S.Y. Cheng1, M. Parziale1, S. Morehouse1, A. Feil1, A. de Muyt2, V. Borde2, A.J. MacQueen1 1) Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT; 2) Institut Curie, PSL Research University, CNRS UMR3664, Paris, France. The synaptonemal complex (SC) is a conserved chromosomal structure that provides a context for the processing of recombination intermediates during meiosis. We recently showed that SC central element components (Ecm11, Gmc2) and the mature budding yeast SC are dispensable for meiotic crossovers (but important for limiting crossover number). However, the SC transverse filament protein Zip1 is essential for normal crossing over. We have initiated a structure-function analysis 62 to identify molecular features of Zip1 that correspond to its SC assembly (synapsis) and crossover functions. Zip1 is predicted to assemble extensive coiled-coil (residues ~175-750), flanked by unstructured terminal regions. Our prior analysis of a Zip1 protein missing residues 21-163 indicates that Zip1’s crossover and SC assembly functions are genetically separable: The “SC- deficient, excess crossover” phenotype displayed by zip1-[Δ21-163] mutants demonstrates that residues 21-163 are essential for synapsis but dispensable for Zip1’s crossover function. Interestingly, we have determined that residues 1-20, which display strong conservation between Zip1 proteins from divergent fungal species, are critical for Zip1’s crossover activity. Strains homozygous for zip1-[Δ2-163] exhibit loss of both SC assembly and Zip1-mediated crossovers. Furthermore, diploid strains homozygous for zip1-[Δ2-9] and zip1-[Δ10-14] alleles, encoding Zip1 proteins that lack residues 2-9 or 10-14, exhibit a striking reciprocal phenotype to zip1-[Δ21-163] mutants: Robust, perhaps premature, SC assembly accompanied by a dramatic deficit in Zip1-mediated crossovers. zip1-[Δ2-9] and zip1-[Δ10-14] mutants also exhibit hyper-SUMOylation of Ecm11, a relatively unusual phenotype that is shared with mutants missing the pro-crossover factor Zip3. Using immunofluorescence on meiotic chromosomes we observed that the Zip1-[Δ2-9] and Zip1-[Δ10-14] proteins fail to interface properly with Zip3 at Zip1 polycomplex structures; chromatin immunoprecipitation furthermore revealed that Zip1-[Δ2-9] and Zip1-[Δ10-14] proteins fail to recruit Zip3 to recombination initiation sites. We propose that Zip1’s N terminal tip promotes crossovers through an interaction with Zip3; we are currently testing whether this interaction is direct. The adjacency of pro-crossover residues to residues that are critical for SC assembly suggests that Zip1's N terminus acts as a molecular linchpin to coordinate early steps in recombination with synapsis.

167 Genome instability in yeast DIS3 exonuclease domain mutants. K. Milbury1, C. Fowler1, B. Paul2, B. Montpetit2, P. Stirling1,3 1) Terry Fox Laborotary, BC Cancer Research Center, Vancouver, British Columbia, CA; 2) Department of Viticulture and Enology, University of California, Davis, CA, USA; 3) Department of Medical Genetics, University of British Columbia. Vancouver, BC, Canada. Chromosome instability (CIN) is characterized by an increased rate of the unequal distribution of DNA between daughter cells. These large changes in chromosome structure or number can occur due to both mitotic defects leading to aneuploidy and DNA damage-induced chromosome rearrangements. Previous large-scale screens for CIN genes in the model organism Saccharomyces cerevisiae identified DIS3, which codes for a catalytic component of the core RNA exosome complex, as a novel CIN gene. Presumed reduction-of-function mutations in human DIS3 have been identified in roughly 11% of multiple myeloma (MM) cases.

We sought to determine the mechanism of CIN in DIS3 mutants, and to recapitulate MM-associated point mutations at conserved sites in yeast cells, in order to understand potential connections of emergent CIN to MM. We have found that while MM-associated DIS3 exonuclease mutations induce DNA:RNA hybrid accumulation and increased rate of CIN, analysis of DNA damage foci by microscopy revealed no increase in double-strand breaks. Yeast DIS3 exonuclease mutants experience growth retardation, temperature sensitivity, and an altered budding index. Microarray analysis of one MM mutant has additionally demonstrated downregulation of cell cycle components, consistent with the potential for mitotic defects, in addition of upregulation of a host of metabolic pathways. Further, genetic interaction profiling by synthetic genetic array indicates MM-associated DIS3 mutations synthetically interact with rRNA processing proteins, as well as a host of mitotic regulators and metabolic pathways, particularly those involved in spindle and kinetochore function. Together, these results demonstrate extensive phenotypic consequences of MM-associated point mutations in DIS3, and support a model for CIN in DIS3 mutants involving defects in cell cycle processes.

168 Investigation of helicases, exonucleases, and TERRA non-coding RNAs in telomere maintenance. T. Momtareen, J. Gallagher Biology, West Virginia University, Morgantown, WV. Telomeres are the nucleoprotein structures that protect eukaryotic chromosomal ends from degradation. Yeast

(Saccharomyces cerevisiae) telomeres are generally characterized as T(G1-3) repeats coated with a variety of structural proteins. There are also replication, processing, and recombination proteins that interact with the telomeres in different stages of the cell cycle to maintain, repair, and protect them. Among these proteins are helicases, Sgs1 and Y’-Help1 and the long noncoding RNAs, TERRA, whose roles in telomere maintenance have not been fully characterized yet. The goal of my project is to understand how Sgs1, Y’-Help1, and TERRA RNAs contribute to telomere maintenance. Telomeres naturally shorten with every cell division but cells can circumvent this in telomerase enzyme dependent and independent mechanisms. Sgs1 is an important protein in telomerase independent maintenance of telomeres. It is the yeast homolog of the bacterial RecQ helicase and the human WRN protein, the mutation of which causes the Werner’s syndrome. Sgs1 has been implicated in the C-strand resection of telomeres along with nucleases Cdc13 and Exo1. However, resection can occur in the absence of all three proteins, indicating the presence of another nuclease. This project will attempt to look for this nuclease and figure out if and how it interacts with Sgs1 to carry out end resection. Another goal of this project is to understand the relationship between TERRA RNA, YRF transcripts, and the Y’-Help1 enzyme and how they potentially affect telomere length. TERRAs (Telomeric Repeat-containing RNA) are long non-coding RNAs transcribed from the telomeres. The transcription start site (TSS) for TERRA is located in the subtelomeric regions. Although, TERRA RNA has been implicated in several telomere maintenance processes, it’s exact function is yet to be discovered. Y’ elements are one of the two types of subtelomeric elements of a chromosome. In the absence of telomerase, increased levels of Y’ transcripts have been observed in yeast cells that have escaped senescence. Moreover, these cells also express the helicase Y’-Help1 encoded by the YRF1 genes located in 63 subtelomeres. Although the TERRA sequence and the Y’ elements overlap, the relationship between them needs to be determined.

169 Involvement of the INO80 Complex in Chromosome Segregation. Jesus Moreno1,2, Ines Pinto1,2 1) Department of Biological Sciences, University of Arkansas, Fayetteville, AR; 2) Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR. Faithful chromosome segregation is an essential property of cell division, required for the maintenance of the genome’s integrity. The main goal of this work is to gain understanding on the role that chromatin and chromatin remodeling complexes play in mitotic chromosome segregation. In an effort to evaluate the proteins that are involved in ploidy maintenance, we carried out a genetic screen of the non-essential deletion library for genes that when mutated caused ploidy increase. Among the mutants that increased ploidy, we encountered members of the INO80 complex. The INO80 chromatin- remodeling complex participates in a variety of biological processes including transcription, DNA repair, DNA replication, and chromosome integrity. It catalyzes the eviction of the H2A.Z histone variant replacing it with H2A. This complex is comprised of 15 subunits, and their specific contribution to chromosome segregations remains largely unknown. The INO80 complex has been implicated in the maintenance of ploidy through the characterization of mutations of the genes encoding the Ies6 and Ino80 subunits, which result in a clear increase in ploidy. To evaluate the contribution of each subunit to chromosome segregation, we tested deletion mutants of all the non-essential subunits as well as a ts allele of ARP4 for benomyl sensitivity and increase-in-ploidy phenotypes. We found that only ino80Δ, arp5Δ, arp8Δ, ies6Δ and taf14Δ caused increase-in-ploidy and increased benomyl sensitivity, and that a shift of arp4ts to restrictive temperature also leads to cells with increased ploidy as well as aneuploidy. Interestingly, the rate of increase in ploidy varied among the subunits tested. We also analyzed genetic interactions between the ploidy-increase causing alleles and SGO1, the gene encoding shugoshin, required for sensing tension and mitotic chromosome stability. High levels of SGO1 show suppression of the ino80Δ phenotypes, suggesting that the ino80 defects can be alleviated by restoring bi-orientation. Ino80 localization at the pericentromere was not affected by the absence of Arp5, indicating that Arp5 is not required for recruitment of the complex. We are currently investigating whether this complex is catalytically active by examining the levels of H2A.Z in pericentrometic regions.

170 Viability in response to heat stress requires shelterin rearrangement and telomere shortening. T. Pohl, C. Webb, Y. Wu, V. Zakian Princeton University, Princeton, NJ. Telomeres are DNA-protein structures that protect the ends of linear chromosomes from degradation, end-to-end fusions, and the loss of genomic DNA that arises from incomplete replication. In all eukaryotes, telomeres consist of tandem copies of a non-coding short DNA repeat that is associated with a number of conserved proteins that together contribute to the capping or end protection functions of telomeres. In Schizosaccharomyces pombe, shelterin, the protein complex that protects telomere ends and regulates access of telomerase to telomere DNA is composed of six proteins; Taz1, Rap1, Poz1, Pot1, Ccq1, and Tpz1. Many of these proteins, especially Tpz1, are homologues of the shelterin components in humans. A large number of stresses are associated with human telomere shortening yet there is little mechanistic data to explain how this shortening occurs. We used fission yeast, whose shelterin-like telomere structure makes it a tractable model for humans to understand the mechanistic basis for reduction of telomere length in response to heat stress. A variety of genetic and molecular techniques were employed to show that S. pombe telomeres shorten by as much as 50% in response to heat stress. The extent of shortening increased with higher temperatures and had no negative effect on doubling time. Even the most extensive shortening was rapidly reversed by return to lower temperatures. Shortening was accompanied by reduced levels of two telomerase subunits, Est1 and Trt1 as well as in reduced abundance and telomere binding of Ccq1 and Tpz1, the ortholog of human TPP1, which is critical for telomerase recruitment and activity. All of these reductions occurred post-transcriptionally. Inability to reduce telomere bound Tpz1 correlated with telomere loss and cell death at high but not low temperatures. We propose that the ability to shorten telomeres by altering the stoichiometry of telomerase and shelterin complexes is an adaptation that makes these hard-to-replicate regions easier to maintain during environmental stress.

171 The histone variant H2A.Z promotes chromosome condensation in Saccharomyces cerevisiae. A. M. Rogers, S. G. Holmes Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT. Chromosome condensation is essential for the fidelity of chromosome segregation during mitosis and meiosis. We previously demonstrated that histone variant H2A.Z is necessary for proper sister chromatid cohesion. To determine if H2A.Z also contributes to chromosome condensation we used a strain expressing Net1-GFP to assess condensation by examining rDNA morphology in metaphase blocked cells. We find that a deletion of HTZ1, the gene for H2A.Z, causes a significant defect in rDNA condensation. Acetylation of H2A.Z’s N-terminal lysines can affect H2A.Z function; however, we find strains expressing a form of H2A.Z that cannot be acetylated do not exhibit rDNA condensation defects. Prior studies found that several phenotypes caused by loss of H2A.Z are suppressed by eliminating Swr1, a key component of the SWR complex that deposits H2A.Z in chromatin. We observe that the htz1Δ swr1Δ strain has near normal rDNA condensation. Surprisingly, we have found that elimination of the linker histone H1 can also suppress the rDNA condensation defect in htz1Δ strains. Strains lacking only Swr1 or H1 exhibit wild type patterns of rDNA condensation. Loss of H2A.Z does not significantly affect transcription of condensin genes. Using ChIP, we find that the loss of H2A.Z does not change condensin association with 64 chromatin. Our results indicate that H2A.Z promotes chromosome condensation, in part by counteracting the function of histone H1.

172 Regulation of Dam1C by the Ras/PKA pathway in Saccharomyces cerevisiae. S. Bikram Shah1, D. Parmiter1, C. S Constantine1, P. Elizalde1, M. Naldrett2, T. Karpova3, J. S Choy1 1) Department of Biology, The Catholic University of America, Washington DC; 2) Proteomics and Metabolomics Facility Center for Biotechnology, Beadle Center, University of Nebraska- Lincoln, Lincoln, NE ; 3) Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, NIH, Bethesda, MD. Faithful segregation of chromosomes during cell division is required for the health and survival of single celled and muticellular organisms. Chromosome segregation relies on stable attachments between the plus ends of microtubules (MT) and the kinetochore (KT) complexes assembled at the centromeres. Dam1c (Dam1 complex) is a heterodecameric complex that forms the outermost part of the KT. Oligomers of Dam1c are thought to form rings that embrace the depolymerizing plus ends of MT’s during anaphase to ensure chromosomes remain stably bound as they move to the opposite spindle poles. We find that overexpression of cAMP-dependent protein kinase A (PKA) catalytic subunits (TPK1, TPK2, TPK3) are lethal in temperature sensitive (ts) dam1 mutants and increases the rate of chromosome loss in otherwise wild-type cells. Previous studies suggested a role for PKA activity in KT function and chromosome segregation. However, the molecular mechanism remains largely unknown. Here we show for the first time that PKA directly phosphorylates Dam1p and regulates its localization to the kinetochore, possibly altering the oligomerization state of Dam1c. In addition, we find that modulating PKA activity by growing cultures under different glucose concentrations leads to a concomitant change to PKA mediated phosphorylation of Dam1p. This work provides a mechanism that joins the Ras/PKA pathway to kinetochore function and points to the possibility that nutrient signaling may directly influence chromosome segregation fidelity.

173 Cdc48 regulates spliceosome assembly and nuclear protein sequestration during genotoxic stress. V. Mathew, K.Y. Jiang, A.S. Tam, P.C. Stirling Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC, CA. Cdc48/VCP is a highly conserved ATPase chaperone that plays an essential role in the assembly or disassembly of protein- DNA complexes, in degradation of misfolded proteins, largely through endoplasmic reticulum-associated degradation. We find that Cdc48 relocalizes during cellular stress to intranuclear protein quality control (INQ) sites. Cdc48 function is required to suppress INQ formation under non-stress conditions. We previously linked the sequestration of a splicing factor, Hsh155 to INQ sites and here we find that Cdc48 physically associates with Hsh155 by co-immunoprecipitation and regulates the assembly of Hsh155 with its partner spliceosome proteins. Cdc48 mutants exhibit splicing defects and show diminished recovery from genotoxic stress treatments. Overall, this study links Cdc48 to the spliceosome assembly/disassembly for the first time, and describes a new role for Cdc48 in nuclear protein quality control aggregate regulation.

174 The yeast core spliceosome maintains genome integrity through R-loop prevention and α-tubulin expression. A. Tam1,2, K. Milbury1,2, V. Mathew1, T. Sihota2, A. Zhang2, P. Stirling1,2 1) Terry Fox Laboratory, BC Cancer Research Centre, Vancouver, British Columbia, CA; 2) University of British Columbia, Vancouver, British Columbia, CA. To achieve genome stability cells must coordinate the action of various DNA transactions including DNA replication, repair, transcription and chromosome segregation. How transcription and RNA processing enable genome stability is only partly understood. Two predominant models have emerged: one involving changes in gene expression that perturb other genome maintenance factors, and another in which genotoxic DNA:RNA hybrids, called R-loops, impair DNA replication. Here we characterize genome instability phenotypes in yeast splicing factor mutants and find that mitotic defects, and in some cases R-loop accumulation, are causes of genome instability. Our data indicates defective splicing affects gene expression of key mitotic genes such as TUB1, which in turn leads to aberrant chromosome segregation. In addition, we also found that defective splicing could indirectly lead to accumulation of R-loops by changing the expression of YRA1, an mRNA export factor that regulates R-loop levels. Taken together, our data suggests differing allele penetrance, and also selective effects on the transcriptome can lead to a range of phenotypes in mutants of the spliceosome. Thus, by altering gene expression, aberrant splicing can create multiple routes to genome instability.

175 Death’s deception: An improved ATAC-seq method to eliminate death signature in mixed-viability samples and new insights into the chromatin structure of aged cells. Bernd J. Wranik, Ilya Soifer, David G. Hendrickson, Griffin Kim, R. Scott McIsaac Calico Life Sciences LLC, South San Francisco, CA. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a powerful method that uses the hyperactive Tn5 transposase and subsequent next-generation sequencing to identify open regions of chromatin. Key to acquiring high quality data is the isolation of intact chromatin from relevant cell types. Genomic DNA from dead cells loses its chromatin structure and can persist in samples for days and in a mixed population of viable/inviable cells, DNA from dead cells results in an artificially open chromatin signature that masks the true accessibility signature of viable cells.

Here we describe a method for eliminating this dead-cell signature by pre-treating the samples with Propidium Monoazide (PMA). PMA is unable to cross the cell membrane of intact cells but can intercalate into free DNA and the DNA of cells with compromised membrane integrity. Exposure to blue light results in a photoreaction that covalently cross-links PMA to the DNA, rendering it non-amplifiable by PCR. As proof of concept, we heat killed yeast cells and combined them with log-phase 65 cells at varying ratios upwards of 1:2 (live:dead); in all cases, the dead-cell signature could be effectively removed from these mixed-viability samples, ‘rejuvenating’ the ATAC-seq trace to that observed in a purely log-phase population.

As an application of this method, we profiled chromatin changes at different yeast replicative ages. In addition to identifying age-associated changes in genomic structure, we find that global nucleosome occupancy does not decrease as a function age as previously reported; when the DNA from aged cells is PMA-treated, the average accessibility of old cells is comparable to that of young cells. We expect this modified ATAC-seq method to be generally useful in situations involving mixed-viability samples.

176 Global Genetic Footprint of an Oncometabolite. Z. Yan1, R. Janke1, M. Kaiser1, B. San Luis2, M. Costanzo2, C. Boone2, J. Rine1 1) California Institute of Quantitative Biosciences, University of California, Berkeley, Berkeley, CA; 2) Donnelly Centre, University of Toronto, Toronto, Ontario,. Human isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate. Cancer-associated IDH mutations abolish this reaction and promote a partial reverse reaction in which alpha-ketoglutarate is reduced to form D-2-hydroxyglutarate (D2-HG). High levels of D2-HG promote the formation of certain cancers. Still, the precise underlying mechanisms of its role in cancer formation, as well as in normal cellular physiology, remain unclear. D2-HG is metabolized by D-2-hydroxyglutarate dehydrogenase, and mutation of the enzymes in humans (D2HGDH) or yeast (Dld2 and Dld3) results in accumulation of high levels of D2-HG. We previously demonstrated that deletion of Dld2 and Dld3 causes accumulation of D2-HG in yeast to levels similar to those observed in cancer cells, and that this resulted in widespread epigenetic changes, namely hypermethylation of histones. Here we utilized synthetic genetic array (SGA) technology available in yeast to study the total impact of D2-HG on cell function. Using this method, we screened the yeast genome for mutations that demonstrated synthetic genetic interactions with high levels of D2-HG in dld2Δ dld3Δ mutants. We identified multiple protein complexes implicated in chromatin remodeling, transcription, DNA repair, and epigenetic regulation. This pattern of genetic interactions has provided us with a broad snapshot of functions that are perturbed by high levels of D2-HG and suggest multiple aspects of chromatin regulation are affected.

177 The phosphorylation of Chromatin Assembly Factor I by CDK and DDK regulates genome stability. Hollie Rowlands, Ashley Cheng, Kholoud Shaban, Barret Foster, Krassimir Yankulov Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada. Chromatin Assembly Factor I (CAF-I) is a histone chaperone, which assembles H3/H4 tetramers in the wake of the replication forks. Together with other histone chaperones CAF-I plays a central role in the transmission of epigenetic state. However, mechanistic insights into CAF-I activity are limited. In mammalian cells CAF-I is essential, but in budding yeast its destruction leads to only marginal loss of fitness, to substantial loss of silencing of subtelomeric genes and to reduced frequency of epigenetic conversions at the telomeres. CAF-I is phosphorylated by CDK (Cyclin Dependent Kinase, Cdc28p) and most likely by DDK (Dbf4-Dependent Kinase, Cdc7p- Dbf4p). Both kinases are essential and regulate key events during the initiation of DNA replication. Recent studies have indicated that DDK could also play a post-initiation role at paused replication forks. In an attempt to better understand the significance of the phosphorylation of CAF-I by CDK and DDK, we introduced multiple point mutations at the phosphorylation sites of Cac1p. Unlike the deletion of CAC1, some of these CAC1 mutants showed severe defects in cell growth, altered morphology and elevated sensitivity to mutagens. Even more, these mutants displayed apparent flocculation-like phenotypes, a feature that is not observed in S288C laboratory strains and its derivatives. Flocculation phenotypes were also observed upon deletion of CAC1 in conjunction with deletions of genes that regulate fork processivity, including DDK. We correlated these phenotypes to de-repression of the FLO family of genes and to frequent recombination between them. In summary, the studies of CAC1 mutants unveiled functions of CAF-I that could not be detected by its destruction. We will present details on how the activity of CAF-I is tuned by protein kinases. We will also highlight the FLO gene loci as a novel model for the study of epigenetic phenomena in S. cerevisiae.

178 Mapping serine tRNA identity elements using mistranslation. M.D. Berg1, J. Genereaux1, J. Zhu1, P. O'Donoghue1,2, C.J. Brandl1 1) Department of Biochemistry, University of Western Ontario, London, Ontario, CA; 2) Department of Chemistry, University of Western Ontario, London, Ontario, CA. tRNAs read the genetic code allowing nucleic acid sequence to be translated into protein. The accuracy of translation requires the ligation of a specific amino acid to the 3’ end of each tRNA. This process is directed by nucleotides or base-pairs within the tRNA, known as identity elements, that are recognized by one of the approximately 20 aminoacyl-tRNA synthetases. For many tRNAs, the anticodon is the main identity element. This is not the case for serine tRNAs. Previously, we identified a serine tRNA containing a proline anticodon that mistranslates serine into proteins at proline codons. Using this mistranslating tRNA, we suppressed a stress sensitive allele of a co-chaperone protein in yeast, tti2-L187P. In this study, we used this mistranslation and suppression system to investigate the functional significance of the nucleotides and base-pairs in tRNASer. Further assessment was obtained by measuring the heat shock response induced by each mistranslating tRNA variant. We find the first and third nucleotide pairs in the acceptor stem and a single base-pair in the D-stem are required for 66 function. Serine tRNAs also contain a unique variable arm between the anticodon and T-arms. We find both the length and the sequence of the variable arm are essential for tRNASer function. Interestingly, incorporating the major alanine identity element—a G3:U70 base pair—into the acceptor stem of the serine tRNA abolishes function, suggesting that it is an anti- determinant. In addition, by taking advantage of the anticodon not being recognized by the synthetase during charging of the tRNA with serine and modulating tRNA function with different secondary mutations, we have generated tRNAs that mistranslate serine at both phenylalanine and arginine codons. In conclusion, our mistranslation and suppression system has allowed the identification of functionally important nucleotides in tRNASer and the generation of mistranslating tRNAs that incorporate serine at any codon without significant toxicity.

179 Building Silenced Chromatin Domains on a Synthetic Chromosome. V.M. Blake, J.D. Rine Molecular and Cell Biology, UC Berkeley, Berkeley, CA. The synthetic eukaryotic genome in Saccharomyces cerevisiae provides an elegant platform to test links of the yet elusive contribution of chromosome structure to transcriptional regulation. By omitting subtelomeric regions, transposable elements, introns, and sequestering transfer RNA genes, synthetic chromosomes are a premier context to define minimal elements that regulate specialized chromatin domains. Epigenetically-heritable silenced chromatin at HML and HMR determines mating-type identity in S. cerevisiae. Silencer sequences that flank these loci recruit Silencing Information Regulators, or SIR proteins, that catalyze assembly of silent chromatin. Studies of these silent loci on a native chromosome are confounded by the potential contribution of repetitive elements that surround these loci, and the potential for long-range effects on silencing beyond that of the Recombinational Enhancer (RE). Therefore, understanding all factors that contribute to building heterochromatic domains on a synthetic chromosome will allow for comprehensive definition of the genetic elements that regulate their epigenetic inheritance and stability of the transcriptionally silent state. I have restored HML and HMR loci to a synthetic chromosome III and found that they are nominally silenced despite the absence of a well-characterized boundary element. I am now monitoring heterochromatin dynamics at single-cell resolution to explore previously inaccessible questions of eukaryotic genome organization with the function-driven discovery tools of genetics.

180 Lagers yeasts and the delicate balance between its hybrid genomes and brewery’s stress. D. Bonatto Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, BR. Lager yeasts (Saccharomyces pastorianus) have been employed in brewery industry for the fermentation of Lager beers, a mass product consumed worldwide. Despite its industrial importance, poorly is known about how S. pastorianus deals with brewery stress (worts with high carbohydrate concentration, low pH, high hydrostatic pressure and dissolved CO2). Additionally, the stress effects induced by brewery’s propagation systems, which employ worts with high nutrient and oxygen concentrations for cell biomass production is also little understood. In this sense, we evaluated the major stress-associated biological mechanisms that are modulated by brewery’s fermentation and propagation conditions by using a transcriptomics and systems biology analyses. Our data indicated that, during propagation, genes associated with DNA repair and meiosis mechanisms are overexpressed, especially those linked to homologous recombination (HR) pathway.

181 Investigating the mechanism of subtelomeric silencing. E. Bondra, J. Rine Molecular and Cell Biology, University of California - Berkeley, Berkeley, CA. Silent Information Regulator (Sir) proteins impart transcriptional silencing through the formation of heterochromatin at the silent mating-type loci HML and HMR, and at telomeres. Transcription of genes at silenced HML and HMR is 1000-fold lower in SIR+ than sir∆ cells. In contrast, silencing of genes near telomeres, while involving the same machinery, appears to achieve an intermediate level of repression, and exhibit no evidence of continuous gradients of repression suggested by past studies of reporter genes. Why such disparities in the degree of silencing at different loci exist and how this tuning is mechanistically achieved remains enigmatic. We hypothesize that silencing at telomeres may be mechanistically distinct from canonical Sir- silencing at HML and HMR, lending plasticity to organisms to better cope with environmental stresses. We aim to answer the outstanding questions regarding subtelomeric silencing that remain after decades of population-based studies at single-cell resolution. Specifically, we will determine whether subtelomeric silencing is a function of distance from telomere, and define the contribution of Sir proteins to this effect. Telomeres in S. cerevisiae comprise telomerase-generated repeats, which contain an abundance of Rap1 binding sites. Repressor/Activator Protein (Rap1) contains a DNA-binding domain, and a large C-terminal interaction domain, which is bound by Sir3 and other partners including Rif1 and Rif2 (Rap interacting factor 1 and 2). Rif1 and Rif2 are negative regulators of telomerase; deletion of either causes a lengthening of telomeres with concomitant increases in Rap1 binding sites. Long telomeres are thought to disrupt silencing at HM loci by titrating away the limiting level of Sir proteins through interactions with Rap1. Utilizing the unmatched sensitivity of the CRASH assay for silencing at HML, we have confirmed massive loss of silencing in a rif1∆, consistent with the proposed model. However, rif2∆ shows no loss of silencing despite having long telomeres. We will utilize genetic analysis to understand the contribution of telomere length on proper maintenance of silencing. In a broader context, we will investigate the spatial co-regulation of subtelomeric loci, providing crucial information regarding the nature of telomere position effect and the mechanism of silencing in subtelomeric domains.

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182 Roles of PCNA in the establishment and maintenance of heterochromatic silencing. M. Brothers, R. Janke, J. Rine Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA. Establishment of silencing de novo at the silent mating type loci HML and HMRa in Saccharomyces cerevisiae requires passage through the S-phase, yet intriguingly does not require replication. Complicating this puzzle further is a dependence ⍺ on replication fork factors. Mutations in POL30, which encodes the homotrimeric DNA polymerase processivity factor PCNA, have a detrimental effect on the cell’s ability to silence the HM loci. Three of these mutants, pol30-6, pol30-8, and pol30-79, have phenotypes indicating that PCNA may be contributing to the establishment and maintenance of heterochromatic silencing in multiple ways. Further characterization of these mutants will help elucidate how replication machinery plays a role in epigenetic inheritance across eukaryotic organisms. Here, epistasis analysis is indicative of two separable roles of PCNA in silencing. Using an assay that can capture transient loss of silencing events in combination with more traditional silencing assays, we explore the connections and differences between PCNA’s effects on the establishment and maintenance of heterochromatic silencing.

183 Creating a “six-pack” strain: expression of lignocellulolytic enzymes in an industrial yeast strain for second- generation bioethanol production. A. Claes, M. R. Foulquié Moreno, J. M. Thevelein Center for Microbiology, KU Leuven - VIB, Heverlee, BE. Second-generation bioethanol is produced from lignocellulosic biomass. This circumvents the current issues concerning usage of fossil fuels and first-generation bioethanol. The major obstacle for this upcoming industry remains the cost of enzymes required for liberating fermentable sugars from lignocellulose. Lowering the use of commercial enzyme cocktails will result in significant savings in production costs. Furthermore, this hydrolysis can be improved to increase the yield of ethanol. This explains the scientific interest in consolidated bioprocessing, a technology in which both lignocellulose hydrolysis and fermentation of hexose and pentose sugars are performed in a single step by one specific microorganism. This requires a robust microorganism that can produce and secrete lignocellulolytic enzymes and ferment the breakdown sugars to ethanol. The current project focuses on conferring lignocellulolytic capacity to an industrial hexose and pentose fermenting Saccharomyces cerevisiae strain. Therefore, several genomic sites are targeted for specific integration of heterologous genes coding for the six essential enzymes for the hydrolysis of lignocellulose. This is obtained by using the CRISPR-Cas9 methodology. Successful genomic integration of multiple copies of a heterologous β-xylosidase and xylanase resulted in efficient fermentation profile of xylan, the main building block of hemicellulose, due to a high observed specific activity of these enzymes. Also, a heterologous β-glucosidase and cellobiohydrolase II are genomically integrated and expressed in this S. cerevisiae strain. High detected specific activity of β-glucosidase results in an efficient hydrolysis of cellobiose into fermentable glucose. Cellobiose is the last breakdown product of cellulose. To completely hydrolyze this fraction, the two remaining enzymes, i.e. endoglucanase and cellobiohydrolase I, are currently being integrated. The resulting “six-pack” strain will be capable of producing and secreting all essential enzymes for the hydrolysis of lignocellulose and fermentation of the liberated sugars to ethanol. This S. cerevisiae strain will be a promising platform microorganism to reach the ultimate goal of consolidated bioprocessing.

184 Engineering and Optimizing Ribosomal Traffic Jams through whole Cell simulations. R. Cohen-Kupiec, H. Zur, T. Tuller Department of Biomedical Engineering, Tel Aviv University, Tel Aviv, IL. One of the crucial aspects affecting cellular growth rate in exponentially growing organisms is the availability of ribosomes during protein translation. It has been shown that in both prokaryotes and eukaryotes the first 50 codons of the ORF contain various signals that slow ribosomal speed in this region, hence affecting the availability of free ribosomes, which in turn limits protein synthesis rates. We developed various biophysical predictive models that link the features of mRNA transcripts to the dynamics of their translation process. Based on comparisons to experimental data, we showed in the past that these models can provide useful predictions related to codon decoding efficiency and ribosome densities, which eventually affect translation rates. Thus, these models can be used for engineering the nucleotide composition of genomes to yield specific dynamics of translation. As a proof of concept, we developed a whole cell simulation for translation in S. cerevisiae based on experimental data. We then developed algorithms for increasing the pool of free ribosomes without affecting the encoded proteins. These methods provided a list of candidate genes in which specific synonymous mutations are expected to affect the cell’s global translation rate, which in turn is expected to directly affect the fitness of the yeast. We used the CRISPR system to mutate the selected genes in haploid WT strains of S. cerevisiae. We examined the growth kinetics of single and double mutants and show that there are mutants that grow better than the WT in lab conditions. We are now performing additional experiments for further validating and understanding the results.

185 Role of UAF in the Transition from pol I to pol II in the Synthesis of rRNA in S. cerevisiae. Heather Conrad-Webb, Kushal Bhatt, Arjuna Vallabhaneni Biology, Texas Woman's Univ, Denton, TX. rRNA synthesis is highly regulated in response to environmental changes, since ribosome synthesis requires a vast majority of cell’s resources. During favorable conditions, Pol I synthesis of the 35S rRNA precursor accounts for ~60% of total RNA synthesis. rRNA synthesis is regulated by the number of open or accessible rDNA repeats and transcription activation of pol I transcription factors: UAF, CF, Rrn3 and pol I. During chronic stress (mitochondrial dysfunction, nitrogen deprivation), Pol II transcribes rDNA to form functional rRNA, a phenomenon also observed in higher eukaryotes, including humans. We 68 propose that the polymerase switch forms a third regulatory level of rRNA synthesis that provides sufficient rRNA synthesis during chronic stress conditions. The binding of UAF stimulates the formation of chromatin to allow maximal Pol I transcription and repression of the switch. Absence of UAF subunits trigger rDNA chromatin alteration and pol II rRNA synthesis. Loss of UAF subunits, but not CF, pol I, or other chromatin modulators, will allow the spontaneous switch to pol II rRNA synthesis and survival of cells in the absence of pol I. Therefore, we hypothesize that UAF is the target of stress signaling pathways that trigger chromatin remodeling facilitating the switch. Furthermore, we propose that UAF facilitates the recruitment of factors known to play a role in rDNA silencing including: HMG protein Hmo1, histone deacetylases Sir2 and Rpd3, methyltransferases Set1 and Set2, and other chromatin modulators. To study this phenomenon, Pol II rRNA synthesis was evaluated using rDNA-LacZ reporter construct. Knockout strains of uaf30 and hmo1 demonstrate an increase in β- galactosidase activity suggesting their role in limiting pol II access to rDNA. Loss of Tor1 pathway components increase activity, while loss of downstream components of stress signaling pathways (rim15, gis1) reduce activity. This suggests that the polymerase switch behaves similar to classic stress responses. To address the effect of stress on UAF binding and the chromatin structure of the rDNA repeat, ChIP assays for differences in occupancy of UAF and Hmo1 on rDNA are being assessed. Secondary modifications of UAF complex members are also being evaluated under non-stress and stress conditions. Identifying the underlying mechanism for this evolutionary conserved phenomenon will help understand cell survival mechanism under various stress conditions.

186 Activity of the RNA Pol II Mediator subunit Gal11 is enhanced by the presence of polyglutamine tracts. D. Cooper, J. Fassler Department of Biology, University of Iowa, Iowa City, IA. Polyglutamine (poly-Q) tract containing proteins are enriched and conserved in eukaryotic transcriptional regulators suggesting a poorly understood functional and/or adaptive role for poly-Q tracts in regulating gene expression. To investigate the role of poly-Q tracts in transcriptional regulators, we study Gal11 (Med15) a poly-Q rich subunit of the RNA polymerase II Mediator complex. Gal11 is an interaction hub with both positive and negative roles in expression of metabolic and stress response genes. The three longest poly-Q tracts of Gal11 display considerable across-strain length variation (10-30 CAG or CAA repeats) which may affect interactions between Gal11 and its partners that would be reflected in the altered expression of Gal11-dependent genes. To study the impact of Gal11 poly-Q variation on stress response and domestication phenotypes we evaluated patterns of tract length variation across sets of beer and wine yeast. The Gal11 poly-Q tract lengths in these strains correspond in part to the phylogenetic relationship between the strains but also reflect shared phenotypes. We propose that Gal11 poly-Q tract lengths are optimized for specific transcription factor (TF) and mediator subunit interactions that have co-evolved within a strain. To investigate potential incompatibilities between native TFs and non-native Gal11s with atypical poly-Q tract lengths, we replaced the lab strain (S288C) GAL11 sequence with GAL11 alleles from diverse wine yeast. The resulting mutant phenotypes are consistent with the hypothesis that poly-Q tract lengths in Gal11 may be non-random. To specifically test the effect of poly-Q tract length variation on Gal11 activity, we generated synthetic Gal11 poly-Q variants in a miniaturized GAL11 (Mini-GAL11) construct that contains a single poly-Q tract and in a full-length GAL11 construct. We used these constructs to examine the effect of naturally occurring and synthetic tract length changes on Gal11-dependent stress phenotypes. The complete removal of a 12-Q tract caused substantial reduction in Gal11 activity. Surprisingly, extensive poly- Q tract length variation (12-47 repeats) was tolerated for all Gal11 activities tested thus far. These data suggest that for poly-Q tracts that are naturally variable in length, the length of the tract may be less important than its presence in influencing the activity of Gal11.

187 Mathematical modeling of small gene regulatory networks reveals key regulators and network properties important for controlling the early response to cold shock in Saccharomyces cerevisiae. K.D. Dahlquist1, B.G. Fitzpatrick2, B.J. Klein1, M.J. ONeil1, L.M. Kelly1 1) Biol, Loyola Marymount Univ, Los Angeles, CA; 2) Math, Loyola Marymount Univ, Los Angeles, CA. A gene regulatory network (GRN) is a group of transcription factors that control the level of expression of genes encoding other transcription factors. Dynamics of GRNs illustrate how expression in the network changes over time. GRNmap, a MATLAB software package (http://kdahlquist.github.io/GRNmap/), uses differential equations to model the dynamics of small- scale GRNs. The software estimates production rates, expression thresholds, and regulatory weights for each transcription factor in the network based on microarray data. Microarray data was obtained from a cold shock experiment where wild-type budding yeast, Saccharomyces cerevisiae, and five strains from which the transcription factors Cin5, Gln3, Hap4, Hmo1, and Zap1 were individually deleted were subjected to cold shock at 13°C for 15, 30, and 60 minutes. Six related GRNs, which ranged from 14-17 transcription factors (nodes) and 25-35 regulatory relationships (edges), were constructed using the microarray data and the YEASTRACT database. GRNmap was then used to estimate production rates, expression thresholds, and regulatory weights for each of these GRNs, followed by visualization of the results with the GRNsight software (http://dondi.github.io/GRNsight/). Forward simulation of the model showed a good fit to the experimental data, particularly in comparison to 30 random networks with the same genes and number of edges. Systematic analysis of edges revealed repeated motifs in the database-derived networks. These motifs included a coherent type I feed forward loop (FFL), an activating regulatory chain, and symmetrical incoherent type I FFLs terminating on the paralogs Yhp1 and Yox1. Multiple regression analysis of the database-derived and random networks demonstrated that model fit to experimental expression data was repeatedly correlated with the eigenvector centrality of individual transcription factors. Better model fit to 69 expression data was observed for transcription factors with high eigenvector centrality, such as Gcn4 and Yhp1. A consolidated network containing 15 transcription factors and 34 edges was constructed from conserved motifs and high eigenvector centrality transcription factors. The consolidated network outperformed all random networks and all but one database-derived network. The consolidated network also featured appreciable overlap with the general environmental stress response, with Hmo1 activating Msn2/Msn4 directly and Yhp1 indirectly.

188 Updated regulation curation model at the Saccharomyces Genome Database. S.R. Engel, M.S. Skrzypek, S.T. Hellerstedt, E.D. Wong, R.S. Nash, S. Weng, G. Binkley, T. Sheppard, K. Karra, J.M. Cherry, The SGD Project Genetics, Stanford University, Palo Alto, CA. The Saccharomyces Genome Database (SGD) provides comprehensive, integrated biological information for the budding yeastSaccharomyces cerevisiae, along with search and analysis tools to explore these data, enabling the discovery of functional relationships between sequence and gene products in fungi and higher organisms. We have recently expanded our data model for regulation curation to address regulation at the protein level in addition to transcription, and are presenting the expanded data on the ‘Regulation’ pages at SGD. These pages include a summary describing the context under which the regulator acts, manually curated and high-throughput annotations showing the regulatory relationships for that gene, and a graphical visualization of its regulatory network and connected networks. For genes whose products regulate other genes or proteins, the Regulation page includes Gene Ontology enrichment analysis of the biological processes in which those targets participate. For DNA-binding transcription factors, we also provide other information relevant to their regulatory function, such as DNA binding site motifs and protein domains. As with other data types at SGD, all regulatory relationships and accompanying data are available through YeastMine, SGD’s data warehouse based on InterMine.

189 Selective export of mRNAs during heat shock in Saccharomyces cerevisiae. L.E. Escalante1, A.P. Gasch1,2 1) Laboratory of Genetics, University of Wisconsin - Madison, Madison, WI; 2) Great Lakes Bioenergy Research Center, University of Wisconsin – Madison,Madison,WI. Yeast must launch multifaceted responses to varied stresses in order to survive in their diverse environments. In addition to launching specific transcriptional changes, mRNAs can also be destined for different fates via nuclear export or retention. Regulated mRNA export can affect gene expression on par with transcription, as nuclear retention of mRNAs prevents their translation. Severe heat shock can lead to selective export of specific mRNAs, as a striking nuclear enrichment of polyA tailed transcripts can be observed quickly after heat shock, yet heat shock transcripts continue to be exported. Surprisingly, it appears that cells continue to synthesize transcripts during stress that are destined for retention. While the selective export of a handful of transcripts has been described, the breadth of this response across the transcriptome and the physiological reason behind it are unknown. Here we are investigating selective export on a global scale by using cellular fractionation combined with RNA sequencing to identify which transcripts are synthesized and retained in the nucleus versus those synthesized and exported. These results will help elucidate a physiological role for selective export versus retention of transcripts synthesized during stress.

190 Failure Modes of Transcriptional Silencing. Marc Fouet, Jasper Rine University of California, Berkeley, CA. In Saccharomyces cerevisiae, the Silent Information Regulator (SIR) proteins are recruited and form a scaffold to induce heterochromatic silencing at the silent mating-type loci HML and HMR. This silent epigenetic state is essential to maintain haploid identity and is inherited from mother to daughter cell with a high stability (10-3 transient losses of silencing / generation). Sir1 plays a role in the establishment of silencing and likely recruits the other SIR proteins (2-3-4) that form a complex. The mechanism by which the SIR complex occupies all of the nucleosomes at HML and HMR is still debated. The mechanism by which silencing is transiently lost is unknown. And while HML and HMR epigenetic states seem to be independent in sir1 or sas2 mutants, silencing of genes residing within the same heterochromatic region (in HML or HMR) was shown to be in the same state. In this study, we ask if silencing loss events occur at the level of the locus, or at the level of individual genes in that locus: specifically, does silencing loss happen simultaneously at HMLα1 and HMLα2, the two genes contained in HML and driven by the same UAS. We took advantage of the sensitivity of the Cre-Reported Altered States of Heterochromatin assay previously developed by our lab, reporting a transient silencing loss event by a Cre-mediated heritable fluorescence color switch. This assay, combined to a mating strategy with a MATα strain lacking MATα1, enable us to monitor silencing loss in both HMLα1 and HMLα2. Our results indicated that silencing loss can occur at either the locus level, or at the single gene level, with HMLα1 expressed while HMLα2 was maintained in a silent state. The quantification of these results at a single-cell level, as well as the observation of regulation (or absence thereof) at the same loci during establishment of silencing using fluorescent reporters and microfluidic monitoring are under investigation.

191 Cell-cycle requirement for gene silencing: an alternate view. Davis Goodnight, Jasper Rine Department of Molecular & Cell Biology, University of California, Berkeley, Berkeley, CA. The HMR and HML loci of Saccharomyces cerevisiae are transcriptionally silenced by the SIR protein complex. A long-standing puzzle is that the transition from an unsilenced state to a silenced state at HMR requires progression through the cell cycle, even though none of the SIR complex components is known to be cell-cycle-regulated. Furthermore, prior work using low- resolution chromatin immunoprecipitation has suggested that SIR complex recruitment and spread at HMR can occur without 70 cell-cycle progression, creating a paradox wherein the machinery known to effect silencing can bind a locus without repressing its transcription. To study silencing establishment in its most native context, without relying on metabolic or temperature shifts, we developed an allele, SIR4-EBD, whose activity is regulated by estradiol. We used the SIR4-EBD allele to characterize the time- and cell-cycle-resolved series of SIR complex binding events associated with silencing establishment using high-resolution ChIP-seq, including the first unambiguous mapping of SIR protein binding to HML and HMR in the same strain using synthetically-generated SNPs to distinguish the two homologous loci. These experiments reveal that, contrary to prior reports, SIR recruitment and spread at both HMR and HML require cell cycle progression. In addition, we show that the only previously-reported mutant capable of bypassing the requirement for cell-cycle passage in establishing silencing, the deletion of a cis-acting tRNA gene adjacent to HMR, does not in fact display this phenotype as we show in two different experimental contexts. This prompted us to design a screen to identify mutants able to bypass the cell-cycle requirement, whose design and initial results we present here.

192 Mutational analysis of 5’-UTR intron revealed that splicing is not necessary for intron-mediated expression enhancement in Saccharomyces cerevisiae. H. Hoshida1,2,3, S. Goto1, M. Kondo1, R. Akada1,2,3 1) Dept Appl Chem, Yamaguchi Univ, Ube, Yamaguchi, JP; 2) Research Center for Thermotolerant Microbial Resource, Yamaguchi Univ, Yamaguchi, Yamaguchi, JP; 3) Biomedical Engineering Center, Yamaguchi Univ, Ube, Yamaguchi, JP. One of the functions of introns is expression enhancement and it is called intron-mediated enhancement (IME). The molecular mechanism of IME is not clear. In S. cerevisiae, we demonstrated that insertion of ACT1 intron into the coding sequence of an yeast-codon optimized secretory luciferase (yCLuc) enhanced secreted yCLuc activity. However, mutational analysis of introns in a coding sequence is difficult, because mutations affecting splicing impair correct translation termination. To avoid splicing requirement, we analyzed intron sequences which were artificially introduced into 5’-UTR for yCLuc expression. In the genome of S. cerevisiae, several 5’-UTR introns are identified. We used three sequences consisting of a promoter and a 5’-UTR containing a intron from RPS25A, RPS26A, and RPS26B genes to express yCLuc. The secreted yCLuc activity was higher compared with the TDH3 promoter and deletion of the intron decreased yCLuc activity, indicating that these 5’-UTR introns enhanced yCLuc expression in S. cerevisiae. To know the effect of the intron position in 5’-UTR, TDH3 promoter and the RPS25A intron region was joined at several positions. As a result, the artificial promoter in which RPS25A intron (327 bp) was directly joined to the full length of TDH3 promoter showed highest yCLuc activity and it was about 50-fold higher than TDH3 promoter alone. Next, several introns from coding regions were joined to the downstream of the TDH3 promoter. All the introns tested enhanced yCLuc expression. The 63-bp intron from QCR10 gene enhanced expression similarly to RPS25A intron. In S. cerevisiae introns, 6 bp at 5’ end, 7 bp around branch point and 3 bp at 3’ end are highly conserved. First, non-conserved region of QCR10 intron was deleted. The complete deletion of the non-conserved 37 bp between 5’ and branch point (7-43del) did not affect enhanced level. Further deletion in the conserved branch point region abolished enhancement. Furthermore, substitutions in the conserved 5’ region in 7-43del also abolished enhancement. However, a mutation of the 3’-end AG, which is the essential sequences for splicing in all eukaryotic introns, abolished splicing but kept enhancing ability. These results suggest that splicing is not absolutely necessary for IME in S. cerevisiae.

193 Epigenetic and cellular signaling mediated by the SMYD lysine methyltransferase Set5. Deepika Jaiswal1, Rashi Turniansky1, James Moresco2, Ganesh Ramaprasad1, Marlene Keisha Kontcho1, Julie Wolf1, John R. Yates III2, Erin Green1 1) Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, MD; 2) Department of Chemical Physiology, the Scripps Research Institute, La Jolla, CA. Genetic and proteomic studies have shown that the post-translational modification lysine methylation regulates key signaling pathways required for growth, differentiation and cell survival. While lysine methylation has been well-studied in the context of chromatin, less is known about signaling via methylation of non-histone proteins. The conserved SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) has been identified as enzymes that target a variety of histone and non-histone proteins, and are key contributors to diverse signaling pathways. In metazoans, SMYD proteins have been implicated in skeletal and cardiac muscle development and differentiation, hematopoiesis and immune system function, and misregulation of their expression is linked to pathological processes, including cardiac diseases and tumorigenesis. Despite significant therapeutic interest in this family of enzymes, our knowledge of their methyl-lysine substrates and functional roles in regulating key signaling pathways is still very limited. Budding yeast enzymes Set5 and Set6 carry the same domain structure as the mammalian SMYD enzymes and are thought to be ancestral members of this protein family. We previously identified Set5 as an H4 lysine 5, 8 and 12 methyltransferase and determined that its function partially-overlaps with the H3 lysine 4 methyltransferase Set1 in repressing genes near telomeres and transposable elements. Set5 localizes to both the nucleus and the cytoplasm, and is therefore predicted to target non-histone substrates for methylation. The mechanisms which influence its localization and methylation activity are still unknown. Here, we have used multiple proteomic assays, including protein microarrays and mass spectrometry, to identify numerous candidate substrates for Set5-mediated methylation. In addition, we have found that Set5 interacts with a diverse set of proteins in the nucleus and the cytoplasm, including proteins involved in transcription, protein quality control pathways and stress responses. Genetic analysis of SET5 mutants has also linked its function to promoting survival in the presence of stress. Further structure-function analysis has revealed that the C-terminal region of Set5, which is similar to C-terminal extensions in metazoan SMYD proteins, is a critical region of the protein for regulation of methylation activity and localization within the cell. We have identified 71 numerous sites of phosphorylation on the C-terminus of Set5 which appear to be central to its regulatory role. Overall, this work provides further insight into how Set5 links methylation at chromatin to broader functions in cellular signaling pathways, which we expect to be highly applicable to metazoan SMYD proteins and open new avenues of investigation regarding their function in both healthy and aberrant signaling pathways.

194 Set4 is a chromatin regulator that promotes cell survival in response to oxidative stress through the regulation of stress response gene expression programs. Yogita Jethmalani, Khoa Tran, Deepika Jaiswal, Erin Green Biological Sciences, University of Maryland Baltimore County, Baltimore, MD. Cells use chromatin-based mechanisms to regulate gene expression rapidly in response to stress, but there are many open questions about how chromatin proteins sense stress and how their activity is fine-tuned in response to stress. In Saccharomyces cerevisiae, the protein Set4 contains both a PHD finger and a SET domain, which are characteristically found in chromatin regulatory proteins. Genome-wide screens indicated that Set4 has low expression under normal conditions, but it is upregulated under stress conditions. Set4 has sequence homology with its yeast paralog, Set3, the fly protein UpSET and the human protein MLL5. MLL5 has been implicated in diverse pathways such as hematopoiesis and spermatogenesis, as well as in disease states including neurodevelopmental disorders and cancer. However, MLL5’s cellular function is still largely unclear. The function of Set4 in budding yeast has not been well-characterized. Based on its potential to be a stress- responsive chromatin regulator, we performed phenotypic characterization of strains lacking or overexpressing SET4. We identified a protective role for Set4 during oxidative stress and determined that the levels of Set4 are tightly regulated to promote cell survival, especially in the presence of oxidative insults. Gene expression analysis showed that Set4 regulates genes responsible for survival during stress, and Set4 directly associates with stress-response genes and acts as a chromatin regulator when cells are treated with hydrogen peroxide. In addition, genetic and protein-protein interaction experiments have revealed potential partners with which Set4 functions to regulate gene expression programs required for proper stress responses. Overall, our data indicates that Set4 is a context dependent chromatin regulator and it has a specific role during stress responses to promote survival. These results further our understanding of mechanisms that protect cells during oxidative stress, and will open new avenues for the investigation of the Set3-Set4 subfamily of SET-domain containing proteins.

195 Characterizing protein interactions promoting heme regulation of the JMJC domain-containing transcriptional regulator Gis1, in yeast. P. Konduri, T. Wang, L. Zhang The University of Texas at Dallas, Richardson, TX. Heme plays versatile and fascinating regulatory roles for fundamental biological processes. Heme serves as a signaling molecule for oxygen levels in yeast, as heme function is entwined with molecular oxygen levels. Heme and oxygen regulate the expression of many genes in eukaryotes by modulating activity of regulatory proteins. In yeast, Gis1 is a novel class of multi-functional heme sensing and signaling DNA-binding transcriptional regulator, belonging to the JMJD2/KDM4 subfamily of demethylases. It is highly homologous to the mammalian JmjC domain-containing protein JMJD2B, which plays an important role in histone demethylation, oxygen regulation, and hormonal signaling. Notably, recent experiments in our lab showed that heme regulates demethylase and transcriptional activities of Gis1. Biochemical studies indicate that heme binds directly to Gis1 (JmjN+JmjC domain, ZnF) and JMJD2B proteins and that heme binding to the ZnF stimulates Gis1 demethylase and transcriptional activities. This study aims to identify and characterize proteins that interact with Gis1 and modulate heme regulation of Gis1 activities. Affinity Purification Mass Spectrometry (AP-MS) was used to identify a series of Gis1 binding proteins under conditions of hypoxia, low heme, and high heme. After a series of computational analyses to rule out the artifacts, genetic and functional analyses identified the role of Mot3 in heme regulation of Gis1 activities. Mot3 binds to Gis1 under the conditions of hypoxia and high heme and promotes heme activation of Gis1 transcriptional activity. Additionally pull-down experiments identified that Mot3 interacts with the ZnF of Gis1. Experiments are underway to assess the role of Gis1 interacting proteins in the heme regulation of Gis1 demethylase activity. Together, our results show that Gis1 represents a novel class of transcriptional regulators, with multiple interacting partners that bind to and regulate Gis1 activities thereby playing a role in mediating heme signaling.

196 Genome-wide transcriptional response to altered levels of the Rpb7 subunit of RNA polymerase II in fission yeast identifies its role in DNA Repair. D. Kumar, N. Sharma University School of Biotechnology, G.G.S. Indraprastha University, Dwarka, New Delhi, IN. Gene expression is primarily controlled at the level of transcription in all living cells. RNA Polymerase II transcribes all protein coding genes and consists of 12 different subunits, Rpb1 to Rpb12. Out of these twelve subunits, Rpb4 and Rpb7 subunits are uniquely present in RNA Polymerase II and form a heterodimer in various organisms. Studies in Saccharomyces cerevisiae have revealed that both these subunits perform some functions independent of each other, while they regulate other processes as a complex. However, a detailed functional characterization of these subunits is lacking in other organisms. Previously published work from our laboratory suggests that the role of these subunits in Schizosaccharomyces pombe may be different from that in S. cerevisiae. Therefore, in the present study, we have used the microarray approach to gain insights into the functions of Rpb7 in S. pombe. We found that low levels of Rpb7 affected multiple biological pathways in S. pombe. KEGG analysis of our microarray data showed that major up-regulated pathways included starch and sucrose metabolism, phagosome, endocytosis, proteasome, oxidative phosphorylation, protein processing in endoplasmic reticulum, various types on N-glycan biosynthesis and spliceosome. Interestingly, the functional annotation clustering of down-regulated genes 72 showed that the most significant cluster consisted of DNA repair genes which demonstrated that Rpb7 affects DNA repair in S. pombe cells. We validated our microarray results through real time PCR, where we confirmed the down-regulation of well-known DNA repair genes including rhp23+, rhp26+, mlh1+, def1+, rad16+ and rpb9+. To further examine the role of Rpb7 in survival of yeast cells under DNA damaging conditions, we next tested the sensitivity of rpb7+ mutant strain in the presence of different DNA damaging agents like UV, MMS and 4-NQO and found that the rpb7+ mutant strain was highly sensitive towards these agents. Taken together, our microarray data shows the role of Rpb7 in important biological pathways, specifically affecting DNA repair processes.

197 Generation of proline accumulating wine yeast strains using CRISPR/Cas9 for enhanced stress protection. Tom Lang, Michelle Walker, Vladimir Jiranek Department of Wine and Food Science, University of Adelaide, Adelaide, South Australia. Yeasts are subject to several physiological stresses during the course of a wine fermentation. These include hyperosmotic stress at the start from the high sugar content of grape must and as sugar is catabolised, stresses associated with increasing ethanol. The yeasts’ ability to adapt and successfully complete fermentation is critical in wine production and for quality. Yeast differ in their stress response to plants, accumulating trehalose and glycerol rather than proline. In plants, proline acts as an osmolyte and reactive oxygen species scavenger, amongst other functions, and thus has the potential to alleviate fermentation stress in winemaking yeasts. Proline accumulation has been studied in Saccharomyces cerevisiae baker’s yeast. Three separate missense mutants identified in the PRO1 encoded gamma-glutamyl kinase, result in the intracellular accumulation of proline [1, 2]. These proline accumulating mutants showed increased resistance to L-azetidine carboxylic acid (AZC), a toxic proline analogue, as well as baking related stresses: freeze-thawing and high sucrose content. Here we report on the use of CRISPR/cas9 editing to introduce proline accumulating mutations into EC1118, a commonly used S. cerevisiae winemaking strain. Some of these mutants displayed a large increase in resistance to AZC, indicating an accumulation of intracellular proline. Further analysis of these strains will determine whether proline accumulation leads to improved performance or viability under ‘wine-like’ fermentation conditions.

1. Tsolmonbaatar, A., et al., Isolation of baker's yeast mutants with proline accumulation that showed enhanced tolerance to baking-associated stresses. International Journal of Food Microbiology, 2016. 238: p. 233-240. 2. Sasano, Y., et al., Proline accumulation in baker's yeast enhances high-sucrose stress tolerance and fermentation ability in sweet dough. International Journal of Food Microbiology, 2012. 152(1-2): p. 40-43.

198 Phosphorylation of the DExD/H box RNA helicase Dhh1 affects Ste12 expression and yeast filamentous growth. Eun Ji Lee, Dae Hee Jung, Jin Mi Kim* Department of Microbiology & Molecular Biology, College of Biological Sciences and Biotechnology, Chungnam National University, Daejeon, KR. Yeast pseudohyphal growth is a stress-responsive transition which is relevant to the virulence in pathogenic fungi. Pseudohyphal growth is regulated by the filamentous growth (FG)-specific MAPK cascade and the cAMP-PKA pathway. Nitrogen deprivation activates a MAP kinase cascade which has the transcription factor Ste12 as its final target. Ste12 was shown to regulate mating and invasive/pseudohyphal growth in Saccharomyces cerevisiae. The P-body component Dhh1 has been shown to be required for pseudohyphal growth.1 During the yeast-to-hyphal transition, Ste12 expression is up-regulated at the translational level and this regulation requires Dhh1. In this study, we analyzed the mutations in the ATPase and RNA- binding domain of Dhh1 and showed their phenotypes in pseudohyphal growth. To identify the signaling networks regulating Dhh1 in pseudohyphal growth, the phospho-deficient and phospho-mimic mutations were introduced at amino acid residues, Thr10, Ser14 and Thr16 of Dhh1. Phosphorylation of Dhh1 appears to decrease pseudohyphal growth and Ste12 expression. P-body localizations of the wild type and mutant Dhh1 during pseudohyphal growth are under investigation. Our results would reveal the critical roles of Dhh1 in P-body accumulation and filamentous growth during stress conditions.

Reference 1. Park, Y. U., Hur, H., Ka, M., and Kim, J. (2006). Eukaryotic Cell, 5(12), 2120-2127.

199 The Nrd1-Nab3-Sen1 transcriptional termination complex is a candidate target of regulatory lysine methylation by Set1. K. Lee1, F. Chowdhury1, K. Biggar2, M. Meneghini1 1) Department of Molecular Genetics, University of Toronto, Toronto, Ontario, CA; 2) Department of Biology, Carleton University, Ottawa, Ontario, CA. MLL (mixed lineage leukemia) is a highly conserved histone lysine methyltransferase that targets histone H3 lysine 4. As its name implies, mutations in MLL cause leukemia, a devastating cancer of the blood. Remarkably, despite the crucial role of MLL in human health and animal development, H3K4 remains the only known enzymatic target of the MLL family. The harbinger exception to this is provided by yeast, in which the Dam1 kinetochore protein is methylated in a SET1-dependent manner. The budding yeast homolog of MLL is called Set1. We previously showed that Set1 can regulate the Nrd1-Nab3-Sen1 (NNS) transcription termination complex in a H3K4me-independent manner. An explanation for our results posited that NNS itself might be a target of inhibitory methylation by Set1. Using LC-MS/MS, we identified 9 lysines distributed amongst Nrd1, 73

Nab3, and Sen1 that were mono-, di-, or trimethylated. Mapping these methylation sites onto the proteins revealed that many of them reside in domains mediating important regulatory functions such as RNA-binding, RNAPII CTD binding, and more. To date, we have optimized methods for measurement of Nrd1-K148me and K171me, as well as of Nab3-K363me. Interestingly, both of the Nrd1 methylations appear to be completely dependent on SET1, while the abundance of Nab3-K363me1 is reduced by approximately 50% in set1∆cells. These findings suggest that Set1 is indeed responsible for Nrd1 methylation on K148 and K171 and that Set1 either acts redundantly with another lysine methyltransferase to accomplish Nab3-K363me or indirectly impacts Nab3-K363me. Nab3-K363 is monomethylated (Nab3-K363me1) and represented an interesting target of methylation because it resides within the RNA-binding RRM domain. Co-crystal structural studies have previously shown that this lysine residue, in fact, makes contact with the RNA backbone. Cells harboring a Nab3 lysine-363 to arginine (Nab3-K363R) amino acid substitution exhibited a profound growth defect revealing a crucial role for Nab3-K363 and suggesting that methylation of this residue may impact Nab3 RNA binding. We uncovered evidence that Set1 directly methylates Nrd1 potentially representing the first-ever identified enzymatic targets of the Set1/MLL superfamily outside of H3K4 or Dam1. Moreover, our findings suggest that Set1 controls at least some of the additional NNS methylations we identified and that these methylations may regulate NNS function through novel mechanistic ways.

200 Yeast Npl3 regulates TERRA transcription in senescing, telomerase-null cells. Julia Lee-Soety, Corinne Merlino, Jennifer McCann, Mark Tingey Department of Biology, Saint Joesph's University, Philadelphia, PA. Telomere maintenance is critical to genome stability. Telomerase-deleted yeast cells (tlc1) undergo replicative senescence and, along with telomere erosion, express non-coding telomere-derived RNA (TERRA). One function of TERRA may be to recruit essential protective proteins to the chromosome ends and prevent the initiation of DNA damage signals. The loss of function in some of these proteins synergistically worsens the tlc1 phenotypes. We are interested in understanding how Npl3, a multi-faceted RNA processing protein, is involved to prevent the faster of senescence of tlc1 mutants. We have measured significantly higher levels of TERRA by qRT-PCR and observed increased dBroccoli-tagged fluorescent TERRA in tlc1 npl3 double mutants compared to tlc1 cells. The increases in TERRA expression is correlated to the rate of senescence. These results suggest that Npl3 may help regulate TERRA expression as telomeres shorten, and we further explored the mechanism of this regulation. To determine if Npl3 may be involved in TERRA transcription elongation, we looked for accumulation of RNA:DNA hybrids (R-loops). Indeed, in the tlc1 npl3 cells, the levels of R-loops were significantly increased compared to tlc1 mutants. More interestingly, R-loop signals colocalized with those of dBroccoli-tagged TERRA at a significantly greater level, especially in the senescent double mutants compared to the tlc1 cells. Thus, Npl3 appears to be critical during the synthesis of TERRA, and the lack of Npl3 causes transcription stall, TERRA R-loop accumulation, and ultimately genome instability. To test this hypothesis further, we overexpressed Sub2 in our cells to see if the TREX complex component and RNA-dependent ATPase/ helicase is able to rescue the npl3 phenotype. Partial rescue of accelerated senescence in tlc1 npl3 cells and of sensitivity to DNA-damage drugs in npl3 suggest that Npl3 may participate in the maturation of TERRA during transcription. The completion of TERRA synthesis may be required for the cell to maintain chromosomal integrity and limit DNA damage responses.

201 A yeast model of CREST proteinopathy: PBP1/ATXN2 modifies CREST aggregation and toxicity. Sangeun Park, Sei-Kyoung Park, Susan W. Liebman Department of Pharmoccology, Univ Nevada, Reno, Reno, NV. Mutations in an increasing number of human genes have been found to cause familial neurodegenerative disease. Proteins encoded by these genes are often soluble in healthy individuals, but form insoluble amyloid-like aggregates that seed further aggregation in the neurons of patients with disease. Yeast has proved to be useful in the study of disease-specific proteins that form prion-like aggregates. When human proteins associated with aggregation in neurodegenerative disease are expressed in yeast, they form aggregates and cause toxicity1. The discovery in yeast that deletion of the ATXN2 ortholog PBP1 reduced TDP-43 toxicity lead to the recent exciting findings that reduction in ATXN2 levels moderates neurotoxicity in ALS mouse models 2, 3. Thus the establishment of new yeast neurodegenerative disease models may lead to the identification of new risk factors for human disease as well as provide a screening platform for drug discovery. Recently, mutations in a new gene, SS18L1, have been shown to cause ALS. SS18L1 encodes the calcium-responsive transactivator (CREST) protein which is an essential component of the nBAF neuron-specific chromatin remodeling complex and is related to the yeast SWI/SNF chromatin remodeling complex4. Overexpression of CREST inhibits neurite outgrowth in cultured neurons and causes retina degeneration in transgenic Drosophila. The CREST protein contains a prion-like domain predicted to be unstructured. Here, we establish a new ALS model in yeast. We show that human CREST expressed in yeast forms largely nuclear, but also some cytoplasmic aggregates. Cytoplasmic aggregation is dramatically increased in the presence of PBP1 overexpression. Also, expression of CREST enhances flocculation (cell clumping) of the yeast cells, inhibits genomic silencing at telomeric regions and causes toxicity, which is increased by the endogenous yeast [PIN+] prion and reduced by deletion of PBP1. 1. Gitler AD, et al. 2017 Dis Model Mech 10, 499-502. 2. Sproviero W et al. 2017 Neurobiol Aging 51, 178 e1- e9. 3. Auburger G et al. 2017 Trends Neurosci 40, 507-16. 4. Chesi A et al. 2013 Nat Neurosci 16, 851-5.

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202 The Regulatory Dynamic of Transcription Initiation in Saccharomyces cerevisiae. Zhaolian Lu, Zhenguo Lin Department of Biology, Saint Louis University, Saint Louis, MO. All regulatory signals of gene transcription are ultimately integrated in the process of transcription initiation in the core promoters. Identification the accurate locations and activities of transcription start sites (TSSs) and core promoters are essential to gain fundamental insights into regulatory mechanisms of transcription initiation. To achieve this goal, we performed non-Amplified non-Tagging Illumina Cap Analysis of Gene Expression (nAnT-iCAGE) and identified genome-wide TSSs in the model organism Saccharomyces cerevisiae across nine growth conditions. Based on integrative analysis of 337 million uniquely mapped CAGE tags, we found that transcription initiation is pervasive in S. cerevisiae. Significant transcription initiation activities were detected at ~1 million sites. Our high-resolution of TSS profiling unraveled the presence of 11,462 core promoters from 5,954 protein-coding genes. Over 50% of genes have at least two core promoters that generate distinct transcript isoforms. Alternative core promoter usage across different growth conditions is prevalent. We also identified multiple novel core promoter elements that are potentially important for transcriptional regulation and valuable for de novo prediction of eukaryotic core promoters. In summary, our work generated the most comprehensive global atlas of transcription initiation and core promoters in the model organism S. cerevisiae, improved the yeast genome annotation and revealed a highly dynamic nature of transcription initiation in yeast.

203 Translational control of lipid synthesis in the cell cycle. Nairita Maitra, Clara Kjerfve, Michael Polymenis Biochemistry and Biophysics, Texas A & M University, College Station, TX. Cell growth is usually balanced with cell division, to ensure that the subsequent generations of cells do not become progressively larger or smaller. Despite its importance, the question of how cells coordinate their growth with their division remains unresolved. Inhibiting Fas1p (beta subunit of fatty acid synthase) arrests the cells in mitosis with abnormally shaped nuclei, suggesting that de novo fatty acid synthesis is necessary for successful passage through mitosis and for remodeling of the nuclear envelope. Recent genome-wide studies from our lab showed an increase in the translational efficiency of mRNAs encoding key regulators of fatty acid synthesis late in the cell cycle. We showed that Fas1p protein oscillates during the cell cycle and peaks in the G2/M phase. We propose that translational control of fatty acid synthesis couples cell growth with nuclear division. FAS1 has two upstream open reading frames (uORFs), 279 and 141 nucleotides upstream of the main open reading frame. To test if the uORFs control the translational efficiency of FAS1, we mutated them and found that in poor nutrients, when the ribosome content of the cell is low, Fas1p levels are doubled, and the nucleus is abnormally shaped. We will present additional evidence for the role of translational control of fatty acid synthesis in the cell cycle and nuclear envelope morphology. Our results suggest that fatty acid synthesis serves as a conduit to link protein synthesis and cell growth with a morphological landmark of cell division, such as nuclear envelope remodeling in mitosis.

204 Adjacent gene co-regulation (AGC) as a strategy for transcriptional control and coupling. M.A. McAlear Dept Molecular Biol & Biochem, Wesleyan Univ, Middletown, CT. Regulons are sets of transcriptionally co-regulated genes that typically function in a common metabolic pathway. Two of the largest, and most consistently regulated gene sets in budding yeast are the ribosomal protein (RP) and the ribosome and rRNA biosynthesis (RRB, also known as ribi) regulons, and together they contain over 400 genes. It was previously discovered that an unusually large fraction (~ 15%) of the members of the two respective regulons exist on the chromosomes as immediately adjacent gene pairs. These paired sets of genes exist in all three tandem, divergent and convergent orientations, and the adjacent gene pairs exhibit tighter transcriptional coupling that the singleton genes of the same regulon. Moreover, in some situations the promoter sequences of one member of the pair have been shown to control the expression of the neighboring gene from thousands of base pairs away. We have dissected the cisand transelements that mediate AGC, and they include promoter consensus motifs, intergenic DNA contributors, and regulators of chromatin structure. Genome-wide analyses have revealed that pronounced AGC exists across varied yeast species, and across numerous, disparate regulons. These findings suggest that AGC may play an important in the evolution of new transcriptional circuits, akin to the operon strategy of prokaryotes.

205 Spt5: Characterizing the roles of the highly conserved KOW domains in S. Cerevisiae. Zachary Morton, Michael Doody, Nancy Sanchez, Jinhua Fu, Grant Hartzog Molecular, Cell, Developmental Biology, University of California, Santa Cruz, santa cruz, CA. Spt5 is a universally conserved eukaryotic transcription factor that associates directly with the RNA Pol II elongation complex. Every organism on earth contains some form of Spt5, and Spt5 is essential for life in all organisms, suggesting it plays an ancient, core role in transcription. Several results implicate Spt5 in overcoming nucleosomal barriers to transcription, recruitment of elongation factors to RNA Pol II, and pre-mRNA processing. For example, spt5 mutants exhibit transcriptional defects such as cryptic initiation, improper polyadenylation site selection, splicing defects and decreased transcription rate and processivity. Despite its central role in gene expression, the mechanisms by which Spt5 exerts its diverse functions are poorly understood. Spt5 is a multi-domain protein with an acidic N-terminus, 5 central KOW domains that lie on the surface of Pol II, and a Carboxy-terminal Repeat region (CTR) similar to Pol II’s CTD. To study Spt5’s role in chromatin regulation, we selected spt5 mutations that confer a cryptic transcription phenotype. These mutations alter residues in a previously unstudied region between Spt5’s acidic and NGN domains, and in several KOW domains. To further study Spt5’s mechanism of action, I purified recombinant fragments of individual Spt5 KOW domains and performed protein 75 affinity chromatography with yeast extracts. Silver stain analysis suggests a different subset of proteins binds each KOW domain. I will identify those proteins that directly interact with Spt5’s KOW domains using MUDPIT mass spectrometry. A similar approach will be taken to identify proteins that interact with the region between Spt5’s acidic and NGN domains. Identification of proteins that interact with individual Spt5 domains will further elucidate the functions of Spt5’s domains, the proteins they interact with and their role(s) in transcriptional elongation.

206 Fine-tuning the stress response of Saccharomyces cerevisiae using CRISPR interference technology. V. Mukherjee, E. Cámara, I. Trollmann, L. Olsson, Y. Nygård Division of Industrial Biotechnology, Biology and Biological Engineering, Chalmers University of Technology, Göteborg, SE. Efficient biochemical conversion of renewable carbon sources is crucial for the transition into an entirely renewable energy system and a resource-efficient society. However, the substitution of fossil based biochemical with its renewable counterpart requires the production to be significantly more efficient and price competitive. Production of second-generation biochemicals (made from lignocellulosic biomass) is challenging due to presence of inhibitors in lignocellulose hydrolysate. Weak acids, furans and phenolic compounds that are formed or released during hydrolysis of biomass are toxic for the producing cells and leads to suboptimal yield and productivity obtained during fermentation. Numerous attempts have been reported to improve the stress tolerance of Saccharomyces cerevisiae by different bioengineering strategies such as deletion/overexpression of genes. However, the inability to achieve a fine balance of the transcriptional expression of the target and the ancillary gene(s) is one of the major factors that impedes the efficiency of many of these strategies. In this project, we apply CRISPR interference (CRISPRi) technology to investigate the potential of fine-tuning the expression of genes that are related to the stress regulation. CRISPRi is a genetic perturbation technique that allows sequence-specific repression or activation of gene expression, achieved by a catalytically inactive Cas9 protein fused to a repressor or activator, which can be targeted to any genetic loci using a sgRNA. Strains with altered regulation will be screened for inhibitor tolerance. Furthermore, transcriptomics analysis of tolerant mutants will be conducted to link superior phenotypes to the transcriptomic landscape. Subsequently, this novel information will be used as a resource to accelerate the design-build-test- learn cycle used for developing industrial yeast strains for efficient conversion of lignocellulosic hydrolysate. Here, we will show data on a methodology that we have developed for studying hydrolysate tolerance, adaptation and ethanol production capacity at microscale, directly in lignocellulosic hydrolysates.

207 The amino- and carboxy-terminal domains of Cth2 protein differentially promote targeted mRNA decay and translational repression in iron deficiency. L. Ramos-Alonso1, A.M. Romero1, M.A. Soler1,2, A. Perea-García1,2, P. Alepuz2, S. Puig1, M.T. Martínez-Pastor2 1) Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), Paterna, Valencia, Spain; 2) Departamento de Bioquímica y Biología Molecular, Universitat de València, Burjassot, Valencia, Spain. Iron is an essential micronutrient and a valuable cofactor for its redox properties, although its bioavailability is frequently limited. Under iron-deficient conditions, Saccharomyces cerevisiae activates the transcription of CTH2, a gene part of the called iron regulon. Cth2 specifically interacts through two Cx8Cx5Cx3H tandem zinc-fingers (TZF) with adenosine/uridine-rich elements (AREs) within the 3’-untranslated region of multiple mRNAs to promote their degradation. Cth2 autoregulates its own ARE-containing mRNA and many transcripts usually related to non-essential iron-consuming pathways. In this study, we performed translation efficiency measurements and polysome profile analyses to demonstrate that, in addition to increase mRNA decay, Cth2 represses the translation of multiple mRNAs in response to iron deficiency. Both Cth2 TZFs and AREs within the target mRNAs are essential to enhance decay as well as for translational repression. A structure-function analysis shows that Cth2 represses translation through both its amino and carboxy-terminal regions, but stimulates mRNA decay only through its amino-terminal domain. Importantly, yeast cells lacking Cth2 carboxy-terminal region display altered levels of multiple iron-containing proteins and a growth defect in iron-depleted conditions. Cth2 post-transcriptional regulation and translational repression facilitate a metabolic remodeling of the cellular iron-dependent pathways to optimize iron utilization.

208 Applying high-throughput genome engineering for industrial strain optimization. Amanda Reider Apel Zymergen Inc., Emeryville, CA. At Zymergen, we have developed a platform that enables bioengineering of microorganisms for the manufacture of a variety of chemicals and novel materials with unprecedented flexibility, efficiency, and reliability. Our platform integrates several core technologies, including: custom software, high throughput laboratory automation, machine learning algorithms, and genome editing tools. We have developed custom scientific computing tools for specifying and tracking the creation of designer microorganisms as well as predicting fermentation performance. High throughput laboratory automation enables more robust and predictable DNA assembly, microbial gene editing, and high throughput assays with exceptional precision for the accurate measurement of desired fermentation metrics. Additionally, throughput allows us to test thousands of design ideas across many library types, in parallel, to identify the ideas and changes that are beneficial to desired phenotypes. Our machine learning algorithms enable efficient navigation of the immensely vast biological search space to find off-pathway targets that further enhances phenotypes. We are applying this modular and iterative approach to engineer a diverse set of microorganisms with improved performance for desired traits.

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209 Natural variation in the fitness effects of high-copy number gene expression in wild isolates of Saccharomyces cerevisiae. D. Robinson1, R. Cai2, A. Gasch2 1) Microbiology Doctoral Training Program, University of Wisconsin-Madison, Madison, WI; 2) Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI. Organisms must respond to external environmental changes to survive in nature. One way organisms cope with these changes in environment is to alter gene expression to increase the fitness of the organism. Gene expression can be altered in numerous ways including changes in gene dosage. However, gene dosage could have different effects on fitness in different genetic backgrounds. Since many organisms use changes in gene dosage to evolve when faced with environmental changes, understanding the extent genetic background contributes to fitness is important. I am examining the fitness consequences of gene overexpression across different lineages of S. cerevisiae using the molecular barcoded yeast open reading frame (MoBY- ORF 2.0) high copy number plasmid library. Based on preliminary results, I have found similarities as well as strain-specific differences in the fitness effects of gene overexpression. It is possible that these fitness effects of gene overexpression could be explained by the underlying variability in the strain’s native gene expression. For this reason, I have conducted RNA sequencing (RNA-Seq) to examine transcript abundance across 20 wild S. cerevisiae strains. I am using this data to look for patterns in gene expression across these strains to gain insight into the physiology of these strains. In the end, I hope to use the results of my experiments test the relationship between variable fitness effects and variable expression of underlying genes across strains.

210 Do nucleosomes carry epigenetic information? D. Saxton, J. Rine Molecular and Cell Biology, University of California Berkeley, Berkeley, CA. Epigenetic information is defined as a phenotypic identity that is carried independently of DNA sequence. Such phenotypes have been frequently associated with specific chromatin states, raising the idea that histone modifications can transmit epigenetic information. Nucleosomes, or more precisely H3-H4 tetramers with their covalent modifications, are reported to be segregated randomly between daughter chromatids during DNA replication. Therefore, if histone modifications carry epigenetic information, we predict that rare events would occur in which all parental marked tetramers segregate to one of the two daughter chromatids, leaving the other with a loss of the chromatin state. Transient losses of heterochromatic silencing at HML occurs in 10-3 cell divisions. Random segregation of parental H3-H4 tetramers, with their associated Sir proteins, would predict that the frequency of silencing-loss events would increase as the shorter deletions of HML. However, by reducing the number of nucleosomes within HML from its native 22 nucleosomes down to 7, we observed no loss of heterochromatin stability, challenging the random segregation of parental H3-H4 tetramers at the replication fork. In the course of these experiments, we discovered an unexpected oscillation of silencing stability as a function of the length of DNA between two nucleosome free regions within HML. I will discuss the impact of mutations that result in a biased inheritance of parental nucleosomes.

211 Live cell analysis of Imd2 expression suggests post-transcriptional regulation in response to intracellular purine levels. E. Schwotzer1, K. Sundling1, M. McClean2, D. Brow1 1) Biomolecular Chemistry, University of Wisconsin- Madison, Madison, WI; 2) Biomedical Engineering, University of Wisconsin- Madison, Madison, WI. IMP dehydrogenase (IMPDH) catalyzes the first committed step of GTP biosynthesis from the purine nucleotide precursor inosine monophosphate, and thus helps regulate the balance of ATP and GTP. Mutations in the human IMPDH1 gene cause autosomal dominant retinitis pigmentosa type 10 (adRP10) and Leber congenital amaurosis type 11 (LCA11), progressive and congenital blindness disorders. Evidence suggests that IMPDH is an RNA-binding protein and the disease mutations are located in its putative nucleic acid-binding domain, not the catalytic domain. In S. cerevisiae, IMPDH is produced from the IMD2, IMD3, and IMD4 genes. Imd2 is uniquely resistant to the IMPDH inhibitor mycophenolic acid (MPA) and its transcription is induced by MPA. Imd2 transcription is regulated by intracellular GTP levels through the use of alternative transcription start sites (TSS) that elicit or bypass transcription termination in the 5’-UTR and early ORF. When GTP is replete, TATA-proximal TSS are used and the transcript contains a Sen1-dependent terminator resulting in attenuated transcripts. When GTP is depleted by MPA treatment, TATA-distal TSS are used, bypassing the terminator and producing full length mRNA. We showed previously that simultaneous treatment with MPA and guanine results in the predominant use of upstream, non-productive TSS, as expected since guanine can be directly incorporated into GMP by HGPRT. Yet live cell microfluidic analysis of Imd2-GFP protein expression reveals that MPA+guanine treatment increases Imd2 protein level beyond that seen by MPA alone. These paradoxical results suggest post-transcriptional regulation of Imd2 expression occurs, possibly in response to ATP level. Intriguingly, Imd2-GFP levels cycle when cells become confluent, and the onset of cycling is delayed by exogenous adenine. Preliminary data also suggest that an adRP10 mutation in IMD2 disrupts the regulation of Imd2 expression in response to both GTP and ATP levels. We are further exploring the complex mechanism of regulation of this key enzyme in nucleotide metabolism.

212 A high-throughput mutational scan of an intrinsically disordered acidic transcriptional activation domain. Max Staller1, Alex Holehouse2, Rohit Pappu2, Barak Cohen1 1) Edison Family Center for Genome Sciences and Systems Biology and Department of Genetics, Washington University in St. Louis School of Medicine, Saint Louis, MO; 2) Center for Biological Systems Engineering and Department of Biomedical Engineering, Washington University in St. Louis, Saint Louis, MO. 77

Transcription factors (TFs) activate gene expression by binding the genome with DNA binding domains (DBDs) and recruiting coactivators with activation domains. While DBDs are phylogenetically conserved, well structured, and bind to related DNA sequences, activation domains are poorly conserved, intrinsically disordered and bind structurally diverse coactivators. These features have made it difficult to identify the amino acid composition features that define activation domains. We have developed a method to measure the activities of thousands of transcriptional activation domains in parallel. We deployed a rational mutagenesis scheme that deconvolves the function of four activation domain sequence features — acidity, hydrophobicity, intrinsic disorder, and short linear motifs — by quantifying the activity of thousands of variants in vivo and simulating their conformational ensembles using an all-atom Monte Carlo approach. Our results with a canonical activation domain from the Saccharomyces cerevisiae transcription factor Gcn4, reconcile existing observations into a unified model of its function: the intrinsic disorder and acidic residues keep two hydrophobic motifs from driving collapse. Instead, the most active variants keep their aromatic residues exposed to the solvent.

In addition, during amino acid starvation, the specific activity of the Gcn4 activation domain increases, a phenomenon we call induction. This induction is in additional to well-established mechanisms of translational induction during amino acid starvation. Mutations that increase activity decrease induction. We propose that Gcn4 activation domain has been selected to have low intrinsic activity and high induction. Our results illustrate how the sequence-to-function relationship of TF activation domains can be revealed by high-throughput rational mutagenesis.

213 A Study of the Functional Complementation Between the ELL and EAF Transcription Elongation Factor Homologs of Human and Schizosaccharomyces pombe. K. Sweta, P. Dabas, N. Sharma University School of Biotechnology, G.G.S. Indraprastha University,Sector 16C, Dwarka ,New Delhi,India. Expression of eukaryotic genes is a highly regulated process. Transcription is the first step of gene expression and it is well accepted that transcription is primarily regulated at the pre-initiation and initiation steps. However, research over the last few years has provided evidence that elongation of transcription is also another key step for controlling expression of genes. A plethora of proteins are involved in regulating the process of transcriptional elongation. ELL (Eleven-nineteen Lysine-rich Leukaemia) and EAF (ELL associated factor) family of proteins are involved in regulating RNA polymerase II pausing during transcriptional elongation in vitro and also a part of different transcriptional elongation complexes in vivo. Homologues of these proteins have been identified in different organisms, including Schizosaccharomyces pombe, Drosophila, C. elegans and humans. However, single homologues of these proteins are present in S. pombe, while multiple human homologs are present. Our previous work on Schizosaccharomyces pombe ELL (SpELL) and EAF (SpEAF) have revealed that S. pombe cells lacking ell1+ or eaf1+ exhibit slow growth under optimum growth conditions and were also sensitive to the various DNA damaging agents. We also identified the amino and carboxyl terminal domain of ELL and EAF respectively to be important for the functioning of these two proteins in S. pombe. So, in the present study we examined the functional complementation of ell1+ or eaf1+ deletion associated phenotype by the human ELL or EAF homologs in S. pombe. Our results demonstrate that human ELL can partially complement the ell1 deletion associated slow growth phenotype of S. pombe cells under normal optimum growth conditions. Similar result was obtained when the rescue of viability of ell deleted S. pombe cells was tested in the presence of DNA damaging conditions. Furthermore, 6-Azauracil sensitivity of ell null mutant was also partially rescued by human ELL.Interestingly, our results show that in contrast to human ELL, human EAF homologs failed to complement the phenotypes observed in the absence of eaf in S. pombe. Truncation mutants of human ELL have also been generated to identify the domain responsible for the partial rescue of SpELL deletion phenotypes. These studies have identified the carboxyl terminal domain of human ELL to be responsible for partial complementation of all the phenotypes associated with ell deletion.Yeast two-hybrid analysis has been used to test the interaction between SpELL and human EAF, as well as between SpEAF and human ELL. All these results taken together suggest that while some of the functions of ELL may be conserved across organisms, but EAF may perform species-specific functions.

214 Pop2 and Ccr4 have distinct and overlapping roles in the translational repression of LRG1 mRNA. A.L. Valderrama1,2, D.L Duy1, Y Suda1,3, K Irie1 1) Department of Molecular Cell Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2) School of Integrative and Global Majors (SIGMA), University of Tsukuba, Ibaraki, Japan; 3) Live Cell Super-resolution Imaging Research Team, RIKEN Center for Advanced Photonics, Wako, Saitama, Japan. Pop2 and Ccr4 are the major subunits of the Ccr4-Not complex involved in mRNA poly(A) tail shortening in Saccharomyces cerevisiae. We have previously shown that both Pop2 and Ccr4 negatively regulate the expression of LRG1 mRNA, encoding GTPase-activating protein for Rho1. LRG1 deletion suppresses the temperature-sensitive growth defect of the pop2∆ and ccr4∆ mutants. We have also shown that the slow growth of the pop2∆ and ccr4∆ is repressed by deleting another gene, PBP1, encoding poly(A)-binding protein (Pab1)-binding protein 1; however, the underlying mechanism remains unclear. In this study, we investigated how pop2∆, ccr4∆, and pbp1∆ mutations influence the length of poly(A) tail and LRG1 mRNA and protein levels during long-term cultivation. During log-phase, ccr4∆ mutant cells have maintained longer LRG1 poly(A) tail and its mRNA level was higher than those in wild-type (WT) cells. Unexpectedly, Lrg1 protein levels in ccr4∆ were comparable with that in WT. In the case of pop2∆, both the mRNA and protein levels were increased significantly in log-phase. During stationary-phase, LRG1 poly(A) tail length was still longer in ccr4∆. This time, both the pop2∆ and ccr4∆ mutant cells have maintained increased levels of mRNA and protein compared to WT. The loss of PBP1 reduced the LRG1 mRNA and protein

78 levels of both the pop2∆ and ccr4∆ mutant cells in the stationary-phase. Our results suggest that Pop2 and Ccr4 have distinct and overlapping roles in regulating LRG1 mRNA and protein levels depending on growth phase and that Pbp1 is involved in the Ccr4-Not complex-mediated regulation of mRNA stability and translation efficiency.

215 Combinatorial barcoding of a multiplex yeast two-hybrid assay. T. Ward1, M. Rich2, S. Fields2,3 1) Molecular & Cellular Biology, University of Washington, Seattle, WA; 2) Genome Sciences, University of Washington, Seattle, WA; 3) The Howard Hughes Medical Institute, Chevy Chase, MD. One-by-all assays can couple genotype to phenotype in order to reveal aspects of protein function or the effects of human genetic variation. Examining variants in two proteins at once to learn about interactions, or examining variants in a large set of genetic backgrounds, is the next step. However, this kind of experiment requires that the two types of variation be coupled in the analysis phase. With one-by-all assays, each cell contains only one variant of a single library; with assays that have two different types of variants per cell, it is not readily apparent which two variants were in the same cell after cell lysis and sequencing preparation steps. The solution to retaining cellular context for variants is combinatorial barcoding. Self-catalytic RNA molecules called ribozymes offer a unique solution to this problem. Ribozymes can execute their splicing reaction in cis or trans and allow for concatenation of sequences that are not from the same transcript. Thus, a barcode for library A and a second barcode from library B could be expressed from separate plasmids and then be spliced together, preserving the cellular context in a paired variant experiment. This ribozyme-mediated barcoding was used in a pilot yeast two-hybrid experiment where activation and DNA binding domains were barcoded as two separate libraries. Using next generation sequencing as a read out, the frequencies of barcode pairs before and after yeast-two hybrid selection provides a way to assess relative fitness of variant pairs.

216 Elucidating the effect of cultivation conditons on the production of an alternative dye replacement. Maren Wehrs1,2,3, Lukas Platz1,2, Jadie Moon1,2, Itay Budin1,2, Aindrila Mukhopadhyay1,2 1) Joint BioEnergy Institute, Emeryville, California, United States; 2) Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States; 3) Institut fuer Genetik, TU Braunschweig, Braunschweig, GER. Non-ribosomal peptides belong to a class of peptide secondary metabolites. This class includes molecules with significant medically relevant properties such as immunosuppressants, antibiotics, anticancer drugs and antiviral compounds as well as molecules with other useful properties that can be leveraged in the dye industry, such as natural pigments. However, the scarcity of natural non-ribosomal peptide production and their structural complexity impede mass production by purification from biological material or chemical synthesis. Moreover, it is often difficult to genetically manipulate the natural producers for production optimization through metabolic engineering. Here, we heterologously express the blue pigment synthase A (BpsA), a member of the non-ribosomal peptide synthase enzyme family from Streptomyces lavendulae in Saccharomyces cerevisiae for the production of the blue pigment Indigoidine. The blue pigment is formed by condensation of 2 molecules of L-Glutamine, synthesized from the TCA cycle intermediate ɑ- Ketoglutarate.

We observed strong correlation of the production efficiency of the blue pigment and the metabolic state of the yeast cell, highlighting the importance of understanding the metabolic characteristics of a given production host.

217 Thiamine (vitamin B1) starvation in Candida glabrata: making do with less. D.D. Wykoff, Emma Lang, Kathleen Selhorst, Anthony Gulotta, Genevieve Hughes, Kristin Barbour, Alison Mody, Meredith Marcotte, Danielle Sens-Castet, Christine Iosue Dept Biology, Villanova University, Villanova, PA. In Saccharomyces cerevisiae, the transcription of thiamine starvation-inducible (THI) genes is regulated by two transcription factors (ScPdc2 and ScThi2) which bind a regulator ScThi3 to upregulate ~20 thiamine biosynthetic genes. Candida glabrata, a closely-related pathogenic species, lacks a THI2 homolog, and lacks half of the biosynthetic pathway, but cells still highly upregulate ~7 biosynthetic genes during thiamine starvation. Both species require PDC2 for this upregulation. ScPDC2 allows for ScPDC1 and ScPDC5 (pyruvate decarboxylase genes) expression during growth in glucose containing medium, making it an essential gene, whereas a Cgpdc2Δ strain is viable. We have used an ADH1 promoted PDC1 gene to suppress the inviability of the S. cerevisiae strain to answer a series of questions. Do Pdc2 and Thi3 from both species function in the other species? Do the transcription factors have different promoter binding specificity and how does Thi2 influence specificity? What are the thiamine responsive DNA elements (TREs) in THI promoters?

To address these questions, we used RNA-seq of a pdc2Δ in both species to assess cross-complementation. We determine that both species’ Pdc2 proteins are required for regulation of THI genes, but CgPdc2 is not capable of complementing the essential function of ScPdc2. This work is the first high-throughput expression analysis of a Scpdc2Δ strain. Additionally, we performed a detailed analysis of THI promoters in both species. We identified two different TREs. One TRE in an evolutionarily recent promoter (CgPMU3) is 10 bp in length, and we have performed both a randomization and scanning mutagenesis to determine the requirements for this 10 bp sequence in the context of a full-length promoter. The other TRE is present in multiple, evolutionarily conserved THI promoters and is 11 bp in length. The two TREs do not share obvious sequence similarity, which is surprising. Placement of the evolutionarily conserved TRE into the CgPMU3 promoter lacking the 10 bp TRE 79 confers thiamine regulation, suggesting both TREs are functionally equivalent. Future studies will determine whether Pdc2 binds these divergent sequences. This work demonstrates that even though C. glabrata has lost some complexity in the transcriptional response to thiamine starvation, it maintains the core evolutionary transcriptional program. It also suggests that new promoters can be easily evolved de novo to be thiamine starvation-regulated.

218 A stress response that allows highly mutated eukaryotic cells to survive and proliferate. R. A. Zabinsky, J. Mares, R. She, M. Zeman, D. Jarosz Chemical and Systems Biology, Stanford University, Stanford, CA. Most mutations are deleterious. Yet many cancers and pathogens can simultaneously survive a high mutation load and harness its evolutionary potential. We use Saccharomyces cerevisiae as a model to investigate this paradox. We propagated defined independent mutator lineages, creating clonal cell populations that harbor thousands of distinct mutations. The fitness cost per mutation was high for initial mutational events, but the phenotypic consequences of later mutations were buffered. The lineages did not share any individual mutations, yet they mounted a concerted transcriptional response to increasing mutation load that is distinct from previously characterized stress responses. Inhibition of this response, which we term EMBR (eukaryotic mutation burden response) eliminated mutational buffering, selectively killing the highly mutated lineages. Homologous pathways are also perturbed in highly mutated human cancers, and their inhibition selectively blocked proliferation of tumor-derived cells. Our data establish that eukaryotic cells can mount a stress response that buffers the cost of accumulating genetic lesions, and suggest that the capacity to survive mutation burden could be targeted therapeutically.

219 Inferring the genetic architecture of expression variation from replicated high throughput allele-specific expression experiments. X. Zhang, J. Emerson Ecology and Evolutionary Biology, University of California, Irvine, Irvine, CA. Variation in gene expression contributes significantly to phenotypic variation. Consequently, natural selection targets not only protein-coding regions, but also gene regulatory elements. The genetic architecture of variation of gene regulation can be decomposed into cis variation and trans variation. Cis-regulatory variation affects the expression difference between two individuals in a non-diffusible manner (eg. a mutation on a promoter region), while trans variation affects the expression difference in a diffusible manner (eg. a coding region mutation on a transcription factor). The cis/trans contribution to expression differences between two individuals can be measured by allele-specific expression in the two individuals and their F1 hybrid. Previous experiments in strains of budding yeasts show that cis variation is the dominant source of expression differences. A popular explanation is that the larger deleterious effects of trans-mutations experience more purifying selection, and therefore are removed from the population.

Previous experiments suffer from two limitations 1) biological replication of gene expression measurement in parental and F1 hybrid strains is not sufficient to estimate over-dispersion, leading to upwardly biased estimates of cis/trans contribution. 2) The cis×trans interaction (eg. the binding of promoters and corresponding transcriptional factors) is not considered in previous models which implicitly assume that cis and trans act independently.

We measure the allele-specific expression in two strains of yeast (yps128: a woodland strain; rm11: a wine strain ) with 10 biological replicates using RNAseq. In order to eliminate the error caused by RNAseq mapping bias, we also assemble the two genomes de novo with long reads using a hybrid metassembly approach. We propose a new statistical model based on normalized allelic RNA-seq read counts in hybrid and parental strains instead of only the allelic expression ratio as in previous models.

We find a significant number of genes that show cis-trans interactions but are classified as lacking cis/trans variation or exhibiting only cis variation according to previous models, indicating that cis-trans epistatic interactions are common. This raises the possibility that hybrid incompatibility is nontrivial even between closely related strains. With a high number of biological replicates, we also explore the model's power and false discovery rate.

220 A novel genome-wide collection of strains for titratable gene expression in Saccharomyces cerevisiae. Y. Arita1, Z. Li1, D. Climie1, M. Costanzo1, D. Botstein2, R. S. Mclsaac2, A. Baryshnikova2, C. Boone1, B. Andrews1 1) Department of Molecular Genetics , University of Toronto, Toronto, ON, Canada; 2) Calico Life Sciences, South San Francisco, CA, USA. A number of genome-wide collections have been constructed for systematic genetic analysis in budding yeast, including arrays of gene deletion mutants, strains expressing temperature-sensitive alleles of essential genes, and various gene overexpression collections. However, none of these collections are ideal for systematic analysis of increased gene dosage. We constructed a collection of strains carrying promoter-replacement alleles of all yeast genes, that render genes responsive to the addition of estradiol, in a dose-dependent manner. When cells are treated with estradiol, a synthetic transcription factor composed of a DNA binding domain, estradiol receptor and VP16 activation domain binds to a modified GAL1 promoter to induce gene expression. The expression level is dependent on estradiol concentration and incubation time. We have systematically characterized the phenotypes of essential genes in our collection whose expression is controlled by estradiol-dependent promoters. We found that ~50% of strains could grow without estradiol, indicating that our estradiol- regulated promoter is not entirely shut-off in many cases. Statistical analysis revealed that the strains that could grow without inducer tended to have estradiol-regulated genes encoding low-abundance proteins. We also tested growth with different 80 concentrations of estradiol and clustered genes by colony size in response to estradiol concentration. Genes encoding proteins which are members of the same complex exhibited a similar behavior in our assays. The estradiol-dependent promoter alleles are likely to be particularly valuable for studies of the effects of gene overexpression, as our studies have identified novel genes that are toxic when overexpressed.

221 MCHM exposure affects multiple cellular pathways in yeast and metazoans. M. Ayers1, M. Perfetto1,2, X. Rong- Mullins1, S. Kirkham1, C. Nassif1, S. Wei2, J. Gallagher1 1) Department of Biology, West Virginia University, Morgantown, WV; 2) Department of Biology, University of Delaware, Newark, DE. Studies often follow chemical spill disasters as we use toxicology to estimate the effects of chemicals on humans and the environment, and the spill of the coal cleaning chemical 4-methylcylohexanemethanol (MCHM) is no exception. We incorporated multiple model organisms to explore the effects on cell biology and metazoan development. MCHM caused cell cycle arrest in treated yeast cells, but decrease in cell viability was dose-dependent, only starting to decrease marginally at the highest doses used. Our study also attempted to uniquely address concerns over how MCHM affected cells by exploring toxicology from an unbiased perspective incorporating RNA-seq data, which revealed expected and novel perturbations. These included expected changes to the pleiotropic drug response and novel changes to the inositol biosynthetic pathway. Multiple lines of evidence show that MCHM can damage cells through the production of reactive oxygen species. While this had been implicated as a result before, our data show the first direct evidence for this potentially toxic effect. In addition to the BY4741 strain-specific experiments above, we also used variation in liquid culture growth phenotypes in MCHM between the S96 and YJM789 strains to perform a QTL analysis. The only significant locus detected was on chromosome IX in a relatively wide peak of approximately 35kb near the centromere. We assayed knockouts of 25 of the genes under this QTL and found four genes whose knockouts in BY4741 improve resistance to MCHM. Furthermore, one gene knockout reduced resistance in BY4741. Further analysis of the alleles of these five genes in the parent strains is underway. Viability of HEK293 human cell line cultures and Xenopus embryos was negatively affected, and similar to yeast cells, addition of antioxidants could alleviate toxicity. Additionally, Xenopus suffered developmental defects as well as reversible paralysis. The organismal implications of changes to such a fundamental cellular pathway point to the importance of continued follow-up study of this chemical.

222 Using trained convolutional neural networks to predict fluorescent signals in unlabeled images of Saccharomyces cerevisiae. Roger Brent1, Laura Boucheron2 1) Division of Basic Sciences, FHCRC, Seattle, Washington 98109; 2) Klipsch School of Electrical and Computer Engineering, New Mexico State University, Las Cruces, New Mexico 88003. During this decade, application of machine learning based on multilayer ("deep") Neural Networks (NNs) has exploded. Much of the current excitement in use of NNs for image analysis was sparked by the demonstration by Hinton and coworkers of the superior ability of trained "deep" Convolutional Neural Networks (CNNs) to correctly label ("classify") objects (eg. cats) depicted in images (Krizhevsky et al. 2012).

Later, a number of researchers trained NNs with different (fully Convolutional) architectures to carry out a different sort of task, to "translate" between cognate images in image pairs. For example, Ronneberger et al. (2015) trained fully convolutional NNs to translate between stacks of EM images and cognate images showing boundaries between cells. The trained network could then predict cell boundaries from EM images alone. Very recently, two groups (Ounkomol et al. (2017) and Christiansen et al. (2018)) used this approach to train NNs to predict fluorescent labels corresponding to mammalian cell states (eg. apoptosis) and cell substructures (eg. nuclei) from brightfield and DIC images.

In the course of other work, we have been applying a similar approach to visualize predicted substructures in budding yeast. We will present examples of such predictions.

Success in this project might have a number of positive consequences. Ability to "visualize" structures and states with normal illumination will reduce overall light exposure and ameliorate consequences of phototoxicity and photomorbidity on normal function. For some labs, ability to "visualize" structures and states using NNs running on smartphones might have a democratizing effect, compensating for lack access to appropriately engineered strains and fluorescent microscopic equipment. In other labs, use of such NNs might free up fluorescent channels that could be used for simultaneous visualization of additional structures and processes.

Christiansen, E. M. et al. (2018) In silico labeling: predicting fluorescent labeling to unlabeled images. Cell 173, 793-803

Krizhevsky, (2012) Imagenet classification with deep convolutional neural networks. In Advances in Neural Information Processing Systems (pp. 1097-1105)

Ounkomol, C., et al. (2017) Three dimensional cross-modal inference: label free methods for subcellular structure prediction. BioRxiv, 216606

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Ronneberger, O. et al. (2015) Unet: Convolutional networks for biomedical image segmentation. In International Conference on Medical Image Computing and Computer-Assisted Intervention (pp. 234–241, Springer, 2015)

223 Chemical genomics in genetically diverse strains of Saccharomyces cerevisiae. Bede Busby1,2, Cristina Viéitez1,2, Marco Galardini2, Athanasios Typas 1, Pedro Beltrao2 1) Genome Biology, European Molecular Biology Laboratory, Heidelberg, Baden-Wurttemberg, DE; 2) European Bioinformatics Institute, European Molecular Biology Laboratory, Hinxton, Wellcome Genome Campus, Cambridge CB10 1SD, UK. Genetic functions have been investigated via chemical genomics in various species, however, these are generally limited to one strain of that particular species. Analysis of different isolates can provide orthogonal information to that provided from classical genetics, particularly for intra-species phenotypic diversity. In this study we used S. cerevisiae genome-wide deletion collections in four genetic backgrounds (S288C, Y55, YPS606, and UWOP587-2421), in order to compare their chemo-genomic profiles, across different conditions. Conditional essentiality shows little conservation across the isolates at the single gene level. However, when we extend this search to entire complexes or pathways, we find that this conservation increases. This could be explained by “switch” genes, which are non-essential in one strain, but conditionally essential in another. In addition, there is a small group of switch genes, that consistently switch across strains. We see that the same complexes are needed for survival in the same conditions within a species, but the ways to activate these complexes vary between individual strains.

224 An Expanded View of the Yeast Interactome Map. A. Desbuleux1,2,3,4,11, Y. Wang1,2,3,11, A. Yadav1,2,3,11, D-K. Kim1,5,6,11, T. Cafarelli1,2,3,11, L. Lambourne1,2,3, C. Pons1,7, I. Kovács1,8, K. Spirohn1,2,3, N. Jailkhani1,2,3, T. Hao1,2,3, P. Aloy1,7, Y. Jacob1,9, Q. Zhong1,2,3,10, B. Charloteaux1,2,3, J-C. Twizere1,4,12, D. E. Hill1,2,3, M. Calderwood1,2,3, F. Roth1,5,6,12 1) Center for Cancer Systems Biology (CCSB), Dana-Farber Cancer Institute, Boston, MA, USA; 2) Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA; 3) Department of Genetics, Harvard Medical School, Boston, MA, USA; 4) Protein Signaling and Interactions Lab, GIGA, University of Liege, Liege, Belgium; 5) Departments of Molecular Genetics and Computer Science and Donnelly Center, University of Toronto, Toronto, ON, Canada; 6) Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; 7) Joint IRB-BSC Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain; 8) Network Science Institute, Northeastern University, Boston, MA, USA; 9) GMVR Unit, Department of Virology, Institut Pasteur, Paris, France; 10) Department of Biological Sciences, Wright State University, Dayton, OH, USA; 11) Co-first author; 12) Co-senior author. Both biophysical and functional interactome maps are essential to understand molecular mechanisms and functional relationships between genes and gene products. In the past decades, multiple efforts have been made to systematically map the biophysical and functional interactomes of Saccharomyces cerevisiae. While functional interactome maps, such as the genetic interaction map, are almost complete, only ~1/6 of the estimated yeast binary protein-protein interactome had been mapped previously. To increase our knowledge of the yeast binary interactome, we expanded the map by covering for the first time all annotated protein-coding genes and by carrying out three binary interaction screens using a modified version of the yeast two-hybrid assay. We also investigated the extent to which current annotations may not have fully captured all protein-coding genes. Having posited and tested an evolutionary model by which functional genes evolve through transitory proto-genes arising through widespread translational activity in non-genic sequences, we included ~1,200 candidate proto-genes in our mapping efforts. To alleviate any assay-specific bias, we confirmed all interactions using two different reporters which identified a set of consistent interactions. After biophysical validation by orthogonal assays, we report ~2,200 interactions, ~7% of them involving proto-genes. The majority of these interactions have not been reported previously, increasing the total number of interactions from systematic efforts by ~70%. Furthermore, we similarly tested all interactions from previous binary maps and integrated the resulting interactions with our map to generate a consolidated yeast binary interaction map. This is the first time that such a comprehensive attempt has been made to compare interactions identified in different assays and in different studies using a uniform platform and to integrate them into a single reference interactome map. We show that this integrated map is comparable in size and functional enrichment to interactions identified by multiple studies in the literature over many years. Our integrated network map can now be explored to understand biological functions of various interactions, investigate relationships between connectivity of proteins, provide information on binary interaction detectability in multiple assays, and suggest functional hypotheses for proto-genes.

225 Integrative analysis of variation in gene expression regulation networks among diverse strains of S. cerevisiae. Barbara Dunn1, Rohith Srivas1, Trupti Kawli2, Lixia Jiang1, Elaine Li1, Kisurb Choe3, Jennifer Gallagher4, Michael Snyder1 1) Dept Genetics, Stanford Univ Med Sch, Stanford, CA; 2) Karius, Inc, Redwood City, CA; 3) Univ Illinois Urbana- Champaign, Dept Microbiology, Urbana, IL; 4) West Virginia University, Dept Biology, Morgantown, WV. Systems biology represents a shift in focus from how biology has been studied in the last century. The ability to identify, characterize and quantify biomolecular components to a high degree of detail on a genomic scale allows us to understand and elucidate global regulatory networks in biological systems. Integration of these datasets represents a start to the eventual goal of understanding how an entire biological system operates, and how such systems vary between genetically differing individuals within a species. The regulation of gene expression is an example of a systems biology problem, as it involves changes in chromatin structure and histone modifications, as well as coordinated action amongst trans-acting transcription factors (TF). Elucidating the extent to which variation in these regulatory mechanisms can influence gene 82 expression, and thus contribute to phenotypic variation, has until recently been limited to examining each regulatory layer in isolation or, in the case of TFs, just one or two factors. Here, we present a unified analysis of the regulatory network in five diverse strains of the yeast S. cerevisiae, including genome-wide measurements of the binding locations of 52 TFs (~25% of all TFs), chromatin accessibility and four histone modifications. We observed a broad range (between 0.1 and 0.75) across all TFs in the fraction of binding sites that differ between strains, with highly conserved and essential TFs displaying significantly lower binding variability. Regions displaying the most variation in both TF binding and chromatin state were enriched for single-nucleotide variants, and coordinated changes across multiple regulatory layers, in contrast to any single data type, were highly correlated with differences in gene expression. Our data provide a unique resource to examine the impact of sequence-level variation across multiple layers of gene regulation.

226 High-throughput screening for food carcinogen resistance genes in yeast underscores DNA damage tolerance mechanisms in conferring carcinogen resistance. Michael Fasullo1, Nick St. John 1, Julian Freedland 1, Cinzia Cera1, Henri Baldino1, Frank Doyle1, Tom Begley2 1) Nanobiosciences, College of Nanoscale Sciences and Engineering, State Univ New York Polytechnic Institute, Albany, NY; 2) RNA Institute, State Univ New York at Albany, Albany, NY. The human response to environmental carcinogens is highly variable. Genetic factors include polymorphic cytochrome P450 and DNA repair genes; however, only a few resistance genes have been described. We used budding yeast as a model organism to determine genetic susceptibility to food-associated carcinogens, including aflatoxin (AFB1) and the heterocyclic aromatic amines (HAAs), such as 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Budding yeast does not contain P450s that metabolically activate these compounds, so we introduced expression vectors that contain specific human P450 and NAT2 genes into the diploid yeast deletion collection. In yeast, either CYP1A2 or CYP1A1 activates AFB1, while both CYP1A2 and NAT2 are required for activation of IQ. To determine resistance genes, we used a high throughput approach for screening the yeast deletion library expressing specific P450 genes or expressing no P450 genes; the illumina platform was used to sequence DNA barcodes and statistical significance was determined for exactly matched barcodes. Screens for AFB1 resistance in the diploid collection expressing CYP1A2 identified 96 genes, including cell-cycle checkpoint, DNA repair, and tRNA modification genes, such as TRM9. Among Gene Ontology (GO) groups, those involved in post-replication repair and DNA damage tolerance were over-represented among AFB1 resistant genes, compared to representation in the genome at- large. Of particular importance, we observed that the CSM2/SHU functions to promote error-free template switching of AFB1- associated DNA adducts, while suppressing AFB1-associated mutations. RAD18, NTG1, FOB1, and yKU70 were identified in screens to identify IQ-resistance genes, suggesting that ssDNA at telomeres and at the rDNA locus may be hotspots for carcinogen-mediated damage. Interestingly, polymorphic alleles of RAD18 and NTG1 have been documented to be risk factors for colon cancer, and RAD18 has been found in multiple screens for carcinogen resistance. These screens provide a novel methodology for identify genes that confer resistance to P450-activated carcinogens and have elucidated mechanisms for DNA damage tolerance. Future experiments are planned to knock-down human orthologues of the yeast genes and conduct similar screens in human cell lines.

227 Modeling a Yeast Cell: Building a Functional Gene Hierarchy. D. Forster1,2, C. L. Myers3,4, B. Andrews1,2, G. Bader1,2,5, C. Boone1,2 1) The Donnelly Centre, University of Toronto, Toronto, ON, Canada; 2) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 3) Department of Computer Science and Engineering, University of Minnesota- Twin Cities, Minneapolis, MN, USA; 4) Program in Biomedical Informatics and Computational Biology, University of Minnesota- Twin Cities, Minneapolis, MN, USA; 5) Department of Computer Science, University of Toronto, Toronto, ON, Canada. The availability of large-scale molecular measurements of yeast cells has provided an opportunity to develop a broad, general model of cellular function. Data integration techniques have been used to unify large-scale data in order to extract meaningful biological insights that may have otherwise been lost when considering a single type of information. By integrating currently available large-scale data, the characteristic strengths of diverse functional information, including protein-protein interactions, gene co-expression, gene product localization, and genetic interactions can be leveraged to provide a unified description of functional organization of the yeast cell. A comprehensive model of the cell can be effectively represented as a hierarchy, where the functional relationships between genes are embedded across a range of specificities - from highly related complexes and pathways, to more broadly related biological processes and cellular components. For example, Pre3 codes for the β1 subunit of the proteasome core particle. In a functional hierarchy derived from integrated data and for sequentially reduced thresholds of functional relatedness, Pre3 can be seen to cluster tightly with other members of the core particle, at a higher level it clusters with a larger group of gene that includes all members of the proteasome (including the regulatory particle), and, at an even higher level, it clusters with members of the protein degradation biological process. In this particular example, physical interaction data provides key data for high-resolution organization of proteasome structure and genetic interaction information contextualizes the proteasome in the wider protein degradation process. Here we show the utility of a functional gene hierarchy for broadly characterizing and organizing yeast genes by reporting improvements in identifying gene-gene relationships over single information types. This integrated model of the cell identifies novel functional roles and, importantly, general functional relationships among genes and their corresponding pathways.

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228 Exploring cell-cycle gene expression using Reporter Synthetic Genetic Array analysis. Hendrikje Goettert1, Helena Friesen1, Adam Rosebrock2, Brenda Andrews1 1) Donnelly CCBR, University of Toronto, Toronto, Ontario, CA; 2) Stony Brook School of Medicine, Stony Brook, NY. We have used the Reporter Synthetic Genetic Array (R-SGA) method, which combines the SGA procedure with a dual-color reporter system, to explore cell-cycle-regulated transcription pathways in budding yeast. In R-SGA screens, a cell-cycle- regulated promoter driving GFP gene expression and a control promoter driving RFP gene expression are introduced into arrays of yeast mutants and fluorescence intensity is measured from colonies on a plate, allowing rapid assessment of the effect of thousands of genetic perturbations on promoter activity. We have generated a dataset covering 27 cell-cycle- regulated promoters, which we have analyzed to survey the effects of gene overexpression, deletion and mutation (for essential genes). We optimized a data normalization pipeline to analyze over 270,000 genetic tests to produce a dataset comprised of about 1,000 mutated or overexpressed genes that influence the activity of one or more cell-cycle-regulated promoters. The resultant dataset provides unique insights resulting from screening overexpression versus loss-of-function alleles. We reasoned that the genes encoding proteins that directly regulate promoter activity may show opposite responses to gene deletion and overexpression. Indeed, the roster of genes that influence cell-cycle-dependent promoters when both overexpressed and deleted includes known direct regulators as well as uncharacterized genes. We explored one such gene, SRL2, and uncovered its function as a repressor of G1 transcription, especially under stress conditions.

229 Growth Rate-Dependent Global Amplification of Gene Expression. D. Gresham Department of Biology, New York University, New York, NY. Regulation of cell growth rate is essential for maintaining cellular homeostasis and survival in diverse conditions. Changes in cell growth rate result in changes in the abundance of rRNA and tRNA, but the effect of cell growth rate on mRNA abundance is not known. We developed a new method for measuring absolute transcript abundances using RNA-seq, SPike in- based Absolute RNA Quantification (SPARQ), that does not assume a constant transcriptome size and applied it to Saccharomyces cerevisiae (budding yeast) cells growing at different rates using chemostats. We find that increases in cell growth rate result in increased absolute abundance of almost every transcript, with significant coordinated changes in absolute abundances among functionally related transcripts. Using RATE-seq, we find that mRNA degradation and synthesis rates increase with increased growth rate, but to differing extents, resulting in net increases in absolute abundance. We present evidence that TORC1 mediates growth rate dependent transcriptome amplification.

230 Small molecule screens in Schizosaccharomyces pombe strains that express mammalian cyclic nucleotide- metabolizing enzymes and PKA-repressed reporters. Charles S. Hoffman Biology Dept, Boston College, Chestnut Hill, MA. The Schizosaccharomyces pombe glucose/cAMP pathway regulates sexual development, growth and metabolism through the transcriptional control of a number of genes, including fbp1, which encodes fructose-1,6-bisphosphatase. This pathway is ideal for small molecule high throughput screens (HTSs) as it is not essential, thus allowing one to replace either the S. pombe adenylyl cyclase (AC) that produces cAMP or the phosphodiesterase (PDE) that hydrolyzes cAMP with genes encoding related proteins from other organisms. In addition, PKA activity can be readily assessed by growth-based or reporter-based assays carried out in 384 or 1536 well microtiter dishes. Our strain collection includes strains expressing 15 of the 21 mammalian PDE genes, all 10 of the mammalian AC genes, and both wild type and mutationally-activated forms of the human GNAS Ga that stimulates the activity of the mammalian transmembrane ACs. Strains expressing an fbp1-ura4 reporter can be used to detect PDE inhibitors by their ability to confer 5FOA-resistant growth, while strains expressing fbp1-GFP and fbp1-luciferase reporters can be used to detect AC and/or GNAS inhibitors that confer increased reporter activity. Compounds identified in these screens are cell permeable and have shown remarkable activity in mammalian cell culture assays. Prior screens for PDE inhibitors have identified PDE4 and PDE7 inhibitors that display anti-inflammatory activity in macrophage and T cells, a PDE4/7 inhibitor that induces apoptosis in CLL cells, a PDE4/8 inhibitor that elevates testosterone production by Leydig cells, and a PDE11 inhibitor that elevates cortisol production by adrenocortical cells. Our most recent HTS has been for inhibitors of GNAS or AC9, as mutationally-activated forms of GNAS are found in McCune-Albright patients, as well as in many patients with pancreatic intraductal papillary mucinous neoplasms and associated adenocarcinomas. A 100,000 compound screen identified 16 validated hit compounds that are being evaluated as to whether they act on AC9, on GNAS, or on GNAS- mediated stimulation of AC9.

231 Systematic Gene-to-Phenotype Arrays enable genome-wide mapping of diverse molecular functions across yeast collections. Philipp Jaeger1,2, Lilia Ornelas3, Cameron McElfresh4, Lily Wong5, Randy Hampton3, Trey Ideker1 1) Department of Medicine, University of California San Diego, La Jolla, CA; 2) BiocipherX Inc., San Diego, CA; 3) Division of Biological Sciences, University of California San Diego, La Jolla, CA; 4) Nanoengineering Program, University of California San Diego, La Jolla, CA; 5) Bioengineering Program, University of California San Diego, La Jolla, CA. In yeast and other microbes, systematic analysis of large mutant collections has been remarkably successful in mapping the genetic architecture of the cell. Such analyses detect alterations in growth caused by genetic mutation, typically by quantifying the sizes of mutant colonies arrayed onto agar or by counting barcode tags within a population of cells after competitive liquid growth. Although colony size and barcode readouts are conducive to screening of cellular fitness, they lack molecular resolution to characterize specific cellular events that fail to induce a growth phenotype. In contrast, optical reporters, 84 including fluorescent probes for pathway activity and tagged proteins, can measure a much larger range of phenotypic readouts but they fall short of throughput of high-density cell colony arrays. We have developed a highly parallel strategy, Systematic Gene-to-Phenotype Arrays (SGPA), to comprehensively map the genetic landscape driving any molecular phenotypes of interest (Jaeger 2018). By this approach, a novel complete yeast genetic mutant array (called SPOCK) harboring mutations for >95% of all yeast genes on a single plate is crossed with specific fluorescent reporters (Jaeger 2018; Neal 2018) and imaged on synthetic membranes at high density and contrast. Importantly, SGPA enables quantification of phenotypes that are not readily detectable in ordinary genetic analysis of cell fitness. We benchmark SGPA by examining two fundamental biological phenotypes: First we explore glucose repression, in which SGPA identifies a requirement for the Mediator complex and a unique bi-modal role for the CDK8/kinase module in transcriptional repression and activation. Second, we examine selective protein quality control, in which SGPA identifies 556 genes including most known quality control factors along with U34 tRNA modification, which acts independently of proteasomal degradation to limit misfolded protein production. Integration of SGPA with other fluorescent readouts will enable genetic dissection of a wide range of biological pathways and conditions. This versatility has far reaching implications for the utility of yeast screening in drug discovery, as large-scale discovery data sets can be generated at low cost and in short time and targeted specifically to phenotypes of interest.

PA. Jaeger, L. Ornelas, et al. (2018). Systematic Gene-to-Phenotype Arrays: A High-Throughput Technique for Molecular Phenotyping. MolCell, 69. S. Neal, PA. Jaeger, et al. (2018), The Dfm1 derlin is required for ERAD retrotranslocation of integral membrane proteins, MolCell, 69.

232 Cell growth defects triggered by the overload of processes regulating protein localization. R. Kintaka1,2, H. Moriya2, B. Andrews1, C. Boone1 1) Molecular Genetics, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, CA; 2) RCIS, Okayama University, Okayama, Japan. In eukaryotic cells, the subcellular localization of proteins is generally governed by regulatory factors such as signal recognition particles, translocators, and chaperones. If the abundance of these regulatory factors is limiting, overexpression of target proteins might exhaust the transport system, leading to a significant growth defect. However, there has been no comprehensive study of the effects of perturbing protein transport by gene overexpression. We previously constructed a series of plasmids with GFP under the control of different protein targeting sequences and measured their upper limit of overexpression in a cell by using the genetic tug of war (gTOW) method (Moriya, et al., 2006). The protein targeting sequences included a secretory signal sequence (SS), mitochondrial targeting sequence (MTS), nuclear export signal (NES), nuclear localization signal (NLS), and cytoplasmic membrane anchoring signal (CC). Each targeting sequence showed a different expression limit, with NES-GFP, SS-GFP, and MTS-GFP being highly sensitive to overexpression. To identify genes required for protein targeting, we introduced the modified GFP plasmids into the deletion and temperature- sensitive (TS) mutant strain collections using the synthetic genetic array (SGA) approach, and slow-growing mutants were identified. Our screen identified genes encoding protein loading factors, transporters and chaperones. To confirm that the defect was due to disruption of the protein transport process, we showed that overexpression of the gene identified in our screen rescued the growth defects induced by overexpression of the modified GFP. We followed up on our assays of growth defects using confocal microscopy and automated image analysis, in order to assess the morphology of subcellular structures in strains overexpressing the modified GFPs. We observed that the structure of the nuclear membrane, ER, and mitochondria changed upon overexpression of NES/NLS-GFP, SS-GFP, and MTS-GFP. These results suggest that overexpression of transport proteins can induce damage to various organelles.

233 Target identification using an integrated chemical genetic approach. S.C. Li1, S. W. Simpkins2, H. Safizadeh2,5, J. Nelson2, M. Yoshimura1, H. Kimura1, Z. Li4, Y. Yashiroda1, Y. Shichino3, S. Iwasaki3, H. Osada1, M. Yoshida1, C. L. Myers2, C. M. Boone1,4 1) RIKEN Center for Sustainable Resource Science, Wako, Japan; 2) Department of Computer Science and Engineering, University of Minnesota-Twin Cities, USA ; 3) RNA Systems Biochemistry Laboratory, RIKEN Cluster for Pioneering Research, Wako, Japan ; 4) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Canada; 5) Department of Electrical and Computer Engineering, University of Minnesota-Twin Cities, USA. We established a high throughput pipeline for chemical-genetic screening in yeast, to link bioactive compounds, including precious natural products, to their cellular targets. The pipeline involves a diagnostic set of barcoded, non-essential deletion mutants, grown in a single pool and scored for compound-specific growth responses using next-generation sequencing, enabling the rapid generation of thousands of chemical-genetic profiles. The global genetic interaction network provides a hierarchical model of cellular function and acts as a key for interpreting chemical-genetic profiles, allowing annotation of compounds to diverse biological processes. To date, we screened ~18,000 compounds from the RIKEN Natural Product Depository and other large compound collections. We identified high-confidence target biological process predictions for ~2000 compounds, determined the functional signatures of various collections, and validated target processes both at the global and process-specific level.

We expanded our target identification framework by developing new screens focused on highly conserved essential genes. 85

We generated three new drug-hypersensitive mutant collections for chemical-genetic screening: 1) a set of ~1000 temperature sensitive (TS) mutants, 2) a set of ~900 heterozygous diploid mutants (HET), and 3) a set of ~1000 strains that overexpress essential genes (MoBY-ORF). These collections were used to generate orthogonal chemical-genetic profiles. The TS mutant profiles were compared to global genetic interaction data for target prediction, while the HET and MoBY-ORF strains directly predicted precise targets. Compounds with congruent diagnostic, TS, HET, or overexpression profiles were prioritized for validation using spontaneous drug-resistant mutant analysis, which often identifies a mutation directly in the target gene.

This approach allowed us to identify precise gene targets for novel compounds. For example, NPD6433 has a precise HET interaction with FAS1 (fatty acid synthase), and a drug-resistant mutant for NPD6433 shows a non-synonymous SNP in the same gene. Supplementation with fatty acids rescues NPD6433 toxicity, so it is likely that NPD6433 directly targets FAS1. In another example, we linked NPD5728 to FRS1, the phenylalanyl tRNA synthetase. Ribosome profiling data supported inhibition of FRS1 by this compound. In conclusion, we present a robust, multifaceted pipeline for identifying true compound- target interactions using chemical genomics.

234 A toolkit for barcode-based combinatorial screening in yeast. X. Liu1,2, Z. Liu1,2, A. Dziulko1,2, D. Francois1,2, R. Morabito1,2, S. Levy2,3,4,5 1) Department of Biochemistry and Cell Biology, Stony Brook University, New York, NY; 2) Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY; 3) Joint Initiative for Metrology in Biology, Stanford, CA; 4) National Institute of Standards and Technology, Gaithersburg, MD; 5) Department of Genetics, Stanford University, Stanford, CA. High-throughput combinatorial screens have become an important tool with a wide variety of applications in basic and applied research. For example, they are commonly used to discover protein-protein and genetic interaction networks, and in protein design and engineering. Early screens, such as the yeast two-hybrid and synthetic genetic array technologies, require that combinations be generated and assayed one-at-a-time, limiting throughput and reproducibility. More recently, amplicon- sequencing-based screens have been developed, greatly improving throughput. For example, CRISPR screens introduce gRNA combinations into cells en masse and use pooled sequencing of gRNAs or associated barcodes as a readout. However, such screens have generally been limited to assaying combinations of small genetic elements that can fit on a single plasmid. Here, we developed a general combinatorial screening platform that uses tandem genomic integration of barcoded plasmids, pooled competitive growth, and double barcode sequencing to quantitatively assay genetic construct pairs. Importantly, genetic constructs can be of any size, from gRNAs to genomes, and, once barcoded, can be easily reused and mixed to form novel pairs. Barcode pairs can be generated either through sequential plasmid insertion or yeast mating, both of which reassemble a split selectable marker and can generate yeast pools containing >107 pairs. To expand the possible applications of this platform, we designed a set of split drug resistance markers (KanMX AI HygMX AI NatMXAI) that allow combinatoric screens to be performed in prototrophic yeast with various genetic backgrounds. We also generated a set of 15 plasmid libraries each containing >100,000 barcodes and two large barcoded yeast strains collections (~12,000 of both MATa and MATα) that can be used for REcombinase Directed Indexing of newly barcoded yeast strains. As a proof-of-principle, we generated a library of ~3000 double barcoded haploid and diploid yeast strains containing different pairs of auxotrophic rescue constructs (MET15, LEU2, HIS3, TRP1, LYS2), and competed cell pools in 12 different environments. We found that the plasmid size has a minimal impact on representation of a construct in our screen and fitness estimation is highly reproducible across barcode replicates and growth replicates. Additionally, this screen revealed new interactions between auxotrophic markers that likely stem from cross-talk in amino acid metabolic pathways. Current efforts are focused on standardizing barcode sequencing and improving the convenience of plasmid library construction by making barcoded plasmids GATEWAY-compatible.

235 Genome-scale protein interaction dynamics using double barcodes. Zhimin Liu1,2, Fangfei Li2,3, Xianan Liu1,2, Danielle Francois1, Sasha Levy2,4,5,6 1) Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY; 2) Laufer Center for Physical and Quantitative Biology, Stony Brook University, Stony Brook, NY; 3) Department of Applied Mathematics, Stony Brook University, Stony Brook, NY; 4) Joint Initiative for Metrology in Biology, Stanford, CA; 5) National Institute of Standards and Technology, Gaithersburg, MD; 6) Department of Genetics, Stanford University, Stanford, CA.

Protein-Protein Interactions (PPIs) are crucial to nearly all cell functions. Thus, fully characterizing the protein interaction network and its dynamics across environments is a central goal in biology. Several large-scale efforts have systematically catalogued PPIs of Saccharomyces cerevisiae in a single benign environment. However, these technologies, which assay one PPI at a time, are too low throughput to reproduce across several environments. Thus, PPIs that are specific to other environments are missing, and how the protein interactome changes across environments is largely unexplored. Here, we attempt to quantitatively assay millions of PPIs across several environments using a highly parallel Protein-Protein interaction Sequencing (PPiSeq) platform that combines the murine dihydrofolate reductase Protein fragment Complementation Assay (PCA), a double barcoding system, pooled cell growth, time-course barcode sequencing, and quantitative fitness measurements. We constructed a pooled PPiSeq library which contains ~7 million barcodes representing ~1.8 million PPIs at ~3x replication. In a benign environment and at low sequencing depths, we could accurately estimate the fitness of ~4.4 million barcodes and 86

~1.5 million PPIs. From this set, we identify 4406 high-confidence PPIs, 3265 of which have not been previously reported. Fitnesses of replicate barcodes representing the same bait-prey PPI pair are highly correlated (Pearson’s r = 0.996). Of the 4406 PPIs identified, 147 have been constructed in two directions (bait-prey and prey-bait). These two versions of the same PPI also show a high fitness correlation (Pearson’s r = 0.70). We are currently characterizing this PPiSeq library in 9 other environments and expect >60 million PPI measurements in total. This data set will likely identify new PPIs that are not present in a benign environment and reveal large-scale changes in the protein interactome across environments.

236 High-throughput phenomics for identifying concentration-dependent chemical interactions and understanding the mechanistic basis of the mixture toxicity. V. Mukherjee1,2, T. Backhaus 3, A. Blomberg2 1) Division of Industrial Biotechnology, Biology and Biological Engineering, Chalmers University of Technology, Göteborg, SE; 2) Department of Chemistry and Molecular Biology, University of Gothenburg, Lundbergslaboratoriet, Göteborg, Sweden; 3) Department of Biological and Environmental Sciences, University of Gothenburg, Göteborg, Sweden. Prevalence of mixtures of synthetic and natural chemicals in the environment is a growing concern for public health and environmental effects. Currently, most chemical legislations around the world are based on the risk assessments carried out on individual substances and theoretical estimates of combination effect. However, exposure to multi-component mixtures may stimulate unpredicted overall toxic response due to interactions in chemical mixtures, which in turn induce unpredictable adverse impacts. Therefore, it is increasingly important to implement a high-throughput experimental approach to identify and to characterize dose-responses of single and mixtures of chemicals. The final output would be to upgrade the current modeling approaches and to obtain more reliable risk assessment. In our project, we are investigating the frequency of interactions in mixtures of chemicals by employing high-throughput yeast phenomics involving high- resolution phenotyping techniques recently developed in our laboratory. Initially we are focusing on five compounds with relatively known specific mode of action. The baker’s/brewer’s yeast Saccharomyces cerevisiae and the marine yeast Debaryomyces hansenii are used in this study as the model organisms to determine the single-substance and the mixtures dose-responses of the chemicals. Thus, we examine organisms at large evolutionary distance hoping to identify generic response of relevance to a vast array of organisms. Our results clearly suggest that both synergistic and antagonistic relationships exist among the tested chemicals and some of these relationships are concentration-dependent. We are also investigating the mechanistic causes of the mixture toxicity by RNA sequencing analysis and metabolomics.

237 Functional genomic and proteomic network analyses of Samoan and Māori traditional medicine. Seeseei Molimau-Samasoni1, Victoria Woolner2, Storm Blockley-Powell2, Pieter Dorrestein3, Paul Atkinson2, Manu Caddie4, Robert Keyzers5, Andrew Munkacsi2 1) Plant and Postharvest Division, Scientific Research Organisation of Samoa, Apia, Samoa; 2) School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand; 3) Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, CA, USA; 4) Hikurangi Bioactives Limited, Ruatoria, New Zealand; 5) School of Chemistry and Physical Sciences, Victoria University of Wellington, Wellington, New Zealand. Natural products are a robust source of drug leads, and medicinal plants have been the source of natural products that are important pharmaceuticals in modern medicine. Psychotoria insularum and Kunzea ericoides are widely used in Samoan and Māori traditional medicine, respectively, for the treatment of various ailments such as inflammation and wounds. However, despite extensive use, the underlying molecular mechanisms of activity are not known. Given the multifaceted range of applications, we predicted each plant will have an epistatic mechanism of action affecting a fundamental cellular function that impacts a wide array of molecular processes. We report herein our genome-wide analyses of leaf extracts conducted in the genetic model Saccharomyces cerevisiae. For P. insularum, sensitivity of strains within the gene deletion library implicated the involvement of iron transport, which was further corroborated with rescue of these growth defects with iron supplementation, altered expression of iron transporter proteins and reduced biologically available heme. To translate results from yeast to mammalian cells, we treated primary murine macrophages with P. insularum extracts and detected an iron-dependent anti-inflammatory response, which correlated the elucidated mechanism of action and Samoan medicinal application. For K. ericoides, sensitivity of strains within the gene deletion library as well as the abundance and localization of proteins in the GFP library identified vesicular transport genes as major mediators of bioactivity. Network analyses of these genomic and proteomic datasets provide chemical genetic interactomes to monitor in bioactivity-guided fractionation of the active components that mediate the anti-inflammatory and wound-healing activities of these traditional medicines. Moreover, these analyses indicate that yeast genetics is a powerful tool to understanding the chemical biology of traditional medicines.

238 Systematic Exploration of Complex HaploInsufficiency (CHI) of Essential Genes in Saccharomyces cerevisiae. T. Nguyen1, M. Rahman2, C. Pons3, M. Costanzo1, C. Myers2, B. Andrews1, C. Boone1 1) Molecular Genetics, University of Toronto, Toronto, Ontario, CA; 2) Department of Computer Science & Engineering, University of Minnesota, Minneapolis, MN, USA; 3) Institute for Research in Biomedicine Barcelona, Barcelona, Spain. Genome-wide Association (GWA) studies have identified many disease-associated variants that explain a portion of heritability of human genetic diseases; however, a large proportion of heritability remains unexplained, an effect known as the “missing heritability problem”. A component of missing heritability can be attributed to genetic interactions (GIs), defined as an observed phenotype with characteristics not explainable by the additive phenotypic effects of mutations in the relevant genes. Systematic analysis of all possible digenic interactions in haploid Saccharomyces cerevisiae (budding yeast) revealed ~1 87 million interactions, providing a wealth of information about the interconnectivity between core biological processes and pathways. While highly informative, GIs in a haploid cell do not account for diploidy or heterozygosity which are important features of mammalian genomes. Heterozygosity in a cell can lead to Haploinsufficiency (HI), a phenomenon where having only one functional allele in a diploid cell leads to a mutant phenotype, and Complex HaploInsufficiency (CHI), which occurs when a new phenotype results from heterozygosity at two more loci in the genome. To screen for CHI in yeast, we developed an automated screening pipeline to generate diploid mutants that are heterozygous for temperature sensitive (ts) mutations, which support growth at permissive temperature, but cause cell death at high temperature, at two loci in the genome. Using colony size as a quantitative metric of cell fitness, we screened all possible essential x essential gene pairs (~1 million) and identified 1500 negative (growth is worse than expected based on single mutant phenotypes) and 900 positive (growth is better than expected) CHI pairs. The fact that CHI are rare relative to haploid GIs suggests that an additional gene copy provides a significant buffer of the effects of loss-of-function mutations in the genome. CHI pairs shared some properties with haploid GI pairs, such as single mutant or single mutant heterozygote fitness being predictive of genetic interaction degree. CHI interaction hubs were enriched for specific bioprocesses, most prominently “ribosome/translation and tRNA processing”, “signaling/stress response” and “polarity/morphogenesis”. We anticipate that further characterization of CHI in yeast will reveal new information about the functional wiring of the diploid cell.

239 High-throughput determination of pathogenic variants of Msh2 and other mismatch repair proteins. A. Ollodart, M. Dunham Genome Sciences, University of Washington, Seattle, WA. Background: Inherited mutations in various components of the DNA mismatch repair (MMR) complex lead to several types of cancer due to an increase in mutation rate in the tumor cells. Assigning cancer risk associated with mutations in these genes is an important goal in personalized medicine. Work has been done to classify pathogenic vs non-pathogenic alleles clinically; however, there continues to be a large gap of alleles which have an unknown effect on mutation rate.

Methods: Saccharomyces cerevisiae also utilize the conserved MMR complex, and are a powerful and cost-effective system in which mutation rate can easily be measured. However, even in yeast, methods for measuring mutation rate have traditionally been low throughput, limiting the utility of these assays for screening large numbers of novel variants. We established a new method that we estimate can determine the mutation rate of hundreds of alleles simultaneously using a continuous culture device, the chemostat, coupled with deep sequencing. We first focused on MMR protein Msh2, as previous work (Gammie et al 2007) allowed us to have a direct comparison of results generated by our new method to more traditional methods. Within a chemostat, an increase in the frequency of resistance to canavanine can only be due to de novo mutation within the CAN1 gene, making mutation accumulation linear over time. This lack of stochastic fluctuation allows us to multiplex mutation rate measurements in mixed cultures by using deep sequencing to count the number of canavanine resistance colonies over time vs the allele’s representation in the bulk culture.

Results: Chemostats individually inoculated with wild type, msh2Δ, and an intermediately functional msh2 allele maintained the fold change in mutation rate found by Gammie. In the pooled variants, I determined the mutation rate of 50 out of 54 alleles, with 4 falling below background. Alleles found to be non-functional in Gammie et al ‘s work were also high mutators in our pooled screen. While low mutators were naturally at low frequency in the canavanine resistant pool, they were still detected by our assay. This result indicates that wild-type alleles can be assayed within the same experiment as non- functional alleles. While we are still optimizing the method to decrease variability, we were able to accurately determine non- functional versus functional alleles of MSH2.

240 Genetic tools for mistranslation. B. Ruiz, S. Zimmerman, S. Fields Genome Sciences, Seattle, WA. Advances in sequencing technology identify growing numbers of missense mutations, but most of these have poorly understood biological consequences. To extend functional studies of missense mutations to the proteome scale, we are developing a technology called Mistranslation Mutagenesis. This technology consists of proteome-wide mistranslation to create a large number of protein variants; a functional selection that is applied to the proteome; and a read-out of the effects of the selection by mass spectrometry. Data from such an experiment are used to measure the enrichment or depletion of mistranslated peptides relative to wild-type peptides. The approach for mistranslation we use is a genetic perturbation in Saccharomyces cerevisiae. We mutate the anticodon of a tRNA to cause amino acid substitution in proteins. However, expression of such a mistranslating tRNA is challenging to regulate because tRNA promoters are often internal to the gene and are part of the mature tRNA sequence. Therefore, we indirectly control the expression of the mistranslating tRNA via genetic tools. In one, we use the yeast rapid tRNA decay pathway to conditionally degrade the mutant tRNA when the mistranslation system is not needed. In another, we use an orthogonal tRNA synthetase and tRNA from E. coli. To measure the utility of each system, we measured cell viability and determined mistranslation levels with mass spectrometric analysis. Preliminary data show that the orthogonal pair system does not produce adequate levels of mistranslation, but that conditional degradation by rapid tRNA decay produces sufficient mistranslation for functional selections.

241 Highly parallel genome variant engineering with CRISPR-Cas9. Meru J. Sadhu, Joshua S. Bloom, Laura Day, Jake J. Siegel, Sriram Kosuri, Leonid Kruglyak UCLA, Los Angeles, CA. Understanding the functional effects of DNA sequence variants is of critical importance; however, measuring these effects 88 in a high-throughput manner is a major challenge. One promising avenue is precise editing with the CRISPR-Cas9 system. We developed a CRISPR-library-based approach for highly efficient and precise genome-wide variant engineering, and showed that it works with high efficiency and accuracy in yeast cells. We used our method to examine the functional consequences of premature-termination codons (PTCs) at different locations within all annotated essential genes. We found that most PTCs were highly deleterious unless they occurred close to the 3' end of the gene and did not affect an annotated protein domain. We further discovered that some putatively essential genes are dispensable, whereas others have large dispensable regions. This approach can be used to profile the effects of large classes of variants in a high-throughput manner.

242 Machine Learning and Computer Vision Approaches for Phenotypic Profiling in Yeast. N. Sahin1,2, M. Mattiazzi Usaj2, C. Boone1,2, Q. Morris1,2,3, B. Andrews1,2 1) Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; 2) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada; 3) Department of Computer Science, University of Toronto, Toronto, ON, Canada. A powerful method to study the genotype-to-phenotype relationship is the systematic assessment of mutant phenotypes using high-content screening and automated image analysis. We have developed a combined experimental-computational pipeline for analysis of the effect of genetic perturbations on subcellular compartments in yeast. Our approach involves using Synthetic Genetic Array (SGA) analysis to introduce markers of various subcellular compartments into yeast mutant arrays to identify comprehensive lists of genes involved in the establishment and maintenance of proper subcellular morphology. Quantitative analyses of these large image datasets require computational approaches such as image recognition, feature extraction and machine learning. We describe a general single cell image analysis pipeline for the quantification of the effect of any genetic or environmental perturbation on subcellular morphology. For the identification of mutant phenotypes, the first step in the computational analysis involves mathematically learning the wild-type morphology of unperturbed cells, and subsequent classification of single cells as having either normal or abnormal morphology. Determining the percentage of cells with mutant morphology in an isogenic population thus allows us to asses the penetrance of a given genetic perturbation. We tested the performance of different outlier detection methods, both traditional machine learning as well as deep learning approaches, by comparing the detected mutant phenotypes with positive controls, and by validating the functional enrichment of high-penetrant genes. To validate the developed pipeline, we queried the S. cerevisiae genome for genes required for proper formation and maintenance of endocytic compartments. We determined the penetrance of approximately 5300 yeast mutant strains for each of the 4 screened endocytic markers. This analysis revealed that mutation of ~10% of the screened genes leads to a morphological phenotype with a penetrance of 50% or greater for at least one of the markers. Mutation of hundreds more genes, mostly connected to more distant bioprocesses, caused moderate but still significant defects in at least one of the major compartments involved in endocytosis. This type of quantitative analysis will allow for the identification of connections between biological processes, prediction of novel gene function, and generation of a clearer understanding of eukaryotic cell biology.

243 Genomic changes resulting from selective pressures of MCHM alter diverse pathways. Z.N. Sherman, A. Ravishankar, A. Pupo Meriño, J.E.G Gallagher Biology, West Virginia University, Morgantown, WV. In 2014 a chemical spill occurred in the Elk River, West Virginia releasing approximately 10,000 gallons of the coal washing chemical, 4-MCHM. The contaminated water affected aquatic organisms, local plant and animal species, and nearly 300,000 residents residing in the Charleston, WV area. In this study we selected yeast with mutations that confer resistance of BY4741 through constant exposure to MCHM. Full genome sequencing and analysis of their DNA sequences detect specific mutations in genes. Our goal is to understand how biochemical pathways were affected by MCHM exposure. Quantitative growth assays in rich and minimal media identified eight MCHM resistant strains. Mutations found in MCHM resistant strains affect proteins multiple pathways highlight the pleiotropic effects of MCHM. Further genetic analysis revealed polymorphisms between biological replicants of the selection. Identical mutations were identified between independently evolved strains. The mutations range from nonsynonymous point mutations to addition/deletion frameshift mutations ~40 base pairs long. In addition, the growth of yeast containing knockouts of candidate genes were tested on MCHM. The mutated genes were involved in various pathways including but not limited to chromosome segregation, cellular growth checkpoints, multiple drug resistance, DNA repair, cellular polarity and budding, metabolism and respiration, etc. By investigating changes in the genome necessary to alter the cellular response to MCHM resistance insights into the effects of MCHM can be extended to other organisms.

244 Towards global mapping of binary protein interactions under diverse conditions. Dayag Sheykhkarimli1,2,3, Dae- Kyum Kim1,2,3, Da Kuang1,2,3, Siyang Li1,2,3, Nozomu Yachie4, Evangelia Petsalaki5, Atina Cote2,3, Jennifer Knapp2,3, Marta Verby2,3, Marc Vidal6,7, David E. Hill6,7, Daniel Durocher1,3, Frederick P. Roth1,2,3,6,8,9 1) Department of Molecular Genetics, University of Toronto, Toronto, Ontario, CA; 2) Donnelly Centre, University of Toronto, Toronto, Ontario, Canada; 3) Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada; 4) Research Center for Advanced Science and Technology, University of Tokyo, Tokyo, Japan; 5) European Molecular Biology Laboratory, European Bioinformatics Institute (EMBL-EBI), Hinxton, UK; 6) Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, Massachusetts, USA; 7) Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA; 8) Department 89 of Computer Science, University of Toronto, Toronto, Ontario, CA; 9) Canadian Institute for Advanced Research, Toronto, ON, Canada. Maps of direct ‘binary’ interactions between proteins (protein-protein interactions or PPIs) have been vital in the modeling and understanding of cellular systems but generally provide only static maps of the global interaction landscape. While some network dynamics can be indirectly inferred from transcript- or protein-level changes, there is no method that can efficiently capture the dynamics of binary interactions, including effects from post-translational modifications and regulations, as well as from environmental changes, at proteome scale. We are engaged in mapping PPIs across multiple conditions, including interactions that are largely inaccessible for currently available methods (e.g. transient interactions or those occurring in cell- cycle arrested cells), but that are of crucial importance to our understanding of biological systems.

Recently, our lab developed the Barcode Fusion Genetics-Yeast two-hybrid (BFG-Y2H) assay, which uses Cre-mediated fusion of DNA barcodes along with high-throughput sequencing to carry out interaction mapping of >2 million protein pairs within a single experiment. Our pilot environment-dependent screen of ~70,000 protein pairs in the presence of the DNA damaging agent methyl methanesulfonate (MMS) captured previously known interactions and many novel DNA-damage specific events. We have modified BFG-Y2H for use in flow cytometry with a fluorescent reporter to uncouple interaction detection from cell growth, making the assay amenable to non-dividing (e.g. cell-cycle arrested) cells. The benchmarking results demonstrate that sensitivity and false positive rate of fBFG-Y2H (fluorescence-BFG-Y2H) is on par with those of current state-of-the-art binary protein interaction assays, including mammalian cell-based systems (e.g. LUMIER). Together with our barcoded collections of yeast and human ORFeomes, fBFG-Y2H enables genome scale dynamic maps of PPIs across diverse growth conditions.

245 Multiplexed precision genome editing with trackable genomic barcodes for quantitative genetics, strain engineering, and synthetic biology. J.D. Smith1,4, K.R. Roy1,2,3,4, S.C. Vonesch5, G. Lin5, C.S. Tu5, A.R. Lederer5, A. Chu1,6, S. Suresh1,6, M. Nguyen1,4, A Tripathi7, W.T. Burnett1,4, M.A. Morgan1,4, W. Wei1,4, R.S. Aiyar1, R.W. Davis1,4,6, V.A. Bankaitis7,8,9, J.E. Haber10, M.L. Salit2,3, R.P. St.Onge1,6, L.M. Steinmetz1,3,4,5 1) Stanford Genome Technology Center, Stanford University, Palo Alto, California, USA; 2) Genome-Scale Measurements Group, Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, Maryland, USA; 3) Joint Initiative for Metrology in Biology, Stanford, California, USA; 4) Department of Genetics, Stanford University School of Medicine, Stanford, California, USA; 5) European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany; 6) Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA; 7) Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, Texas, USA; 8) Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas, USA; 9) Department of Chemistry, Texas A&M University, College Station, Texas, USA; 10) Rosenstiel Basic Medical Sciences Research Center and Department of Biology, Brandeis University, Waltham, Massachusetts, USA. Our understanding of how genotype controls phenotype is limited by the scale at which we can precisely alter the genome and assess phenotypic consequences of each perturbation. Here we describe a CRISPR/Cas9-based method for multiplexed accurate genome editing with short, trackable, integrated cellular barcodes (MAGESTIC) in S. cerevisiae. MAGESTIC uses array- synthesized guide-donor oligos for plasmid-based high-throughput editing and features genomic barcode integration to prevent plasmid barcode loss and to enable robust phenotyping. We demonstrate that editing efficiency can be increased >5- fold by recruiting donor DNA to the site of breaks using the LexA-Fkh1p fusion protein. We performed saturation editing of the essential gene SEC14 and identified amino acids critical for chemical inhibition of lipid signaling. We also constructed thousands of natural genetic variants, characterized guide mismatch tolerance at the genome-scale, and ascertained that cryptic Pol III termination elements substantially reduce guide efficacy. MAGESTIC will be broadly useful to uncover the genetic basis of phenotypes in yeast and engineer strains for improved traits.

246 Identification of novel genetic elements for improving acetic acid tolerance in an industrial yeast strain for second-generation bioethanol production. M. Stojiljkovic, M.R. Foulquié Moreno, J.M. Thevelein Center for Microbiology, KU Leuven - VIB, Heverlee, BE. The main requirements for conversion of biomass into second-generation bioethanol by yeast are high xylose fermentation capacity and high inhibitor tolerance. Acetic acid is among the most important inhibitors. Therefore, the goal of our work is to find novel alleles/SNPs conferring high acetic acid tolerance and introduce them in a strain for second-generation bioethanol production. In previous work, we used pooled-segregant whole-genome sequence analysis for QTL mapping and identified five genes linked to high tolerance to acetic acid (Meijnen et al. 2016). The K11 strain displays high acetic acid tolerance but contains all five inferior alleles. We transformed its genomic DNA into the inferior ER18 strain used in the previous work. Around 60 transformants with higher acetic acid tolerance were isolated. The most tolerant transformant, MS164, contained only 7 SNPs compared to its parent, and was tolerant to 0.9% acetic acid, instead of 0.6% for the parent (growth on YPD solid medium at pH 4.7). MS164 was crossed with the unrelated haploid acetic acid tolerant strain 16D and about 30 segregants isolated with high fermentation performance in liquid YPD medium with 1.2% acetic acid. They were pooled and subjected to pooled- segregant whole-genome sequence analysis for QTL mapping. This analysis revealed 4 major and 5 minor QTLs linked to either parent strain, indicating that both parents contain superior alleles for conferring acetic acid tolerance, but all QTLs were largely unlinked to the 7 SNPs. We are currently identifying the causative elements in these QTLs and testing all 7 SNPs for their effect on acetic acid tolerance. 90

We can conclude that QTL mapping by pooled-segregant whole-genome sequence analysis is able to identify the genomic positions of novel causative genetic elements present in the superior parent strain and likely also in the inferior parent strain, irrespective of the presence of the SNPs introduced by the whole-genome transformation. Whole genome transformation itself turns out to be a powerful technique for strain improvement, although side-effects caused by spurious mutations cannot be ruled out. Once identified, the causative genetic elements from both approaches will be combined into a single industrial strain for second-generation bioethanol production to further improve acetic acid tolerance and further optimize the performance of the strain in hydrolysates with high levels of acetic acid.

247 Systematic assessment of genetic context-dependent gene essentiality. Jolanda van Leeuwen1,3, Carles Pons2, Jason Wang1, Guihong Tan1, Jochen Weile1, Frederick Roth1, Patrick Aloy2, Brenda Andrews1, Charles Boone1 1) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada; 2) Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain; 3) Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland. The functional consequence of a mutation can depend upon the genetic background in which it occurs. In the most extreme case, a gene can be essential in one genetic background, but have little or no effect on cell growth in another, which means gene essentiality is both context-dependent and evolvable.To systematically identify the set of yeast genes whose essential roles are dependent on genetic context, we isolated spontaneous mutations that could overcome the lethality associated with deletion alleles of essential genes, and we identified the genetic alterations driving the suppression. In total, we examined 732 different essential genes, and we discovered a subset of 124 (~17%) that showed context-dependent essentiality. These dispensable genes frequently played a role in transport or signaling processes, and tended to be less well conserved across species than other essential genes. Although for most essential complexes, either all or none of the subunits were dispensable, in a few cases only loss of specific submodules of the complex could be compensated for. The genetic variants driving the suppression phenotype commonly involved aneuploidies or gain-of-function mutations in essential suppressor genes. When multiple independent suppressor genes were identified for a query gene, they often encoded members of the same pathway or complex. In summary, we produced a validated resource of the drivers of context-dependent essentiality, which revealed how the rewiring of cellular processes can tolerate the deletion of genes that are normally indispensable for cellular viability.

248 Mechanistic insights into genetic suppression through aneuploidy. J.Zi Yang. Wang1,2, J. van Leeuwen1, G. Tan1, J. Hou1, C. Boone1,2, B. Andrews1,2 1) Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 160 College Street, Toronto, Ontario M5S 3E1, Canada; 2) Department of Molecular Genetics, University of Toronto, 160 College Street, Toronto ON M5S 3E1, Canada. Recent analysis of human genome sequence data has revealed that some individuals carrying ‘lethal’ mutations in their genomes survive, presumably due to other genome variants or suppressors. However, there are no systematic studies of how often complete loss-of-function of an essential gene can be bypassed by mutation elsewhere in the genome. We addressed this question by screening for spontaneous suppressors of the lethal growth phenotype caused by deletion of 732 essential genes and found that 124 (~17%) genes were dispensable in certain genetic conditions. We observed two major mechanisms for bypass of essential gene function: most suppressors mutations were point mutations in genes that were functionally related the deleted essential gene (217/380 suppressors). However, some essential gene deletions were suppressed by full or partial chromosome aneuploidies. To investigate the mechanisms by which aneuploidies can suppress the loss of essential genes, we used automated, systematic gene overexpression to discover genes on the aneuploid chromosome that, when overexpressed, can overcome the lethality associated with deleting an essential gene. Out of 61 query genes screened, we found that 21 queries were suppressed by overexpression of a single gene on the aneuploid chromosome, and that the suppressors were often functionally related to the deleted essential gene. Our results suggest that partial or whole chromosome duplication is a significant mechanism of genetic suppression.

249 Global analysis of Saccharomycopsis predator yeasts. Klara Junker1, Ana Hesselbart1, Yeseren Kayacan2, Jürgen Wendland1,2 1) Bioengineering Sciences, Vrije Universiteit Brussel, Brussels, BE; 2) Carlsberg Research Laboratory, Copenhagen, Denmark. Yeasts of the genus Saccharomycopsis are present in several niches around the globe. Besides their potential usefulness in fermentations they exhibit a unique predacious behaviour, which allows them to feed on and kill suitable fungal prey cells. This behaviour, known as necrotrophic mycoparasitism, can be induced upon starvation for methionine. Early steps in predation apparently include prey recognition, attachment to prey cells and formation of a penetration peg, a drill with which a prey cell is penetrated and killed. The host range is very broad and includes yeasts, such as Saccharomyces cerevisiae and diverse Candida species, but also filamentous fungi. Comparative genomics based on the the S. schoenii, S. fermentans and S. fodies genomes identified the cause of methionine auxotrophy as the loss of genes required for sulphate utilization. Ser Interestingly, genomic signatures, such as a tRNACAG and CTG positions at conserved Ser/Thr sites in e.g. Candida albicans or S. cerevisiae suggest that Saccharomycopsis species, like C. albicans, have reassigned translation of CTG codons from leucine to serine. Further evidence supporting this codon reassignment was derived from proteomics analyses that identified peptides of several hundred proteins in which CTG codons were translated into serine instead of leucine. We now aim at understanding the biology of predation and the identification and characterization of molecular pathways and 91 genes required for successful killing of prey cells. We have identified several promoters that are functional both in S. schoenii and S. cerevisiae and have generated a suite of functional analysis plasmids. These include resistance genes against G418, hygromycin and nourseothricin/clonNAT. As reporters we have introduced lacZ, and various fluorescent proteins (GFP, Venus, Cherry). Genome profiling indicated the presence of multiple copies of full-length transposons and of multiple gene families encoding proteins that may play major roles for the yeasts’ predacious behaviour. These include genes encoding for cell-cell adhesins, so called flocculins; genes for cell wall degrading enzymes, e.g. chitinases; and glucosidases, as well as a large number of protease encoding genes. These genes as well as genes involved in the formation of penetration pegs offer excellent targets to analyze predatory behavior in Saccharomycopsis. Transcript profiling using RNAseq under various conditions, e.g. with or without prey, revealed several gene sets upregulated under specific conditions as well as genes upregulated during predation, for example protease genes. We are currently setting up tools to generate targeted gene deletions in S. schoenii to initiate functional analyses of these genes. Hesselbart A. et al. Genome Announc. 2018 Jan 11;6(2). Junker et al. Genome Announc. 2017 Nov 16;5(46).

250 Exploring the mechanisms of synthetic lethality in S. cerevisiae. C. Yeung1,2, H. Lian1,2, G. Tan2, H. Fang2, M. Masinas2, J. Hou2, H. Friesen2, C. Boone1,2, B. Andrews1,2 1) Department of Molecular Genetics, University of Toronto, 1 King's College Cir, Toronto, ON M5S 1A8; 2) Donnelly Centre for Cellular and Biomolecular Research, 160 College St, Toronto, ON M5S 3E2. Genetic interactions (GI) occur when an unexpected phenotype arises from combining mutations in two or more genes, and likely underlie many complex phenotypes. Systematic analysis of digenic interactions involving deletion alleles of non essential genes and conditional alleles of essential genes in S. cerevisiae revealed ~ 1 million interactions, enabling construction of a reference genetic interaction map. However, while digenic interactions have been catalogued, the mechanistic underpinnings of synthetic lethality (SL), and other types of genetic interaction, remain poorly understood. We have established an experimental pipeline to enable systematic exploration of phenotypes associated with SL interactions (most negative GIs) involving non-essential genes. The pipeline involves: 1) creation of a novel collection of yeast strains carrying temperature-sensitive alleles of non-essential genes; 2) utilizing high content screening (high-throughput fluorescent microscopy combined with automated image analysis) and; 3) systematic identification of dosage suppressors that rescue SL. As a proof-of-principle, we focussed on constructing ts-alleles of BNI1, which encodes a formin that nucleates actin filaments, and 6 of its SL partners, whose roles encompass different biological processes, including chromosome segregation, cell wall maintenance and cytokinesis. Both high-throughput fluorescence microscopy screening and dosage suppression mapping suggest that different mutant gene pairs give rise to SL phenotypes through different mechanisms. For example, a mutant strain carrying the conditional gene pair bni1 shs1ts (SHS1 encodes a gene that encodes for a subunit of the septin ring) results in several defects not seen in the single mutants, including phenotypic abnormalities in the septin ring, plasma membrane, endosomes and inner nuclear membrane. Moreover, complementary dosage suppression mapping in this gene pair identified functionally related suppressors with established roles in cell polarity (BEM3, SYP1) as well as more functionally diverse suppressors (FAA3, TEC1, RPL11A). We anticipate that our analyses will reveal the underlying biology of synthetic lethality involving specific gene pairs, and general principles governing negative genetic interactions.

251 Diversity and dynamics of copy number variation during adaptive evolution. Grace Avecilla1,2, Stephanie Lauer1,2, Pieter Spealman1,2, Sasha Levy3, David Gresham1,2 1) Center for Genomics and Systems Biology, New York University, New York, NY; 2) Department of Biology, New York University, New York, NY; 3) Joint Initiative for Metrology in Biology, National Institute of Standards and Technology, Stanford University. Copy number variation (CNV) is a common but complex class of mutations with broad implications in evolution, adaptation, and disease. Despite the importance of CNVs in adaptive evolution, their variable size and structure, compounded by the challenge of detecting them at low frequencies, has made it difficult to elucidate their dynamics and diversity in evolving populations. To investigate the dynamics of CNVs in adaptive evolution, we studied the general amino acid permease GAP1 in Saccharomyces cerevisiae populations evolving under selection. We constructed a barcoded lineage tracking library in a GAP1 CNV reporter strain, and performed experimental evolution in glutamine-limited chemostats for hundreds of generations. Using flow cytometry, we evaluated the proportion of the population with a GAP1 CNV and found remarkably reproducible dynamics of GAP1 CNV formation and selection in replicate populations. To quantify the number of unique CNV lineages in each population, we used FACS isolation of CNV lineages and Barseq. We find that hundreds of individual CNV lineages arise and compete during the early stages of adaptive evolution, but that diversity rapidly declines with prolonged selection. Although the early stages of CNV dynamics are reproducible between populations, later dynamics differ between replicate populations. Sequence characterization of the selected CNV lineages reveals diverse structures. We quantified differences in fitness between CNV lineages, and find evidence that CNV size and GAP1 copy number underlie fitness differences between lineages that acquire GAP1 CNVs. Our study provides unprecedented resolution of the dynamics and diversity of CNVs in evolving populations.

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252 The Yeast Atlas, diversity of wild yeast collected from North and South America. Jordan Barney, Matthew Winans, Mahmoud Summers, Jennifer Gallagher Biology, West Virginia University, Morgantown, WV. The ecology of wild strains of Saccharomyces cerevisiae from around the globe is poorly understood. Early domestication by human kind and ubiquitous use of a select few strains has led to an ambiguous origin, although relatively new data suggests an out of Asia theory and importance of insect vectors in yeast mobility. Glyphosate is an herbicide which is commercially used as the main ingredient in Roundup®. Prolonged use has increased glyphosate resistant plants, which may affect human health. In this study, urban and rural isolates were obtained through phenol-chloroform extraction of genomic DNA and amplification of their internal transcribed spacer (ITS) of the ribosomal DNA. Sanger sequencing was employed and used in conjunction with NCBI databases for identification of the genus. Using species specific multiplexing primers, species level identification has been resolved. Development of The Yeast Atlas as a resource to better understand mechanisms of response, adaptation, and evolution to stimulants such as chemicals including glyphosate is a tool for yeast researchers to harness the genetic diversity of wild strains with ease. This effort is currently on going; genetically diverse wild yeast are being both processed and identified. Once identified, the collected S. cerevisiae samples will be categorized by location and evaluated for phenotypic responses to chemicals. Greater than 623 samples have been amassed and 538 isolates recorded. PCR amplification of 421 amplicons aided in 329 species being matched in the NCBI database. Phylogenetic diversity of the isolates varies to include Pichia, Lanchancea, Candida, and more genera of yeast other than Saccharomyces. North and South American isolates of S. cerevisiae will be assayed against chemical stressors, including glyphosate, as a screening process for phenotypic traits of wild yeast.

253 Wine can increase fitness? Just ask yeast! J.R. Bellon1, C.M. Ford2, A.R. Borneman1, S.A. Schmidt1 1) Australian Wine Research Institute, Glen Osmond, SA, Australia; 2) School of Agriculture, Food & Wine, University of Adelaide, Urrbrae, SA, Australia. Utilising interspecific hybridisation between industrial Saccharomyces cerevisiae wine yeast and other members of the Saccharomyces species has helped winemakers differentiate their wines through the production of novel phenotypes that shape wine flavor and aroma. In previous work an interspecific wine yeast strain was successfully generated to incorporate the targeted Saccharomyces uvarum parental trait of low acetic acid production into a S. cerevisiae wine yeast. However, the resultant hybrid had less robust fermentation kinetics than is desirable for an industrial wine yeast. We used wine production, repeatedly, to enhance its fitness. An adaptive evolution series of sequential grape juice fermentations exposed the yeast to cyclic stresses of high sugar/low ethanol followed by low sugar/high ethanol environmental conditions. Natural Saccharomyces interspecific hybrids from industrial fermentation sources have lost chromosomal material from one or both parental lineages, with chromosomal aneuploidy and genome rearrangements common. These changes lead, presumably, to greater fitness for the hybrid in the environment they were isolated from. Traditional approaches to adaptive evolution of yeast strains utilise end-point sampling after hundreds of generations, carrying the risk of selecting for collateral mutations that shape phenotypes in addition to that which is targeted. Identifying a mutant with increased fitness from an early timepoint in the evolutionary process reduces this risk. Our approach was to progressively screen isolates from evolving populations using species-specific PCR-RFLP chromosomal markers designed to each arm of all 16 Saccharomyces chromosomes. This way we were able to track transient changes and the sequence of events that occurred on a chromosomal-species basis. The fitness of isolates with chromosomal patterns that became highly represented were compared to the original hybrid parent. An evolved S. cerevisiae x S. uvarum isolate with the most represented endpoint chromosomal complement, but from an early time point, displayed improved fitness status in a competitive growth assay and improved fermentation kinetics while retaining the desirable fermentation trait of reduced volatile acidity production. Genomic stability studies on newly-generated Saccharomyces interspecific hybrids provides insight into events that occur in the incipient stages of species formation following interspecific hybridisation events.

254 Oxygen and the evolution of multicellular size: hypothesis testing via long-term evolution. G.O. Bozdag1, Chris Reinhard2, William Ratcliff3 1) School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA; 2) School of Earth & Atmospheric Sciences, Georgia Institute of Technology, Atlanta, GA; 3) School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA. The long delay and sudden appearance of complex multicellular life on Earth presents one of the most challenging puzzles in biology. The ‘oxygen control hypothesis’ suggests that the increase of the atmospheric oxygen concentration around ~635- 541 million years ago (i.e. the period right before the Cambrian) lifted a constraint on multicellular size by allowing O2 to diffuse more deeply into nascent multicellular organisms. There is still no consensus as to whether changes in environmental oxygen levels have been the only, or even key, factor for the evolution of larger multicellular size. Moreover, this fundamental relationship has never been directly tested experimentally. To address this key knowledge gap, we have generated snowflake yeast (i.e. a synthetic model of early multicellularity) that obligately ferment, obligately respire, or facultatively respire, and evolved them for over 1000 generations with selection for faster settling, providing strong artificial selection for larger size. Surprisingly, the yeast growing anaerobically were able to respond to this selection best, evolving to be dramatically larger clusters than those capable of facultative or obligate respiration. We hypothesize that this is the result of evolutionary feedbacks arising from competition for limiting resource, O2, that favor reduced size. Furthermore, 2/15 experimental lines evolved much larger groups that are visible to the naked-eye. By whole genome sequencing, we started to identify candidate

93 genes that are likely to change properties of single cells (e.g. cell wall proteins). We suggest that changes in cell-level properties translate into emergent multicellular properties. In sum, our results highlight an interesting caveat to the ‘oxygen control hypothesis’: providing limited O2 actually drives the evolution of smaller multicellular size than those growing entirely anaerobically.

255 Goldilocks and the Three Genotypes: Characterizing the Prevalence of Overdominance in Adaptive Mutations that Arise in Diploids. V. Chen1, M. Sanchez2, L. Herissant1, D. Yuan2, S. Hoelscher3, D. Petrov2, G. Sherlock1 1) Genetics, Stanford University Medical School, Stanford, CA; 2) Biology, Stanford University, Stanford, CA; 3) Chemistry, Stanford University, Stanford, CA. When evolving diploids acquire mutations, they are generally heterozygous. Some of these heterozygous mutations provide a fitness benefit, making the mutants more fit than their unmutated counterparts (also called the reference). When the heterozygous mutant (Aa) is more fit than either the homozygous mutant (aa) or homozygous reference (AA) genotype, that mutation is overdominant. A well-known example of overdominance is sickle cell anemia, in which heterozygous individuals (Aa) have no or mild anemia and resistance to malaria infection while homozygous individuals (aa) with two copies of sickle cell allele have severe anemia and the homozygous individuals (AA) with two copies of the healthy allele are susceptible to malaria. Because of heterozygote advantage, all three genotypes are maintained in the population, due to balancing selection; thus sickle cell anemia is common in populations exposed to malaria despite its severe symptoms. However, it is unclear how many other disease-causing alleles have undiscovered fitness benefits and how common the phenomenon of overdominance is. Theoretical predictions suggest that a substantial fraction of beneficial mutations that arise in diploids will be overdominant, but to date few studies have systematically determined whether such mutations are in fact overdominant. In this research, we are determining the prevalence of overdominance both using data from the yeast deletion collection, and by measuring the fitness of all three genotypes (homozygous, heterozygous, and reference) of adaptive mutations that arose during diploid evolutions. This research will provide insight on the characteristics of overdominance and potentially how this phenomenon may have affected evolution and selection of various traits including heritable diseases.

256 Identifying the set of genes in Saccharomyces cerevisiae. Grahame Evans, Fred Dietrich Molecular Genetics and Microbiology Department, Duke Univ, Durham, NC. With the sequencing of the genome of S. cerevisiae strain S288c in the 1990’s, it was possible to identify the set of protein coding genes in that strain. Now that a large number of strains of this species have been sequenced, it is possible to take a more careful look at the set of genes found in the species S. cerevisiae, in contrast to that of strain S288c. For each chromosome there is a central portion of the chromosome that is highly conserved, and generally co-linear with the lab strain S288c. We have identified the 32 genes that approximate the ends of this conserved central portion of the genome. Beyond the ends of the central portion of each chromosome gene content is quite variable, and at times exchanged between chromosomes, lost, or duplicated. All known essential genes are within the central portion of the genome. Within the central portion of each chromosome most genes are part of the “core set” of genes found in all, or nearly all strains of S. cerevisiae. In some strains there are alleles of these “core genes” that contain stop codons and or frame shifts. These are alleles of these genes, not pseudogenes. S. cerevisiae typically lives as a diploid in the environment, so having an apparently null allele of a “core gene” may often not be detrimental, and could possibly even be beneficial. There is also a smaller set of strain specific central genes. There are numerous cases of gene duplication, falling into four main categories. 1) There are ancient duplicated genes that are also duplicated in related species, remnants of the whole genome duplication or of single gene duplication events. 2) There are more recent tandem duplicated genes. 3) There are cases of sub-telomeric duplication/loss resulting in gene duplication. 4) There are cases of core genes where a duplicate copy is attached at a telomeric end. In some of these cases the residual terminal telomeric repeat can be seen at the site where the duplicate sequence was attached. In addition to gene duplications and null alleles, there are also some pseudo genes, which are defined as additional segments of DNA attached at a location where the donor gene is not found. There are cases of pseudo genes derived from mitochondrial genome sequence, derived from rDNA, and derived from protein coding genes. These are generally in the sub- telomeric regions. There are relatively few genes of unknown function within the core set of S. cerevisiae genes. We are undertaking a project to identify function of these remaining genes of unknown function, starting with the approximately 30 S. cerevisiae genes of unknown function with human orthologs.

257 Eukaryotic acquisition of a bacterial operon. D.T. Doering1,3, J. Kominek1,2, D.A. Opulente1, X.-X. Shen4, X. Zhou4, J. DeVirgilio5, A.B. Hulfachor3, C.P. Kurtzman5, A. Rokas4, C.T. Hittinger1,2,3 1) Laboratory of Genetics, Genome Center of Wisconsin, Wisconsin Energy Institute, J. F. Crow Institute for the Study of Evolution, University of Wisconsin-Madison, Madison, WI; 2) DOE Great Lakes Bioenergy Research Center, University of Wisconsin-Madison, WI; 3) Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI; 4) Department of Biological Sciences, Vanderbilt University, Nashville, TN; 5) Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, US Department of Agriculture, Peoria, IL. Encompassing roughly 500 million years of evolution, the budding yeast subphylum (Saccharomycotina) displays a remarkable level of genetic, phenotypic, and metabolic diversity. Systematically exploring this diversity by whole-genome 94 sequencing through the Y1000+ Project has led to the first-in-class discovery of a horizontal operon transfer event of a catecholate-class siderophore biosynthesis pathway. Through phylogenetic hypothesis testing, we demonstrate that the operon was transferred roughly 50 million years ago from an Enterobacteriaceae lineage into the ancestor of the Wickerhamiella/Starmerella clade, an under-studied group of yeasts that diverged from their common ancestor with Saccharomyces cerevisiae roughly 300 million years ago. Additionally, despite the fundamental differences between bacteria and eukaryotes in genome organization and Central Dogma processes, we show that all transferred and retained operon genes are actively expressed, exhibit both bacterial and eukaryotic transcriptional features, result in the production of the siderophore enterobactin, and enable the sequestration of iron from the environment. After transfer to the eukaryotic host, several genetic changes occurred, including structural rearrangements, insertion of additional eukaryotic genes, and secondary loss of the horizontally-acquired genes in some lineages. We conclude that the operon genes were transferred from the bacterial lineage into the yeast ancestor, underwent structural changes for eukaryotic gene expression, and were maintained by selection to adapt to a highly-competitive, iron-limited environment.

258 A genome-wide screen for genes that affect mutational robustness. J. Frickel1,2, K. Verstrepen1,2 1) Center for Microbiology, VIB , Heverlee-Leuven , BE; 2) Center of Microbial and Plant Genetics, KU Leuven , Heverlee-Leuven , BE. Whereas organisms require mutations to evolve, only a small fraction of mutations are potentially beneficial. This implies that organisms are under pressure to balance the negative and positive consequences of genetic mutations. An increasing body of research suggests that populations can harbor cryptic genetic variation, i.e. genetic variation that is not phenotypically expressed under normal conditions, but which may lead to phenotypic differences in times of stress. This might suggest that organisms harbor mechanisms to suppress the negative consequences of mutations (mutational robustness) when they are not needed, but allow expression when the organisms are not optimally adapted to their environment. Such mechanisms could, in theory, increase evolvability by allowing the accumulation of mutations until conditions arise in which the mutations might be favorable (e.g. during environmental perturbations). However, with the exception of a potential role of the central chaperone Hsp90 (involved in protein folding), little is known about the molecular mechanisms that could drive such systems.

As a first step to identify such mechanisms, we conducted a high-throughput genome-wide screen to identify genes that alter the fitness effect of random mutations. Using automated microscopy, we measured the fitness of around 200 individual cells for each of the mutants in the yeast deletion collection. Concurrently, we also measured the fitness of 200 cells from each of the deletion strains with each cell containing different random mutations. In doing so, we were able to identify genes that strongly influence the fitness effect of random mutations. These genes are thus candidates involved in buffering of random genetic variation and their phenotypic consequences. Interestingly, the set of candidates are enriched for genes involved in protein folding and chromatin remodeling, which have previously been suggested to be involved in mutational robustness. Our ongoing work uses experimental evolution and phenotyping assays to investigate in detail how such mechanisms contribute to the balancing act between the negative effect of mutations and evolvability.

259 Systematic humanization of the yeast cytoskeleton reveals functionally equivalent human orthologs. Riddhiman GARGE1, Jon Laurent2, Aashiq Kachroo3, Edward Marcotte1 1) Center for Systems and Synthetic Biology, Molecular Biosciences, University of Texas at Austin, Austin, TX; 2) Institute for Systems Genetics, Department of Biochemistry and Molecular Pharmacology, New York University Langone Health; 3) The Department of Biology, Centre for Applied Synthetic Biology, Concordia University, Montreal. Saccharomyces cerevisiae (Baker’s yeast) and Homo sapiens (humans) are separated by a billion years of evolution, yet recent studies from our lab and others have demonstrated that nearly half of tested essential yeast genes can be replaced by their corresponding human ortholog(s), reflecting the deep conservation of essential gene function in highly diverged organisms. Replaceability was observed to be a modular property, i.e genes belonging to shared pathways or complexes all had the same tendency to be replaceable or not. To further understand gene replaceability in core cellular processes, we are currently focusing on the cytoskeleton, including actins, myosins, tubulins and septins. These gene families have expanded in humans in contrast to either one two members in yeast. Despite their functional conservation among higher eukaryotes, specific roles are unclear for many members of these expanded families. Using a combination of classical yeast genetics, microscopy and CRISPR genome editing we systematically substituted ~80% of the human cytoskeletal genes for their yeast counterparts to assay if they could functionally substitute. We found one or more of the human genes within a particular cytoskeletal gene family functionally complementing the lethal defect induced by loss of the yeast ortholog. We characterized additional phenotypes beyond growth and observed differential abilities of human cytoskeletal orthologs to rescue cell morphology, meiotic and non-essential cytoskeletal roles. I will discuss our findings from the cytoskeletal humanization experiments and their utility in structure-function studies of human genes within expanded gene families.

260 Differential Expression of Whole Genome Hybridization Paralogs in S. Cerevisiae following PKA inhibition. B. Heineike1, H El-Samad2 1) Bioinformatics Graduate Program, University of California San Francisco, San Francisco, CA; 2) Biochemistry and Biophysics Department, University of California San Francisco, San Francisco, CA. One of the unique characteristics of Saccharomyces Cerevisiae (S.Cer) is that it performs fermentation when glucose is present even in the presence of oxygen (i.e. the Crabtree Effect). When glucose runs low in S.Cer, if a respiratory carbon 95 source (such as ethanol) is present, the cell undergoes a number of physiological changes and shifts to a primarily respiratory metabolism. The cAMP/PKA pathway is a key player in this transition; when glucose levels drop, PKA is inhibited, triggering a signaling and transcriptional cascade which contributes to this metabolic shift.

The evolution of the Crabtree Effect in yeast largely coincides with a Whole Genome Hybridization (WGH) event approximately 100 Million Years Ago (Mya) in which a new species was formed from two ancestral strains which diverged tens of millions of years previously. This hybrid species contained orthologous genes (WGH paralogs), over 500 of which survive in S.Cer today.

We wanted to understand how gene expression programs downstream of PKA evolved in the S.Cer branch following the WGH by comparing gene expression profiles following PKA inhibition between S.Cer and Kluyveromyces Lactis (K.Lac) (which shares an ancestor with one of the parental strains in the WGH). We noticed that the subset of genes that were expressed in S.Cer but not in K.Lac under PKA inhibition was enriched for metabolic genes, and was also enriched for WGH paralogs. For most genes possessing a WGH paralog that was activated in S.Cer under PKA inhibition, the other paralog tended to be constituively expressed at a relatively high level. In other words the paralog pair was differentially expressed. For most of these differentially expressed paralogs, their shared K.Lac ortholog tended to have constitutive high expression.

We further investigate the regulatory basis for the loss of constitutive high expression and gain of PKA induction using bioinformatic analysis of binding sites and genetic perturbations.

261 Dissecting the genotype-to-phenotype map in eukaryotes: Molecular determinants of complexity, heterosis, pleiotropy, and epistasis in heritable traits. C.M. Jakobson1, R. She1, D.F. Jarosz1,2 1) Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA; 2) Developmental Biology, Stanford University School of Medicine, Stanford, CA. The intrinsic complexity of heritable traits has been known for over a century: Fisher assessed the statistical impact of polygenicity, dominance, heterosis, pleiotropy, and epistasis on heredity in 1918. Although the prevalence of these phenomena is clear in patterns of inheritance, the molecular variation that is responsible, such as changes in protein coding sequences or regulatory regions, remains obscure. To fully exploit the recent explosion in the availability of genome sequence information, we must understand in great detail how genotypes map to expressed traits.

Using a panel of more than 18,000 fully genotyped F 6 diploid yeast derived from a cross between two wild Saccharomyces cerevisiae isolates, we identified thousands of genetic variants responsible for over a dozen complex traits. The unprecedented statistical power of the extra-large segregant panel allowed us to identify hundreds of genetic loci underlying each quantitative trait, frequently with single-nucleotide resolution. Moreover, we measured the precise molecular variation that underlies genetic nonlinearities and interactions. We developed a detailed picture of the diverse mechanistic contributors to complexity, heterosis, pleiotropy, and epistasis. Previously, these measurements have been made comprehensively only for the most radical type of genetic variant: the gene deletion. We found that, in fact, all types of natural molecular variation, including synonymous variants, make significant contributions to the genetic architecture of complex traits. The space of possible genotypes, even for simple eukaryotes, is unimaginably vast, effectively precluding explicit investigation of each variant and combination thereof. Elucidating the architecture of complex traits in high throughput at the level of single nucleotides will therefore be crucial for understanding heritability in the context of evolution and disease.

262 Evolutionary Implications of Genomic Responses to Stress. Karen Schmidt2, William Sexton1, Quinn Dickinson1, Jacob Childress2, Frank Rosenzweig1, Eugene Kroll1 1) School of Biological Sciences, Georgia Institute of Technology, Atlanta, GA; 2) Division of Biological Sciences, University of Montana, Missoula, MT. Knowledge of genetic mechanisms that enable populations to adapt to novel environments is required to explain their evolutionary fate. We have developed an experimental approach to study these mechanisms using starvation as a proxy to environmental conditions whose overall effect is to lower mean population fitness. We use pulsed-field gels, advanced sequencing and mapping methods, competition assays and modeling to associate genomic and phenotypic evolutionary changes. We find that the incidence of genomic rearrangements in surviving members of starved cells is several-fold greater than that among non-starved cells; by contrast, the incidence of point mutations does not significantly differ between these conditions. Notably, survivors with restructured genomes are more resilient to starvation than their common ancestor. Genome restructuring is so pronounced in some isolates that they have become reproductively isolated from their ancestors. Because both resilience to starvation and reproductive isolation were strongly correlated with genomic restructuring, we suggest that severe environmental stress may promote incipient speciation. Here, we will describe the dynamics of starvation-associated genomic restructuring, analyze distributions of genomic events across different subgroups of cells in stressed populations, and report on a nascent clustered/multicellular phenotype in the genetic background of a wild Saccharomyces yeast originally isolated from champagne viticulture.

263 Spontaneous Changes in Ploidy Are Common in Yeast. Yaniv Harari1, Yoav Ram2, Martin Kupiec1 1) School of Molecular Cell Biology and Biotechnology, Tel Aviv Univ, Tel Aviv, Israel; 2) Efi Arazi School of Computer Science, 96

Interdisciplinary Center (IDC), Herzliya, Israel. Changes in ploidy are relatively rare, but play important roles in the development of cancer and the acquisition of long-term adaptations. Genome duplications occur across the tree of life, and can alter the rate of adaptive evolution. Moreover, by allowing the subsequent loss of individual chromosomes and the accumulation of mutations, changes in ploidy can promote genomic instability and/or adaptation. Although many studies have been published in the last years about changes in chromosome number and their evolutionary consequences, tracking and measuring the rate of whole-genome duplications have been extremely challenging. We have systematically studied the appearance of diploid cells among haploid yeast cultures evolving for over 100 generations in different media. We find that spontaneous diploidization is a relatively common event, which is usually selected against, but under certain stressful conditions may become advantageous. Furthermore, we were able to detect, quantify and distinguish between two different mechanisms of diploidization, one that requires whole- genome duplication (endoreduplication) and a second that involves mating-type switching despite the use of heterothallic strains. Our results have important implications for our understanding of evolution and adaptation in fungal pathogens and the development of cancer, and for the use of yeast cells in biotechnological applications.

264 Describing and understanding the fitness landscape of a yeast tRNA gene. Chuan Li1, Jianzhi Zhang2 1) Biology, Stanford University, San Jose, CA; 2) Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, MI. The fitness landscape of a gene maps each mutational variant of the gene to the fitness of the organism carrying the variant. It allows explaining as well as predicting evolutionary trajectories and is therefore of fundamental biological importance to understand evolution. Notwithstanding, past measures of FLs typically probe a limited number of variants in one environment, because even the characterization of a small fraction of the FL of one gene under one environment had been a formidable challenge until recently. Using a high-throughput method that combines precise gene replacement with next-generation sequencing, we quantified fitness landscape of a tRNA gene and its variation across four different environments comprising >23,000 genotypes. We are interested in elucidating the underlying mechanistic determinant of the observed fitness landscape. We measured and calculated multiple parameters that are likely affecting the fitness landscape of the tRNA gene and their relative contribution of these factors, providing a mechanistic explanation to the observed fitness landscape.

265 The complex underpinnings of genetic background effects. M. Mullis, T. Matsui, R. Schell, R. Foree, I. Ehrenreich Molecular and Computational Biology, University of Southern California, Los Angeles, CA. Spontaneous and induced mutations commonly show different phenotypic effects across genetically distinct individuals. Although these background effects are known to result from epistasis between mutations and the polymorphisms already present in a population, their underlying genetic architecture remains poorly understood. Here, we genotyped 1,411 wild type and mutant segregants from the same budding yeast cross, and measured their growth in 10 environments. We then mapped genetic interactions between each of seven different gene knockouts and segregating loci. In total, we detected 1,086 interactions between a knockout and one or more loci. 95% of these interactions involved higher-order epistasis between a knockout and multiple loci. When collapsed into unique sets of epistatic loci, 65% of these interactions were found in only a single knockout background, while 35% were detected in multiple knockout backgrounds. Loci that were found in one knockout background almost exclusively exhibited larger effects in mutants than wild type individuals. In contrast, loci that were detected in multiple knockout backgrounds tended to show smaller effects in mutants than wild type individuals. Analysis of these loci across growth conditions revealed that most of the interactions between the knockouts and segregating loci were specific to a particular environment. Our results clarify the genetic complexity, types of epistasis, extent of pleiotropy, and role of the environment in background effects.

266 Subtelomeric gene presence polymorphisms disproportionately contributes to yeast trait variation. K. Persson1, S. Stenberg2, E. Aspholm1, M. Leduc-Darras1, F. Salinas3, G. Liti4, J. Warringer1 1) University of Gothenburg, Department of Chemistry & Molecular Biology, Gothenburg, SE; 2) Centre for Integrative Genetics (CIGENE), Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences (UMB), Ås, Norway; 3) Center of Studies in Sciences and Food Technology (CECTA), Universidad de Santiago, Santiago, Chile; 4) Institute for Research on Cancer and Ageing, Nice (IRCAN), University of Nice, Nice, France. The domesticated baker's yeast shows twice the phenotype polymorphism despite possessing half the single nucleotide polymorphism of its closest relative. We hypothesized that this seeming contradiction was due to a disproportionate contribution to trait variation from chromosomal rearrangements, leading to gene presence/absence and gene copy number variations in accessory genes. Supporting this hypothesis, such polymorphisms have recently been found to be much more frequent in the domesticated baker’s yeast with 30% of QTLs mapping to subtelomeric regions. We evaluated this hypothesis using a reverse genetics approach. First, we mimicked subtelomeric gene presence variants by deleting accessory genes in strains where they were present. Second, we mimicked gene duplications by inserting an extra copy in a strain where the gene was naturally present in a single copy. Third, we mimicked SNP based allele variants using pairs of diploid hybrids that were reciprocally hemizygotic for particular allele pairs. We exhaustively explored the effect of each genetic variation on growth rates across >100 different environments, using a high resolution growth phenomics platform and a randomized well replicated design. Establishing growth rate distributions of each class of genetic variation, we found each subtelomeric gene presence/absence variation, but not each gene duplication, to contribute more to trait variation than SNP based differences 97 between allele pairs. We conclude that subtelomeric gene presence/absence variation have a disproportionate influence on fitness associated trait variation, and presumably evolution and natural adaptation. Because of the challenges of assembling low complexity subtelomeres, this variation will escape detection in GWAS and linkage associated studies unless long read sequencing is employed and may contribute substantially to missing heritability.

267 Resistance to glyphosate-based herbicides in S. cerevisiae does not pertain only to the shikimate pathway. A. Ravi Shankar, J. Gallagher Biology, West Virginia University, Morgantown, WV. S. cerevisiae isolated from different environments are subject to a wide range of selective pressures whether intentional or by happenstance. Due to their close contact with plants, insects and other animals in its immediate surroundings, chemicals such as herbicides, fungicides and antibiotics, can affect non-target organisms. First marketed as RoundUp™, glyphosate is the most widely used herbicide in the world due to its versatility, ability to control a wide spectrum of weeds, and its minimal toxicity to mammals. In plants, glyphosate inhibits EPSPS, of the shikimate pathway, which is present in many organisms but lacking in mammals. The shikimate pathway produces chorismate which is the precursor to all the aromatic amino acids, para-aminobenzoic acid, and Coenzyme Q10. Sequence comparison between the target proteins, i.e., the plant EPSPS and the yeast orthologous protein Aro1, predicted that yeast would be resistant to glyphosate. However, S. cerevisiae has a wide-range of glyphosate-based herbicide resistance that does not pertain only to the shikimate pathway, but arises from its exposure to the other chemicals present in the commercial formulations along with the active ingredient. Using QTL mapping we discovered that the growth variation seen in the subset of yeast tested was not due to polymorphisms within Aro1, instead, it was caused by genetic variation in genes coding for transporter proteins, the amino acid sensor system, mRNA splicing proteins and some mitochondrial proteins. Using genetic variation as a probe into glyphosate response, we uncovered mechanisms that contribute to glyphosate transport and response.

268 The effect of immigration on cooperator-cheater dynamics: defectors can rescue otherwise doomed populations. P. Samani1, G. Sherlock2, F. Rosenzweig1 1) Georgia Institute of Technology; 2) Stanford University Medical School. Evolutionary rescue (ER) is the persistence of stressed populations via genetic adaptation. Over the past decade a number of demographic, genetic, and eco-evolutionary factors have been described that can impact the likelihood of ER in stressful environments. However, the role of immigration remains obscure. Theory suggests that the effect of immigration on ER depends on the Allee effect (density-dependent growth). Using experimental yeast populations, we investigated this issue in both short-term and long-term experiments. During the short-term experiment we investigated the effect of density- dependent growth on the population dynamics of cheaters and cooperators; in the long-term (evolutionary) experiment we explored the effect of immigration on the likelihood of evolutionary rescue. We used the cheater/cooperator yeast system (Cooperator: SUC2, his3Δ1, MET17; Cheater: suc2Δ::KanMX4. HIS3, URA3; both constructed in BY4741; Gore et al. 2009) that utilizes sucrose as the sole carbon source. Different mixtures of cooperators and cheaters were grown at 30°C in synthetic medium (minus histidine) supplemented with 5% sucrose and 4µg/ml histidine. Cooperators produce and secrete invertase into the periplasmic space. Invertase breaks down sucrose to glucose and fructose, after which both of these monosaccharides become available to all cells in the culture. Cooperators’ growth, unlike cheaters, is limited by the low level of histidine (4µg/ml) in the medium. Cheaters however, do not produce invertase, and therefore do not contribute to the production of monosaccharides, which are the common goods in this system. Preliminary results from the short-term experiment point to a possibility of altruistic behavior emerging under special cheater migration regimes. Our data suggest that cheaters may be secreting small amounts of histidine, which augments cooperators’ growth when cheaters are abundant. Mixed populations of cooperators and cheaters produced greater biomass than populations in which cooperators were grown alone, and cooperators themselves grew faster and sometimes to higher yields than cheaters. Currently, we are running complementary assays (MicroMolar Histidine Assay Kit and HPLC) to validate these striking observations. Overall, our results suggest that a yeast cooperator-cheater system can switch into an altruistic relationship under special demographic circumstances.

269 Fitness effects of mutations emerging under nitrogen limitation. K. Schwartz1, D. Aggeli1, J. Blundell3, D. Fisher4, S. Levy2, G. Sherlock1 1) Department of Genetics, Stanford Univ School Medicine, Stanford, CA; 2) Joint Initiative for Metrology in Biology (JIMB) National Institute of Standards and Technology (NIST) Stanford University, CA; 3) Hutchison-MRC Research Center, Cambridge Biomedical Campus, Cambridge, UK; 4) Department of Applied Physics, Stanford, CA. We evolved barcoded haploid populations of S. cerevisiae via serial batch culture in the presence of limiting nitrogen, and identified beneficial mutations by sequencing hundreds of adaptive clones. We observed genetic changes in a relatively small subset of genes controlling the nitrogen catabolite repression system. We also found that transposition events, resulting in beneficial mutations, occur much more frequently under nitrogen limitation, compared to glucose limitation. The fitness advantage of these mutants was modest, and not as high as for mutations selected in limiting glucose evolutions. The time point chosen for mutational analysis (generation 192) was late enough in the experiment to allow for multiple adaptive mutations to occur in the same lineage, yet we did not observe clones with multiple mutations. That led us to hypothesize that there are genetic interactions that function at both the transcriptional and post-translational level. Through mating and re-barcoding we are measuring fitness of double mutants to test all pairwise genetic interactions among a subset of adaptive alleles of the most frequently mutated genes (GAT1, PAR32, MEP1, MEP2 and MEP3). We expect to uncover epistatic 98 interactions between different members of the nitrogen metabolic pathway, and better understand how different parts of the pathway function together.

270 Population structure among wild S. cerevisiae over decades of evolution in southeastern Pennsylvania. J.A. Shapiro, Y. Guo, J. Liu Biology, Bryn Mawr College, Bryn Mawr, PA. The genomic diversity of Saccharomyces cerevisiae has been studied extensively in a global context, through comparisons among strain isolates from a variety of natural, industrial, and clinical settings. These global patterns of diversity, however, are limited in their ability to elucidate the patterns of evolution that are taking place in the species within more local environments and on shorter times scales. To investigate patterns of evolution observed at these more limited scales, we conducted targeted sampling of S. cerevisiae from oak trees in southeastern Pennsylvania. In addition, we took advantage of historical isolates of S. cerevisiae from the same region that were collected nearly twenty years ago in order to assess the possible changes in the population over a relatively short time scale (though one that could include up to thousands of generations). Whole-genome sequencing and analysis of single nucleotide polymorphisms of 72 strains revealed the presence of three distinct clades of S. cerevisiae coexisting among the oak trees in the region, with members of each clade observed in both current and historical samples. To investigate the potential for effects of structural variation on the apparent isolation among the three clades, we performed nanopore long-read sequencing on six strains, two from each clade. Numerous structural variants were observed, aligning well with the previously identified clade divisions. Ongoing and future work will investigate the extent of phenotypic variation among clades, as well as the potential for intra-clade reproductive isolation that may contribute to the observed population structure and allow the continued coexistence of these distinct clades within a small geographic area.

271 Evolutionary engineering of recombinant Saccharomyces cerevisiae cells for improved xylose consumption and fermentation. Wallace M. Pereira1, Margareth Patiño1, Dominika Wloch-Salamon2, Barbara Dunn2, Gavin Sherlock2, Boris U. Stambuk1 1) Department of Biochemistry, Universidade Federal de Santa Catarina, Florianopolis, Santa Catarina, BR; 2) Department of Genetics, Stanford University, USA. S. cerevisiae is the preferred microorganism used in the production of bioethanol, but lacks the ability to utilize xylose, a carbon source present in the hemicellulose fraction (the second most abundant polysaccharide in nature) of lignocelluloses hydrolysates. A major focus in metabolic engineering for xylose fermentation has been in the area of establishing and improving an intracellular xylose catabolic pathway. However, independently of the xylose-utilizing pathway used, the uptake of xylose across the yeast plasma membrane occurs through a large family of hexose transporters that has been reported to have substantial metabolic flux control over xylose fermentation. We have recently analyzed the importance of individual HXT transporters in xylose fermentation by an hxt-null strain with high xylose reductase, xylitol dehydrogenase and xylulokinase activities (Gonçalves et al., Enzyme Microb. Tech. 63: 13–20, 2014). In order to obtain recombinant strains of S. cerevisiae with increased ability to ferment xylose, we performed an evolutionary engineering experiment with this hxt-null strain overexpressing the moderately high-affinity HXT2 transporter. While this strain could hardly grow in minimal medium with xylose as unique carbon source, after repeated 48 h transfers to new xylose media we were able to selected two evolved strains (9C-MA and 10A-MA) capable of growing in xylose (and fermenting this carbon source) as efficiently as with glucose. Since the underlying mutation(s) responsible for this new phenotype were not present in the HXT2-overexpressing plasmid, we performed whole-genome sequencing of the parental and evolved strains using the Illumina HiSeq2000 platform. Only one mutation was found in both evolved strains, changing a thymine into an adenine in the STB5 gene, which was confirmed by traditional Sanger sequencing. This gene encodes for a zinc finger transcription factor with dual activator/repressor functions required for pentose phosphate pathway regulation and NADPH production. Our data also indicates that the strains underwent self-diploidization, and further amplification of the STB5 gene. We are currently analyzing how the mutation in STB5 (which changes a cysteine into a serine) affects the activity of this transcription factor that may be responsible for the improved xylose-utilizing phenotype of the evolved recombinant S. cerevisiae strains. Financial Support: CAPES, CNPq, FINEP (Brazil) and NIH (USA).

272 Loss of canonical budding yeast sulfate transporters selects for co-option of the sulfonate transporter SOA1 in low-sulfate conditions. Bryce Taylor, Aaron Miller, Ivan Liachko, Anna Sunshine, Erica Alcantara, Maitreya Dunham Genome Sciences, University of Washington, Seattle, WA. When a critical cellular function is lost, existing genes may be co-opted to serve this function. In this study, we investigate adaptive routes two species of yeast take to grow in sulfate-limited media after loss of the canonical sulfate transporter genes SUL1 and SUL2. We performed five independent evolution experiments using sulfate transport-deficient (sul1∆ sul2∆) strains of two yeast species, Saccharomyces cerevisiae and Saccharomyces uvarum, grown in sulfate-limited media. All five independent evolution experiments yielded sulfate transport-proficient populations capable of dramatically improved growth in this condition. Clones from all five populations were found to contain missense mutations in the 2nd MFS domain of a sulfonate transporter, SOA1. Three of these five mutations altered the same codon of SOA1. Introduction of the three SOA1 alleles from our S. cerevisiae experiments in a sul1∆ sul2∆ soa1∆ strain was sufficient for growth on sulfate-limited media, whereas the wild type allele was not, demonstrating that they had become efficient sulfate transporters. Recent work demonstrates that the ancestral SOA1 has a high affinity for sulfonates isethionate, and vastly lower affinity for sulfate. The three evolved alleles all resulted in improvements in transport of sulfate. One allele appears to have a pleiotropic effect on 99 transport of isethionate, dramatically reducing its affinity for this sulfur source. We are in the process of investigating whether natural isolates of S. cerevisiae possess alleles of SOA1 that have altered transport profiles. To achieve this, we are querying libraries of SOA1 alleles from 1011 yeast isolates under conditions limiting for several sulfur sources.

273 Identifying positive genetic interactions using experimental evolution. R. Vignogna, S. Buskirk, G. Lang Department of Biological Sciences, Lehigh University, Bethlehem, PA. Laboratory evolution enriches for genotypes that promotes rapid growth. This continuous selective pressure, exerted over thousands of generations, selects for positive genetic interactions between evolved mutations. We identified a strong positive genetic interaction between mutations in KEL1 and HSL7 in one experimentally-evolved population. We used this population as a basis for identifying additional variants that have positive genetic interactions with kel1 by replaying the evolution of this population. We performed hundreds of evolutionary replay experiments by restarting the evolution of our focal population when it contained the kel1 variant, but not the hsl7 variant. We tracked the fate of the kel1-lineage in each replay population. We performed whole-genome sequencing to identify the other variants that arose in the background of our kel1 variant. We identify additional variants of hsl7, as well as variants of known kel1-interactors, like hsl1 and lte1. In addition, we observe several other genes that are mutated recurrently in our replay experiments, providing a list of putative and previously unknown kel1 interactors. We are currently reconstructing these mutations individually and in combination with the kel1 variant to validate these inferred genetic interactions. Our results shed light on a previously under-explored region of the yeast genetic-interaction network. More broadly, this work demonstrates the power of experimental evolution to identify genetic interactions that are not easily detected by other methods.

274 Genetic dissection of an ancient divergence in yeast thermotolerance. Carly Weiss1, Jeremy Roop1,2, Rylee Hackley1,3,4, Julie Chuong3, Igor Grigoriev1,5, Adam Arkin1,6, Jeffrey Skerker1,6, Rachel Brem1,3 1) UC Berkeley, Berkeley, CA; 2) Fred Hutchinson Cancer Research Center, Seattle, WA; 3) Buck Institute for Research on Aging, Novato, CA; 4) Duke University, Durham, NC; 5) US Department of Energy Joint Genome Institute, Walnut Creek, CA; 6) Lawrence Berkeley National Laboratory, Berkeley, CA. Some of the most unique and compelling survival strategies in the natural world are fixed in isolated species. To date, molecular insight into these ancient adaptations has been limited, as classic experimental genetics has focused on interfertile individuals in populations. Here we use a new mapping approach, which screens mutants in a sterile interspecific hybrid, to identify eight housekeeping genes that underlie the growth advantage of Saccharomyces cerevisiae over its distant relative S. paradoxus at high temperature. Pro-thermotolerance alleles at these mapped loci were required for the adaptive trait in S. cerevisiae and sufficient for its partial reconstruction in S. paradoxus. The emerging picture is one in which S. cerevisiae improved the heat resistance of multiple components of the fundamental growth machinery in response to selective pressure. This study lays the groundwork for the mapping of genotype to phenotype in clades of sister species across Eukarya.

275 Fine tuning Acetyl-CoA Carboxylase 1 (ACC1) activity through localization: Functional genomics reveal a role for the lysine acetyltransferase NuA4 and sphingolipid metabolism in regulating Acc1 activity and localization. K.K. Baetz, T.T. Pham, S. Huard Ottawa Institute of Systems Biology, Univ Ottawa, Ottawa, ON, CA. Acetyl-CoA Carboxylase 1 (Acc1) catalyzes the conversion of acetyl-CoA to malonyl-CoA, the rate-limiting step of de novo fatty acid synthesis. As a master-regulator of lipid synthesis, Acc1 has been proposed to be a therapeutic target for numerous metabolic diseases including diabetes, obesity and cancer yet its regulation remains poorly characterized. We have shown that Acc1 activity is reduced in the absence of lysine acetyltransferase NuA4 in Saccharomyces cerevisiae. This is not through modulation of protein levels or direct acetylation of Acc1, but through localization. In wildtype cells Acc1 is localized throughout the cytoplasm in small punctate and rod-like structures. However in NuA4 mutants, Acc1 localization becomes diffuse. To uncover the mechanisms regulating Acc1 localization we perform a high content microscopy screen to identified other deletion mutants that impact Acc1 localization and then measured the activity of Acc1 in these mutants through chemical genetics and biochemical assays. Three unique phenotypes were identified. Mutants with hyper-active Acc1 form one or two rod-like structures centrally within the cytoplasm. In contrast, mutants with mid-range reduction in Acc1 activity displayed diffuse Acc1 localization, while the mutants with the lowest Acc1 activity (hypomorphs) formed thick rod-like Acc1 structures at the periphery of the cell. All the Acc1 hypomorphic mutants were implicated in sphingolipid metabolism or very- long chain fatty acid elongation and in common, their deletion causes an accumulation of the short chain fatty acid palmityl- CoA. Through exogenous lipid treatments, enzyme inhibitors and genetics, we determined that increasing palmityl-CoA levels inhibits Acc1 activity and remodels Acc1 localization. Further our studies suggest that NuA4 has novel role in regulating sphingolipid flux and palmityl-CoA levels. Together this study suggests yeast cells have developed a dynamic feed-back mechanism in which downstream products of Acc1 can fine-tune the rate of fatty acid synthesis.

276 ABC transporters and protein sorting of transporters as mediators of glyphosate resistance. S. Carlson, J. Blaize, A. Ravishankar, A. Pupo Meriño Biology, West Virginia University, Morgantown, WV. Chemical herbicides are intensively used in modern agriculture. These herbicides also unfortunately induce non-target effects such as declining plant diversity. The popular herbicide glyphosate, commonly known as Roundup, is extremely 100 effective through its non-selective inhibition of the shikimate pathway, which is responsible for the production of aromatic amino acids and other useful biochemical compounds. S. cerevisiae, a species of yeast highly susceptible to environmental selective pressures, was used as a model organism to study glyphosate resistance. Previous research has found transporters to be critical components in the development of resistance to glyphosate. Here, this mechanism was further investigated using in-lab evolutions to select and sequence glyphosate resistant yeast and assessing yeast containing gene knockout growth in the presence of glyphosate. An insertion in SRP72 provided an interesting new course for research. Srp72 is a component of the signal recognition particle, which targets integral membrane proteins to the plasma membrane via co- translational translocation. Additionally, mutations were found in multidrug transporter genes, QDR1 and YCF1, in glyphosate resistant strains. Using the limited data from this study, ensuing research around genetically acquired glyphosate resistance was given future direction.

277 Investigating the effects of nutrient availability on genome stability in S. cerevisiae. L Henry, K Shroff, S Shah, A Akal-Strader, J.S. Choy Department of Biology, The Catholic University of America, Washington, DC. Nutrient signaling pathways integrate environmental cues and metabolic activities with cell cycle progression and growth. There has been a general focus on identifying the mutations that contribute to metabolic reprogramming and how metabolic changes can accommodate the nutrient demands of proliferating cells. However, it is not well understood how the changes in metabolic processes influence cellular responses to DNA damage, DNA replication and mitotic spindle stress. Toward a systematic analysis of the genetic reprogramming that accompanies nutrient availability, we investigated how glucose and oxygen affects the DNA damage response (DDR) in yeast cells. Using the budding yeast, S. cerevisiae, a collection of mutant alleles representing nearly 50% of all essential genes were screened for their ability to grow in the presence of different drugs that cause replication stress, spindle stress, or DNA breaks. We identified and confirmed a set of genes that are required to repair DNA damage only under limiting glucose conditions. These genes have functions in a variety of pathways indicating that nutrient availability can impact the regulation of the DDR through diverse mechanisms. Among these genes, we are focused on elucidating the role of a highly conserved MAP kinase pathway in directing the DDR when glucose is limiting. This work serves as a foundation to garner new knowledge about differences in the DDR when cells are faced with nutrient limitation and point to potential vulnerabilities that may be useful targets in anti-cancer therapies.

278 Tools for improving secondary metabolite production via heterologous expression in yeast. Angela Chu1, Curt Fischer1, Sundari Surresh1, Justin Smith1, Molly Miranda1, Joseph Horecka1, James Li1,2, Ronald Davis1,2,3, Lars Steinmetz1,3,4, Colin Harvey1, Maureen Hillenmeyer1, Robert St. Onge1 1) Stanford Genome Technology Center, Stanford University, Stanford, CA; 2) Department of Genetics, Stanford University, Stanford, CA; 3) Department of Biochemistry, Stanford University, Stanford, CA; 4) EMBL, Heidelberg, Germany. Synthesis and heterologous expression of fungal natural product gene clusters in yeast offers tremendous potential for discovery of new molecules for medicinal and industrial applications. Here, we describe tools and resources that leverage the genetic tractability of yeast for improving production of secondary metabolites produced via heterologous expression. Using an optimized S288C strain with improved respiratory fitness and sporulation efficiency (DHY), we established a robust locus for expression of large synthetic gene clusters. This locus is compatible with Recombinase Directed Indexing (REDI), which enables high-throughput genetic modification and subsequent characterization of these gene clusters. We compare compound production from the integration locus to that of plasmid-based gene clusters. We also target native yeast metabolic genes for repression using CRISPR interference (CRISPRi), with the goal of diverting metabolic flux towards secondary metabolite production. High-throughput LC/MS analysis identified several yeast genes that, when repressed, significantly improved compound production, thereby revealing genetic targets for further optimization. Finally, to circumvent the limited throughput of LC/MS-based detection, we developeda novel growth-based,small-molecule “biosensor.” This biosensor enables high-throughput screening of native yeast genes, or gene cluster variants, to identify factors that influence productivity.

279 Structure/Function Analysis of the Hif1 Histone Chaperone in Saccharomyces cerevisiae. N.S. Dannah Chemistry and Biology, Ryerson University, Toronto, ON, CA. Understanding how eukaryotic cells assemble their chromatin is a significant research subject in part because several human pathologies including cancer are associated with defects in chromatin assembly. Transporting of newly synthesized histones H3/H4 occurs in a stepwise fashion and is regulated by a variety of protein factors including histone chaperons. In the budding yeast Saccharomyces cerevisiae, the Hif1 protein is an evolutionarily conserved H3/H4-specific histone chaperone and a member of the nuclear Hat1 complex that catalyzes the deposition-related acetylation of newly synthesized histones H4. Hif1 as well as its human homolog NASP have been implicated in an array of chromatin-related processes including histone H3/H4 transport, chromatin assembly, DNA repair and telomeric silencing. In this study, we elucidate structural and functional aspects of Hif1. Through targeted mutational analysis, we demonstrate that the acidic region of yeast Hif1 which interrupts the TPR2 is essential for physical interaction with the Hat1-complex. We also demonstrate that Hif1 requires its C- terminal basic patch for nuclear localization. Furthermore, we provide evidence for the involvement of Hif1 in regulation of histone metabolism by showing that cells lacking HIF1 are both hypersensitive to histone H3 overexpression. Finally, we describe a functional link with a transcriptional regulatory protein Spt2 possibly linking Hif1 to transcription-associated

101 chromatin reassembly. Taken together, our results provide novel mechanistic insights into Hif1 functions and establish it as a key player in various chromatin-associated processes.

280 Biochemical interaction between the MAPK Slt2 of the PKC1-MAPK pathway and Grx3/Grx4 glutaredoxins influence the oxidative stress response in budding yeast. Nuria Pujol-Carrion, Maria Angeles de la Torre-Ruiz Dep. Basic Medical Sciences-IRBLleida, University of Lleida, SPAIN. This study demonstrates that both monothiol glutaredoxins Grx3 and Grx4 physically interact with the MAPK Slt2 forming a complex involved in the cellular response to oxidative stress. The simultaneous absence of Grx3 and Grx4 provokes a serious impairment in cell viability, Slt2 activation and Rlm1 transcription in response to oxidative stress. Both in vivo and in vitro results clearly show that Slt2 can independently bind either Grx3 or Grx4 proteins. Our results suggest that Slt2 form iron/sulphur bridged clusters with Grx3 and Grx4. We present evidence demonstrating that Cys38, Cys139 and Cys165 of Slt2 are important for binding to Grx4 whereas only Cys165 plays a role in binding to Grx3. Both active sites of Grx3 and Grx4 are required for binding to Slt2 since mutations in Cys174 from Grx4 or in Cys221 from Grx3 seriously impair binding to Slt2. Oxidative stress and iron availability promote Slt2 dimerisation through Cys165 residue. Our results contribute to extend the functions of both monothiol glutaredoxins to the regulation of a MAPK relevant for the oxidative stress response.

281 Biochemical and genetic characterization of two basidiomycete biosynthetic gene clusters by assembly and expression in yeast. Curt R. Fischer1,3, Mancheng Tang2, Angela Chu3, Sundari Suresh3, Molly Miranda3, Justin Smith3, Colin J.B. Harvey3, Maureen E. Hillenmeyer3, Yi Tang2, Robert St. Onge2 1) ChEM-H, Stanford University, Stanford, CA; 2) Department of Chemical Engineering, UCLA, Los Angeles, CA; 3) Stanford Genome Technology Center, Stanford University, Stanford, CA. Biosynthetic gene clusters (BGCs) from yeast's deeply divergent sister clade, the Basidiomycota, are an underexplored source of bioactive specialized metabolites. Basidiomycota, which include mushrooms, puffballs, smuts, rusts, and crusts, aren’t easily cultivable in most labs. To study basidiomycete BGCs, we developed a heterologous expression platform to synthesize, assemble, express, and re-engineer fungal biosynthetic gene clusters in yeast. Here we here report biochemical and genetic characterization of polyketide synthase (PKS) genes from the white-rot fungus Punctularia strigosozonata (tree bacon) and from the brown-rot fungus Hydnomerulius pinastri (spiny dry rot).

Expression of an autoinducible, single-copy, genomically-integrated P. strigosozonata PKS resulted in accumulation of acylated amino acids in the culture supernatant. We hypothesized that these polyketide/amino acid hybrid molecules were formed directly by the PKS, and that the thioesterase domains of these enzymes have evolved to use amino acids as the polyketide- releasing nucleophile. The observed products of the P. strigosozonata PKS were N,S-disorbyl cysteine and N,S-disorbyl homocysteine. Using a targeted CRISPRi screen of 96 yeast essential genes, we identified host (i.e. yeast) genes whose repression modulated the relative proportion of these two products, implying that the host, not the heterologously expressed PKS gene, can control the product spectrum of this PKS. We also identified unusual sequence characteristics of the P. strigosozonata PKS thioesterase domain that may be responsible for its ability to acylate both the N and S position of cysteine and homocysteine. These sequence characteristics occur widely in fungal thioesterase domains. We also a expressed a PKS from H. pinastri, and it also yielded an amino acid/polyketide hybrid molecule.

In conclusion our study shows that yeast is an attractive host for basidiomycete biosynthetic gene clusters, and that polyketide / amino-acid hybrid molecules can be synthesized by polyketide synthase enzymes acting alone.

282 A yeast producer’s perspective – developing new yeasts fit for industrial applications. J. Fisher, M. Oeser, A. Argyros Strain development, Mascoma LLC/ Lallemand, Lebanon, NH. Saccharomyces cerevisiae, an extremely efficient fermenter capable of withstanding an array of harsh conditions, has long been the workhorse of the fermented products industries. Genetic tractability and the availability of modern molecular tools position this organism extremely well to bring the innovations of synthetic biology to industrial scale. However, any scientist wishing to successfully deploy a new industrial yeast product must consider several key factors. Strains developed for an industrial purpose must be genetically and phenotypically stable, well-suited for their application, and amenable to large- scale production.

Here we present bioethanol-producing yeast as a case study for developing an industrial yeast product. We outline the most important processes in which a strain is expected to perform consistently, and we discuss some of the challenges present in each step. We discuss ways in which industry and academia can work together to develop yeast-based products of the future.

283 Evidence for a moonlighting role of the N-acetylglucosamine kinase from Yarrowia lipolytica. CL Flores, C. Gancedo, I. Arroyo Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM, Madrid, Madrid, ES. N-acetylglucosamine (NAGA) kinase catalyzes the first intracellular step of NAGA metabolism in those organisms that use this sugar. In the yeast Yarrowia lipolytica the transcription levels of the genes encoding the NAGA catabolic enzymes are elevated during growth in NAGA and very low during growth in glucose. Disruption of the gene YlNAG5 that encodes NAGA kinase causes inability to grow in NAGA and changes in the transcription pattern of NAGA catabolic pathway genes. In 102 an Ylnag5 mutant the transcription levels of these genes are elevated during growth in glucose, reaching levels similar to those found when the wild-type strain grows in NAGA. At least two possibilities might account for this behaviour: a) the YlNAGA kinase is a moonlighting protein that participates in the control of transcription b) intracellular NAGA, likely derived from chitin turnover, is an inducer of transcription and lack of NAGA kinase allows its build-up. If the YlNAGA kinase is a moonlighting protein there is a possibility that expression of a heterologous NAGA kinase might complement only one of the two functions of this protein; either the metabolic or the regulatory one. Several heterologous NAGA kinases have been expressed in an Ylnag5 mutant and among them the human NAGA kinase failed to complement the absence of growth of the mutant in NAGA while it restored the repression of the genes of the NAGA catabolic pathway during growth in glucose. As a reporter of the transcription state of the genes of the pathway we used the enzymatic activity of the glucosamine-6P deaminase encoded by the gene YlNAG1. We also tested the hypothesis of the internal NAGA being the inducer of the transcription of the NAGA catabolic pathway genes. In the absence of NAGA in the culture medium hydrolysis of chitin during remodelling of the cell wall could be a source of this sugar. This NAGA needs to be internalized by the NAGA transporter YlNgt1. If NAGA were the inducer of the transcription, in an Ylngt1 nag5 mutant that cannot internalize the sugar the NAGA catabolic pathway genes should be repressed. However, we found that in such a mutant the glucosamine-6P deaminase is derepressed. Our results are consistent with the idea of NAGA kinase from Y. lipolytica being a moonlighting protein. Acknowledgments.- This work was supported by grant CIVP18A3896 of the Fundación Ramón Areces (Madrid, Spain).

284 Construction and characterization of a strain of Saccharomyces cerevisiae able to grow on glucosamine as carbon and nitrogen source. C. Gancedo, CL Flores Instituto de Investigaciones Biomédicas "Alberto Sols" CSIC-UAM, Madrid, ES. The aminosugar glucosamine is taken up by Saccharomyces cerevisiae and phosphorylated by hexokinase but is not metabolized further. A search of genomic sequences in S. cerevisiae showed that there is no gene similar to those encoding enzymes catalyzing the deamination-isomerization reaction leading from glucosamine-6-phosphate to fructose-6-phosphate. Such enzymes are present in yeasts, such as Yarrowia lipolytica, able to use N-acetylglucosamine as carbon source. We have cloned the gene YlNAG1 encoding glucosamine-6-phosphate deaminase from Y. lipolytica and introduced it in S. cerevisiae. The transformed S. cerevisiae strain grew in media with glucosamine as only carbon and nitrogen source. Growth in glucosamine required a functional respiratory chain as shown by the lack of growth in this sugar in the presence of antimycin A. A petite derived from the strain was also unable to grow on the sugar. During growth in glucosamine the imbalance between the carbon and nitrogen provided and that required by the yeast resulted in excretion of ammonium to the medium. We have constructed strains overexpressing only one of the main hexose transporters from S. cerevisiae and found that those harbouring HXT1, HXT2, HXT3 or HXT4 did grow in glucosamine, while those overexpressing only HXT5 or HXT6/7 did not. Glucosamine caused catabolite repression of different enzymes but, in some cases, to a smaller extent than glucose. The availability of a strain of S. cerevisiae able to grow in glucosamine opens the possibility to study the effects of this important sugar in a well documented organism. Acknowledgments.- This work was supported by grant CIVP18A3896 of the Fundación Ramón Areces (Madrid, Spain).

285 Structural & functional analysis of yeast Aqr1 involved in amino acid excretion. G. C. Kapetanakis1, C. Gournas1, M. Prevost2, B. André 1, I. Georis3 1) Molecular Physiology of the Cell, Université Libre de Bruxelles, 6041 Gosselies; 2) Structure and Function of Biological Membranes, Université Libre de Bruxelles, 1050 Brussels, Belgium; 3) Labiris, Brussels, Belgium. Amino acids are the most abundant nitrogenous compounds in yeast and all other organisms. Their uptake into yeast cells involves a wide set of plasma membrane permeases which have been intensively investigated. Yeast cells are also capable of excreting amino acids. This is most readily detectable in strains overproducing a particular amino acid and when the permeases mediating its uptake are nonfunctional. For instance, cells expressing a feedback-insensitive mutant form of aspartate kinase, an enzyme involved in homoserine and threonine biogenesis, overproduce both compounds and detectably excrete them when the Gap1, Agp1, and Gnp1 permeases catalyzing their re-uptake are inactive. This strain has been exploited to identify the Aqr1 transporter as a major actor of this excretion (1). The protein belongs to the multidrug- resistance family of the Major Facilitator Superfamily (MFS). Our current work aims at further characterizing Aqr1 as an amino-acid excretion protein. Structural models of Aqr1 have been built using the solved structures of several bacterial MFS transporters as templates, and amino acid residues potentially involved in substrate recognition have been identified by docking calculations. Novel excretion assays based on yeast cells entrapped in alginate beads have been set up. We also analyze the intracellular traffic of Aqr1 to assess the proposed model that the protein releases amino acids into the external medium by first accumulating them into small vesicles, which then fuse with the plasma membrane via exocytosis (1). Finally, conditions have been set up to investigate excretion by yeast of two additional amino acids, namely tryptophan and lysine. The progress state of this project aiming at better understanding the mechanisms of amino acid excretion in yeast will be presented. (1) Velasco, I., Tenreiro, S., Calderon, I. L., & André, B. (2004). Saccharomyces cerevisiae Aqr1 is an internal-membrane transporter involved in excretionof amino acids. Eukaryotic Cell, 3(6),1492–1503.

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286 New mitochondrial function assay technology for yeasts. In Iok Kong, Barry Bochner Biolog Inc., Hayward , CA. Saccharomyces cerevisiae is an important model organism for human biology research because the ease of genetic manipulation facilitates studies of gene and organellar function. The mitochondrion is an organelle of particular interest because functional defects can lead to wide-ranging energetics-related human disorders including cancer, ageing, neurological disorders, metabolic disorders, and immunodeficiencies. The standard method for assaying mitochondrial function in yeast involves a laborious and multistep procedure of removing the cell wall to generate spheroplasts, followed by purification of mitochondria. We sought a simpler approach to permeabilize the yeast membrane and avoid the multistep procedure. After examining several options, we found one method using digitonin that worked well and can effectively permeabilize the plasma membrane. Digitonin sequesters membrane sterols and instantaneously creates pores, allowing the mitochondria to be assayed in situ. The pores allow extracellular small molecules to enter the cell while intracellular small molecules leak out. Our assay approach has allowed us to utilize the PM1 MicroPlate™, a panel of 95 biochemical substrates, to examine both the mitochondrial metabolism using permeabilized cells and the live cell metabolism using unpermeabilized cells. Yeast strains are cultured in lactate and glycerol-containing medium, harvested, washed, and resuspended. The cells can then be optionally permeabilized with digitonin and monitored for metabolism using a tetrazolium redox dye mix that picks up electrons at the distal end of the mitochondrial electron transport chain and turns purple. In wells that contain biochemical substrates that are metabolized, electrons flow from those substrates to NADH or FADH2 and then down the electron transport chain to the redox dye. Thus the assays simply involve pipetting cells into the wells of the PM1 MicroPlate™ and following the kinetics of purple color formation to measure either live cell or mitochondrial metabolism of substrates. Although Saccharomyces cerevisiae metabolizes a limited set of fermentable sugars in the PM1 MicroPlate™, the metabolism of the mitochondria in permeabilized cells is entirely different and much more complex. With minor variations, the same protocol can be used with other yeasts, such as Schizosaccharomyces pombe and Candida albicans, to compare their live cell and mitochondrial substrate metabolisms.

287 Substantial increase of isocitric acid-yield by the deletion of YHM2, a novel mitochondrial carrier for citrate in the oleaginous yeast Yarrowia lipolytica. Z. Kövesi, M. Gatter, G. Barth Institute of Microbiology, Technische Universität Dresden, Sachsen, DE. Background: Isocitric acid (ICA) is a biologically active compound involved in the tricarboxylic acid cycle. ICA is suitable for nutritional use, but it is medicinal applicability is also gaining increasing attention. Although the hemiascomycetous yeast Yarrowia (Y.) lipolytica is a natural producer of ICA, it mainly secretes citric acid (CA). This imbalance in favor of CA highly diminishes the ICA-yield. In the past, there have been several attempts to genetically modify this whole-cell biocatalyst in order to enhance the production of isocitric acid. Results: In this study, we identified a putative mitochondrial transporter protein responsible for the export of citrate out of the mitochondrion into the cytosol. This carrier protein is encoded by the novel gene YHM2. By deleting YHM2 the ICA/CA product ratio could be increased from 12 % to 92 % compared to the wild-type strain. Within five days up to 71.3 g l-1 ICA (ICA/CA ratio: 86 %) with sunflower oil as the carbon source and 15.5 g l-1 ICA (ICA/CA ratio: 95 %) with glucose as the sole carbon source could be achieved under production conditions in a 600 ml-bioreactor. Additional overexpression of the aconitase encoding gene ACO1 in the YHM2-lacking strain led to a drastic decrease in the ICA/CA ratio (27 % & 32 %). A single-copy overexpression of YHM2 resulted in a slight increase in the ICA/CA product ratio to 22 % while simultaneously reducing the total acid production. An ICA/CA ratio of 98 % could be achieved by cultivating in medium containing 25 M itaconic acid, a strong inhibitor of the isocitrate lyase in Y. lipolytica. Conclusion: Within this work, the novel transport protein YHM2 has been identified in Y. lipolytica. This putative mitochondrial citrate and oxoglutarate carrier represents an interesting metabolic target for the production of isocitric acid with this yeast. Although the productivity rates for ICA do not reach commercially viable levels yet, near CA-free supernatants could be achieved by the addition of itaconic acid to the production medium.

288 Utilization of competing lipid biosynthetic pathways induced by rescue of met4∆ with nutritional supplementation or opi1∆. M. A. Kukurugya, T. Mahatdejkul-Meadows, B. J. Wranik, R. S. McIsaac, B. D. Bennett Calico Life Sciences LLC, South San Francisco, CA. Saccharomyces cerevisiae lacking Met4p, a regulator of S-adenosyl methionine synthase (SAM2), cannot grow without supplementation of methionine or S-adenosyl methionine (SAM). SAM is the methyl-donor for many metabolic reactions, including synthesis of the membrane forming lipid phosphatidylcholine (PC). PCs are synthesized via two main pathways: three subsequent methyl group additions to phosphatidylethanoamines (PEs) from SAM (the methylation pathway) or condensation of diacylglycerol and CDP-choline (the Kennedy pathway). Here we determined the relative levels of over 300 metabolite and lipid species in met4∆ and met4∆opi1∆ mutants relative to WT via liquid chromatography-mass spectrometry. This analysis allowed us to catalog changes in sulfur and phospholipid metabolism concurrent with rescue of the growth defect of a met4∆ strain. We examined supplementation of yeast nitrogen base without amino acids with methionine, choline, SAM, or methionine + choline, as well as knock out of OPI1 for their ability to rescue phospholipid biosynthesis in the met4∆ background. We found that the met4∆ strain could be rescued in both growth and lipid profile by supplementing SAM in minimal medium, or by supplementation of choline + methionine, but not by supplementation of choline alone. With methionine supplementation, PC biosynthesis was rescued by increased utilization of the methylation pathway. In the case of 104 methionine + choline supplementation, we found increased usage of the Kennedy pathway. We also found that met4∆opi1∆, which was previously known to rescue the growth of met4∆, dramatically increased the utilization of the methylation pathway. Despite the fact that the met4∆ strain is limited in growth rate by SAM production, and SAM is required to make PCs in the absence of choline, the PC content of the met4∆ strain was the same as the WT strain. In the met4∆opi1∆ strain, we found significantly increased levels of PCs relative to wild type and met4∆, and decreased levels of monomethyl- and dimethyl- phosphatidylethanoamine intermediates in the methylation pathway, with no effect on growth rate, regardless of the media supplementation. Taken together, these findings suggest that the WT level of PCs is near the minimal acceptable levels for normal growth, but increased levels of PCs in the membrane are tolerable for a similar growth rate.

289 Regulation and function of TORC1 complex during development of yeast colonies. J. Marsikova1, D. Wilkinson1, L. Vachova2, Z. Palkova1 1) Department of Genetics and Microbiology, Charles University, Faculty of Science, Biocev, Vestec, CZ; 2) Institute of Microbiology of the CAS, v.v.i., BIOCEV, Prague, Czech Republic. Yeast growing on solid surfaces are able to develop organized multicellular colonies, composed of cells with different physiological and morphological features. In our laboratory we characterized two major cell subpopulations: U-cells localized to upper colony regions and L-cells localized to the colony interior [1, 2]. On one hand, U-cells possess biosynthetic capacity controlled by nutrient sensing pathways and are metabolically active cells with longevity features. On the other, U-cells behave similarly to starving cells (e.g. activating autophagy and accumulating glycogen). L-cells are starving and stressed. U- cells activate a unique metabolism that is important for survival of these cells. U-cells activate TORC1, as identified using a strain expressing GFP-labeled Gat1p. The Gat1p transcription factor is present in the cytosol in active TORC1 kinase conditions and enters the nucleus upon TORC1 kinase inactivation. We observed that a majority of U-cells exhibit rapamycin- sensitive cytosolic Gat1p-GFP localization, while predominantly nuclear localization of Gat1p-GFP was detected in L-cells [1, 2]. The evolutionarily conserved TORC1 plays a central role in promoting cell growth, biosynthesis and proliferation, while negatively regulating catabolic processes catabolic processes (including autophagy) and the general stress response. The purpose of this work is to identify processes, potentially regulated by the TORC1 pathway in colony cell subpopulations and thus extend our knowledge of yeast colony development. We performed genome-wide transcription profiling by RNA sequencing of cell subpopulations isolated from different areas of colonies (lower, upper and margin regions) and in different developmental phases (acidic- and alkali-phase colonies). The results showed large expression differences between individual cell subpopulations. Gene ontology (GO) analysis of genes, differentially expressed between particular subpopulations allowed us to propose a model of metabolic reprogramming that occurs in individual colony subpopulations [3]. The data obtained contributed, among other things, to identifying metabolic processes potentially regulated by the TORC1 complex. The work was supported by GAUK 912218.

290 Magnesium uptake by connecting fluid-phase endocytosis to an intracellular inorganic cationfilter. S Klompmaker, K. Kohl, N. Fasel, A. Mayer Department of Biochemistry, University of Lausanne, Epalinges, CH. Cells acquire free metals through plasma membrane transporters. But, in natural settings, sequestering agents often render metals inaccessible to transporters, limiting metal bioavailability. Here we identify a pathway for metal acquisition, allowing cells to cope with this situation. Under limited bioavailability of Mg2+, yeast cells upregulate fluid-phase endocytosis and transfer solutes from the environment into their vacuole, an acidocalcisome- like compartment loaded with highly concentrated polyphosphate. We propose that this anionic inorganic polymer, which is an avid chelator of Mg2+, serves as an immobilized cationfilter that accumulates Mg2+ inside these organelles. It thus allows the vacuolar exporter Mnr2 to efficiently transfer Mg2+ into the cytosol. Leishmania parasites also employ acid- ocalcisomal polyphosphate to multiply in their Mg2+-limited habitat, the phagolysosomes of inflammatory macrophages. This suggests that the pathway for metal uptake via endocytosis, acidocalcisomal polyphosphates and export into the cytosol, which we term EAPEC, is conserved.

291 The white willow bark extract delays yeast chronological aging because it remodels lipid metabolism to decelerate the age-related onset of liponecrotic cell death. Y. Medkour1, K. Mohammad1, P. Dakik1, M. McAuley1, N. Medkour2, T. Taifour1, V. Titorenko1 1) Concordia University, Montreal, PQ, CA; 2) McGill University, Montreal, PQ, CA. The yeast Saccharomyces cerevisiae is a unicellular eukaryote that has been successfully used as a model organism for the discovery of signaling pathways and chemical compounds that modulate cellular aging and define organismal lifespan not only in yeast but also in various multicellular eukaryotes. Using a robust quantitative assay, we screened a library of plant extracts for extracts that can delay chronological aging in S. cerevisiae. Our screen identified the white willow bark extract PE21 as the most potent aging-delaying pharmacological intervention yet described. The longevity-extending efficiency of PE21 greatly exceeds those of currently known natural anti-aging compounds, such as resveratrol, rapamycin, spermidine, caffeine, and lithocholic acid. We demonstrated that PE21 slows yeast chronological aging because it attenuates Sch9, a protein kinase stimulated by both the nutrient-sensing TORC1 and the sphingolipid-dependent Pkh1/2 signaling pathways. Our mass spectrometry-based quantitative lipidomic analyses revealed that PE21 markedly remodels cellular lipidome of chronologically aging yeast. We found that PE21 alters the age-related chronology of changes in the concentrations of different lipid classes as follows: 1) it decreases the concentrations of free fatty acids (FFA) and triacylglycerols (TAG); and 2) it lowers the concentrations of cardiolipins and increases the concentration of phosphatidylethanolamine, both of which are synthesized in mitochondria. We showed the following: 1) genetic interventions that reduce the concentration of FFA by attenuating the lipolysis of TAG enhance the aging-delaying efficiency of PE21, delay the age-related onset of liponecrotic 105 regulated cell death (RCD), and increase cell resistance to liponecrotic RCD elicited by an exposure to exogenous palmitoleic acid (POA); and 2) genetic interventions that increase the concentration of FFA by weakening the incorporation of FFA into phospholipids lower the aging-delaying efficiency of PE21, accelerate the age-related onset of liponecrosis, and increase cell susceptibility to POA-induced liponecrotic RCD. Our findings suggest a mechanism through which PE21 slows yeast chronological aging by specifically remodeling lipid metabolism in the endoplasmic reticulum, lipid droplets, peroxisomes, and mitochondria. The resulting changes in the intracellular concentrations of several lipid classes decelerate the age-related onset of liponecrotic RCD.

292 Flavin-based metabolic cycles are integral features of growth and division in single yeast cells. R. O'Laughlin1, B. Baumgartner2, M. Jin3, L. Tsimring3, N. Hao4, J. Hasty1,3,4 1) University of California, San Diego, Department of Bioengineering; 2) Booz Allen Hamilton; 3) BioCircuits Institute, University of California, San Diego; 4) Molecular Biology Section, Division of Biological Science, University of California, San Diego. The yeast metabolic cycle (YMC) is a fascinating example of biological organization, in which cells constrain the function of specific genetic, protein and metabolic networks to precise temporal windows as they grow and divide. However, understanding the intracellular origins of the YMC remains a challenging goal, as measuring the oxygen oscillations traditionally associated with it requires the use of synchronized cultures growing in nutrient-limited chemostat environments. To address these limitations, we used custom-built microfluidic devices and time-lapse fluorescence microscopy to search for metabolic cycling in the form of endogenous flavin fluorescence in unsynchronized single yeast cells. We uncovered robust and pervasive metabolic cycles that tightly coupled to the cell division cycle (CDC) and oscillated across different nutrient conditions. We then studied the response of these metabolic cycles to chemical and genetic perturbations, showing that their coupling with the CDC can be altered through treatment with rapamycin, and that metabolic cycles continue even in respiratory deficient strains. These results provide a foundation for future studies to investigate the physiological importance of metabolic cycles in processes such as CDC control, metabolic regulation and cell aging.

293 Elucidating the effects of MCHM on yeast metabolism. Amaury Pupo Merino1, Michael Ayers1, Kang Mo Ku2, Jennifer Gallagher1 1) Department of Biology, West Virginia University, Morgantown, WV; 2) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV. On January 2014 approximately 10,000 gallons of crude 4-Methylcyclohexanemethanol (MCHM) and propylene glycol phenol ether were accidentally released into the Elk River, West Virginia, contaminating the tap water of around 300,000 residents. Crude MCHM is an industrial chemical used as flotation reagent to clean coal. At the time of the spill, MCHM's toxicological data were limited, an issue that have been addressed by different studies focused on understanding the immediate and long term effects of MCHM on human health and the environment. Using yeast as a model organism we study the effect of acute exposition to crude MCHM on metabolism. BY4741 cells were treated with MCHM 0.055% on YPD for 30 minutes (60, 90 and 120 minutes long treatments were also tested). Polar and lipid metabolites were extracted from cells by a chloroform-methanol-water mixture, following a modified Bourque and Titorenko’s protocol. The extracts were dried and derivatized (MTFSA for polar and BSTFA for lipid compounds) and analyzed by GC/MS. Polar fraction compounds were identified against a characterized set of primary metabolites by targeted metabolomics. This identification was complemented by queries against NIST database (untargeted metabolomics). For the lipid fraction compounds only the untargeted metabolomics approach was used. 32 and 46 compounds were identified and quantified from the polar and lipid fraction, respectively, for a total of 66 unique compounds. MCHM affects amino acid metabolism (A, D, E, R, P, G, S, T, V, L and I), glutathione and inositol phosphate metabolisms and steroid biosynthesis. These results partially confirm and enrich previous RNA-Seq and flow cytometry data and provide evidence of the early effect of MCHM on yeast metabolism.

294 Next generation platforms for strain optimization. L. Raetz, A. Meadows, D. Abbott, J. Hung, C. Reeves, A. Singh, B. Kaufmann-Malaga, Y. Zhang, J. Lerman, D. Dougherty, A. Kilbo, S. Mantovani, C. Yu, E. Wilson, O. Erbilgin, S. Chandran Amyris, Emeryville, CA. The natural products world is unparalleled in its molecular diversity and wide application space. There are however numerous challenges associated with realizing the full potential of these molecules. Optimization of microbial production of any natural product requires repeated iterations of the design-build-test-analyze engineering cycle. The rate at which any team can execute the engineering cycle directly affects the development time for any product. Similarly, the magnitude of strain improvement in each cycle impacts overall costs and development time. At Amyris, advanced tools for strain engineering, high throughput screening, analytics, and bioinformatics have been developed over the years to rapidly accelerate the engineering cycle and reduce the number of necessary iterations. With these capabilities, scientists at Amyris can efficiently optimize multiple natural product pathways simultaneously. This presentation will cover details of the next- generation strain engineering platforms that enable Amyris scientists to rapidly iterate through multiple cycles of the strain optimization process, as well as the engineering of metabolic pathways to convert naïve yeast strains into commercial scale production hosts for various natural products.

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295 Cloning and characterization of truncated sugar transporters from Spathaspora yeasts isolated from rotting wood. Marilia M. Knychala, Leonardo G. Kretzer, Angela A. dos Santos, Belisa B. de Sales, Boris U. Stambuk Department of Biochemistry, Universidade Federal de Santa Catarina, Florianopolis, Santa Catarina, BR. Saccharomyces cerevisiae is the main microorganism employed for bioethanol production, but it is unable to use xylose as a carbon source. One bottleneck for the fermentation of xylose is the lack of efficient xylose transporters, and thus xylose- fermenting yeasts isolated from rotten wood may represent a good source of new sugar transporters. The genome sequence of the xylose-fermenting yeasts Spathaspora passsalidarum and Spathaspora arborariae contain several genes that encode for sugars transporters. Although we have cloned six of them, none of the permeases allowed xylose fermentation by recombinant S. cerevisiae, despite that one of them (XUT1) clearly showed 14C-glucose and 14C-xylose transport activity when expressed in yeast. Since cloned heterologous sugar transporters may suffer endocytosis after ubiquitination of lysine residues, we have truncated the genes removing K residues from the N- or C-terminal domains of the cloned permeases. We used the DLG-K1 strain of S. cerevisiae, which lacks the major hexose transporters (hxt-null) and has high activity of the xylose reductase, xylitol dehydrogenase and xylulokinase enzymes allowing xylose utilization, and transform it with plasmids containing genes encoding sugar transporters from S. arborariae (SaXUT1) or S. passalidarum (SpXUT1). These genes were also amplified removing the last 22 C-terminal residues of SpXUT1, including 3 lysine residues predicted to be ubiquitinated (T- SpXUT1), or the last 18 C-terminal residues as well as the first 17 N-terminal residues of SaXUT1, removing 4 lysine residues that could be ubiquitinated (T-SaXUT1). While the full-length SaXUT1 or SpXUT1 permeases could not allow the recombinant yeast strains to grow on or consume glucose or xylose, the truncated (and K-less) transporters (T-SaXUT1 and T-SpXUT1) allowed not only growth and glucose fermentation, but also xylose fermentation by recombinant S. cerevisiae. Our results indicate that the expression of C- or N-terminal truncated (and lysine-less) sugar permeases is a promising strategy to discover novel xylose transporters from xylose-fermenting yeasts. Financial support: CAPES, FINEP, FAPESC and CNPq.

296 A Saccharomyces cerevisiae genetic platform to functionally characterize genetic and biochemical redundancy in the catalytic components of the fatty acid elongase complex. K.E. Stenback1,4, A.A. Campbell1,3,4, K. Flyckt1,4, T. Hoang1, B.J. Nikolau1,2,3,4 1) Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA; 2) W.M. Keck Metabolomics Research Laboratory, Iowa State University, Ames, IA; 3) Center for Metabolic Biology, Iowa State University, Ames, IA; 4) NSF Engineering Research Center for Biorenewable Chemicals, Iowa State University, Ames, IA. Fatty acids that are longer than 18 carbons in length, or very long chain fatty acids (VLCFAs), play essential roles in a number of crucial cellular processes. The biosynthesis of VLCFAs occurs by the iterative extension of pre-existing fatty acyl-CoAs through a series of four-reactions catalyzed by four distinct enzymes that are components of the fatty acid elongase (FAE) complex. The first reaction in FAE is the Claisen condensation between malonyl-CoA and a fatty acyl-CoA primer. Two distinct classes of enzymes catalyze this condensation reaction, either a plant-specific 3-ketoacyl-CoA synthase (KCS) or an ELO enzyme that is prevalent among eukaryotic organisms, including plants, fungi, and animals. Multiple gene paralogs of both KCS and ELO enzymes occur in plant genomes (e.g., 27 and 6 respectively, in the maize genome), which contributes the greatest genetic and biochemical complexity within the overall FAE system. This complexity, along with the fact that the FAE system is an integral membrane complex, are major barriers to its characterization. To overcome these challenges, we have created a Saccharomyces cerevisiae platform, which comprises a series of strains each individually expressing plant FAE components. We have shown that each of the individual plant FAE components are capable of competently replacing the yeast homologs. The substrate specificity of KCS enzymes as well as the function of additional FAE components is being explored further by reconstituting yeast strains that are void of their native FAE components, which have been replaced by ‘pure’ plant FAE systems. The construction of these yeast strains will eliminate genetic and biochemical redundancy and allow for the biochemical characterization of each enzyme component.

297 Yeast Trk transporters mediate potassium uptake and contribute to cell pH homeostasis and fitness. Hana Sychrova, Hana Elicharova, Vicent Llopis-Torregrosa, Klara Papouskova Dept Membrane Transport, Inst Physiology CAS, Prague 4, CZ. The regulation of ion and pH homeostases is an essential process critical for cell viability. The maintenance of high intracellular concentrations of potassium and neutral pH is important for a variety of cellular functions including cell volume, DNA integrity, protein modification and trafficking. It is becoming increasingly evident that the coordination between primary H+-ATPases and transport systems involved in the influx and efflux of potassium allows this pH maintenance to occur. Genes encoding three types of potassium uptake systems have been found in yeast genomes (Trk1 uniporters, Hak1 proton-K+ symporters, Acu1 ATPases). Heterologous expression of these transporters in S. cerevisiae lacking its own potassium uptake system revealed that the activity of Trk transporters is crucial for the control of membrane potential, intracellular pH and cell fitness. Deletion of TRK genes results in an increase of membrane potential and decrease of intracellular pH in both osmotolerant and pathogenic yeast species. In C. glabrata, it results also in a change of cell surface properties, decreased virulence and pathogenicity. Taken together, our results find the Trk-type potassium uptake systems in Candida cells to be a promising target in the search for their specific inhibitors and in developing new antifungal drugs. This work was supported by grants from the Czech Science Foundation (GA CR 16-03398S) and from the FP7-PEOPLE-2013- ITN ImresFun (606786).

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298 Control of mitochondrial protein synthesis machinery in response to cellular energy requirements in Saccharomyces cerevisiae. Y. Verma, U. Mehra, A. Nair, K. Datta Department of Genetics, University of Delhi South Campus, New Delhi, IN. Budding yeast generates ATP either through fermentation or respiration depending upon availability of carbon source. Nuclear gene regulation in response to glucose is understood to some extent; however mitochondrial gene expression and its regulatory factors remain unexplored. Mitoribosomes maturation is a well-organized process that involves products encoded from both nuclear and mitochondrial genome. Many proteins are conserved from bacteria to humans; however few of them have evolved to function in a species-specific manner. Maturation of mitoribosomes is abetted by numerous accessory factors like GTPases, helicases, rRNA modifiers, chaperons etc. We have examined the role of two GTPases, MTG3 and MRX8 in ribosome maturation/translation. MTG3 belongs to a circularly permuted class of GTPase, conserved from yeast to humans. Deletion of MTG3 leads to defects in the utilization of glycerol as sole carbon source and accumulation of 15S rRNA precursor indicating a role in small subunit biogenesis. We have shown that MTG3 associates with both small and large subunit of the mitochondrial ribosome. Cells harboring mtg3ts mutants at non-permissive temperature have aberrant levels of the large mitochondrial subunit. Our studies also indicate that nucleotide binding is essential for it's in vivo function but dispensable for its association with mitoribosomes. Mtg3p co-precipitates with large subunit assembly factor Mtg2p, indicating a role in its assembly/maturation. MRX8, a YihA class of GTPase, predicted to function in translation, has an orthologue in bacteria, yeast and in vertebrates including humans. The mitochondrial orthologs, all have an extension at their N-terminus in comparison to their bacterial counterpart. We have shown Δmrx8 cells have compromised cellular respiration. Mrx8p is localized to the mitochondrial matrix and associates with the 74S monosome irrespective of the nucleotide occupancy state. Although mrx8 mutants compromised for GTP and GDP binding are non-functional in vivo, however, mrx8 mutants predicted to be either preferentially bound to GTP or GDP in vivo are functional. Taken together this indicates that MRX8 might communicate changing NTP/NDP pools to mitochondrial ribosomes. Interestingly, the human orthologue of Mrx8p restored cellular respiration in Δmrx8 cells indicating functional conservation.

299 The Use of the Gene Ontology to Describe Biological Function at SGD. S. Aleksander, B.L. Dunn, S.R. Engel, T. Jackson, K.A. MacPherson, R.S. Nash, P. Ng, M.S. Skrzypek, E.D. Wong, J.M. Cherry, The SGD Project Department of Genetics, Stanford University, Stanford, CA. The Saccharomyces Genome Database (SGD; www.yeastgenome.org) is a comprehensive resource of curated molecular and genetic information on the genes and proteins of Saccharomyces cerevisiae. Since 2001, SGD has used the Gene Ontology (GO) to annotate the functions of gene products in budding yeast. The GO comprises three sets of structured, controlled vocabularies, or “ontologies”: the Molecular Function ontology describes activities of gene products; the Biological Process ontology places these molecular functions in a biological context; and the Cellular Component ontology indicates the subcellular localizations of gene products. Expert curators select GO terms to apply to gene products based on published scientific literature. At SGD, results from traditional experimental methods are the primary sources of evidence used to support GO annotations. In addition, results from comparative sequence and genomic studies, as well as analyses of functional genomic and proteomic data, have provided valuable insights into the biological roles of gene products, and these data are incorporated into SGD as well. SGD has several web interfaces and analysis tools that display and use these data. The Locus Summary lists each manually curated and high-throughput GO annotation and indicates when computational GO annotations are available. The GO Term Finder searches for significant shared GO terms used to describe the genes in an input list to aid in discovery of potential gene similarities. The GO Slim Mapper maps annotations of a group of genes to more general terms and/or bins them into broad categories, also known as “GO Slim” terms. Gene Ontology annotations are also incorporated into YeastMine, SGD’s multifaceted search and retrieval environment that provides access to diverse data types. Searches can be initiated with a list of genes or a list of Gene Ontology terms. These interfaces and tools are important as part of SGD's ongoing mission facilitate research, education, and discovery using the Gene Ontology.

300 Beyond S288C: Incorporating new S. cerevisiae strain genomes and their associated not-in-S288C ORFs into SGD. B. Dunn, S. R. Engel, G. Binkley, S. R. Miyasato, T. K. Sheppard, J. M. Cherry, The SGD Project Department of Genetics, Stanford University, Stanford CA. Over the past ~10-15 years the world has seen a breathtaking increase in the speed, ease, and efficiency of genome sequencing technology along with an equally breathtaking decrease in the associated costs. This has led to an explosion in the number of available high-quality whole-genome sequences of S. cerevisiae strains, isolated from a wide variety of geographical locations (all continents except Antarctica) and environmental niches, including natural, human-associated and industrial sources. As its name implies, the Saccharomyces Genome Database (SGD; www.yeastgenome.org) began as a repository of the genome sequence of S. cerevisiae, specifically the S288C lab strain, which represented the first wholly- sequenced eukaryotic genome. There are currently >1500 different whole-genome sequenced S. cerevisiae strains that have been described in a publication and deposited in sequence databases, with many more likely to be added in the coming months and years. We see both challenges and opportunities for SGD during the process of incorporating these large numbers of new genomes and curating the associated information; an example is the existence of ORFs in these strains that do not exist in the S288C reference genome. We will present ideas about various ways to store these data, as well as how to present strain-specific information and how to present information gleaned from comparison across all the whole-genome sequenced strains. These may include the addition of many more “not-in-S288C” Locus Summary Pages, the identification and 108 labeling of “core” ORFs (i.e., those shared by virtually all whole-genome sequenced strains) vs. “variable” ORFs, the display of sequence variation in ORFs across many strains, and the creation of “Strain Pages” for sequenced strains, showing relevant isolation and phenotypic information and links to the genome sequence. We hope that the addition of these strain genomes and associated information will be of great use to the yeast community.

301 Downloading Data from SGD. Felix Gondwe, Travis K. Shepperd, Ajay Shrivatsav Vichanthangal Prathivadhib, Shuai Weng, Pedro H. R. de Assis, Gail Binkley, J. Michael Cherry, The SGD Project Genetics, Stanford University, Palo Alto, CA. Data at the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) is accessed through a dynamic faceted search backed by Elasticsearch technology, a full-text search and analytics engine. Elasticsearch provides a powerful window into SGD’s complex data. Traditionally, SGD users used the File Transfer Protocol (FTP) server to download data files, but we are moving towards a more flexible approach by leveraging our search tool. File metadata has been loaded into the database and integrated into the greater SGD search. As a result, users can search for files the same way they might search for a gene or phenotype data. Results are matched based on metadata, including the file name, description, keywords, and the PMID if the file is associated with a published reference. The files themselves are stored using the AWS (Amazon Web Services) S3 storage service. Our new approach to downloading SGD data files provides our users with a more customized, end-to-end experience.

302 Yeast orphan gene project: Finding a place for ORFans to GO. P. Hanson1, T. Tobin2, E. Strome3, M. Miller4, D. Aiello5, J. Keeney6 1) Biology Department, Birmingham-Southern College, Birmingham, AL; 2) Biology Department, Susquehanna University, Selinsgrove, PA; 3) Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY; 4) Biology Department, Rhodes College, Memphis, TN; 5) Biology Department, Austin College, Sherman, TX; 6) Biology Department, Juniata College, Huntingdon, PA. Course-based undergraduate research experiences (CUREs) have numerous positive impacts on students, including increased knowledge of course content, independence, and interest in related subject matter. CUREs may also have potential to increase diversity in STEM by removing barriers that reduce access of underserved groups to more traditional, mentored research. However, the number of widely adopted, easily transferable CUREs is relatively small. Here we describe development, preliminary assessment, and propagation of a collaborative CURE that aims to determine the function of as yet uncharacterized Saccharomyces cerevisiae genes. More than 20 years after the sequencing of the budding yeast genome, nearly 10% of open reading frames (ORFs) are still considered uncharacterized. Thus, we formed and are growing a consortium of undergraduate researchers and faculty at primarily undergraduate institutions (PUIs) to collaborate in assigning functions to these orphan genes (ORFans). In summer 2017, 18 faculty members and 3 students from 18 institutions attended the first ORFan workshop where they were introduced to the ORFan bioinformatics work-flow and basic laboratory techniques. Participants reported that the time given to network/collaborate/talk about teaching, the bioinformatics sessions, and the wet-labs were clear strengths of the workshop. In fall 2017 over 300 students conducted research on ORFans, learning important skills in bioinformatics, genetics, and molecular biology, as they mined genomic datasets, designed and conducted gene-specific experiments, and interpreted their findings. Pre-post-tests confirmed that students gained both (1) an understanding of the Gene Ontology (GO) system for describing gene function and (2) knowledge regarding the use of bioinformatics to assign gene function. These findings support a published study showing that the ORFan bioinformatics modules increase the self-reported learning and confidence of students.

303 YeastMine: An Advanced Search and Analysis Tool for SGD Data. Kalpana Karra, Robert Nash, Kevin MacPherson, Gail Binkley, Michael Cherry, The SGD Project Stanford University, Palo Alto, CA. The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) provides high-quality curated genomic, genetic, and molecular information on the genes and gene products of the budding yeast Saccharomyces cerevisiae. YeastMine provides a sophisticated tool for searching SGD data with a list of genes, GO terms and other data types. Pre-made queries called templates can be used to get results for most commonly asked questions. For more complex questions, one can use multiple templates, intermediate steps and list operations to get answers. Registered users can store their queries and results in MyMine. Regulatory relationships, human homolog and disease connections, and protein complex data are the most recent data types loaded into YeastMine. YeastMine is built using the open source software InterMine (http://www.intermine.org), a data warehouse solution for biological data. Proposed authors: Kalpana Karra, Robert S. Nash, Kevin A. MacPherson, Gail Binkley, J. Michael Cherry, and The SGD Project Department of Genetics, Stanford University, Stanford CA 94305, USA

304 Saccharomyces Genome Database: Outreach and online training services. K.A. MacPherson, K.S. Dalusag, O. Lang, S.T. Hellerstedt, S.R. Engel, E. Wong, R. Nash, M. Skrzypek, J.M. Cherry, the SGD Project Department of Genetics, Stanford University, Palo Alto, CA. The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is the leading community resource for the budding yeast S. cerevisiae. SGD provides high-quality, manually curated information on the yeast genome and offers a wide variety of tools and features that have made it an indispensable resource for many researchers. To inform our user community about new developments at SGD, improve familiarity with SGD features and tools, and increase public awareness 109 of the importance of yeast to biological and biomedical research, SGD has engaged in a variety of online training services and outreach efforts. Here we present the SGD Webinar Series, a series of interactive webcasts aimed at demonstrating the SGD website and the value of yeast as a model organism, and the SGD YouTube channel, which currently provides over 50 useful help videos and quick tutorials (http://www.youtube.com/SaccharomycesGenomeDatabase). We will continue to develop these services to provide outreach to students and scientists on the significance and beauty of biology, and facilitate greater use and understanding of the resources made available by SGD. This work is supported by a grant from the NHGRI (U41 HG001315).

305 Associating Yeast Genes with Human Disease-related Genes at SGD. R.S. Nash, S.R. Engel, K.A. MacPherson, K. Karra, G.A. Binkley, T.K. Sheppard, E.D. Wong, J.M. Cherry, SGD Project Dept of Genetics, Stanford University, Palo Alto, CA.

The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org) is a comprehensive resource for curated, molecular and genetic information on the genes and proteins of S. cerevisiae. Model organism genetics holds great promise for advancing our understanding of human gene function and involvement in disease. Elucidating the biology of yeast genes has in many cases provided valuable insight into the function of their homologous human counterparts. With the goal of making connections between yeast genes, their human homologs and associated diseases, we have undertaken a project to collect and display this information at SGD.

At the start of this project, yeast-human cross-species functional complementation results were collected from the literature and stored in the YeastMine data warehouse where the data can be accessed using preformed template queries. Relevant information was also added to the respective Locus Summary Page descriptions. A subset of these human homologs have been determined to be disease associated. For this subset, the corresponding disease ontology (DO) terms were identified and associated with both the human gene and the corresponding yeast homolog, along with supporting information. Diseases associated with human genes that have a computationally determined yeast homolog have also been included in this set. Disease pages have been designed that include the following pieces of information: disease name, ID and definition from DO, yeast systematic and ORF names, human HGNC-derived gene names, annotation type (manual vs HTP), evidence code, reference, source and relevant links. A disease summary has been generated at SGD and will be included on relevant Locus Summary pages with a link to the browsable Disease page. It is our hope that making this information available to our users will facilitate studies aimed at understanding the biological functions of these genes and the role these genes play in the pathology of disease.

306 Comparative Genomics at the Saccharomyces Genome Database. Patrick Ng, Stacia Engel, Gail Binkley, Travis Sheppard, J. Michael Cherry, The SGD Project Genetics, Stanford University, Palo Alto, CA. Saccharomyces cerevisiae, a model organism fundamental to our understanding of eukaryotic cell biology, has long been utilized for the exploration of the biology described by omics technologies. The canonical sequence information for this valuable research data is hosted by the Saccharomyces Genome Database (SGD), a publicly available resource with manually and high-throughput curated data regarding function, expression, and interaction of genetic loci and their products. SGD also houses comparative analysis tools to explore homology relationships in humans, model organisms, and species across the fungal clade. In addition, SGD facilitates queries into the genetic and peptide sequence divergence among several strains of S. cerevisiae. The curated datasets of fungal homologous proteins and annotated whole genome sequences of S. cerevisiae isolates that provide the basis for these tools are available for direct download from SGD. SGD seeks to accumulate sequence data from geographically and ecologically diverse S. cerevisiae strains for the benefit of the research community; these are provided by resources such as the 100-Genomes Strains and 1002 Yeast Genomes Project. This work is supported by a grant from the NHGRI (U41 HG001315).

307 Getting Data: API Access to SGD and The Alliance of Genome Resources. Travis Sheppard, Gail Binkley, Felix Gondwe, Kalpana Karra, Ajay Shrivatsav Vichanthangal Prathivadhib, Shuai Weng, Pedro H.R. de Assis, J. Michael Cherry, SGD Project Genetics, Stanford University, Stanford, CA. The Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) has long provided comprehensive integrated biological information for the budding yeast Saccharomyces cerevisiae in the form of websites designed for presentation to the human user. Additionally, SGD now offers all data available on the website via a machine-readable API (application programming interface). The API is documented at https://github.com/yeastgenome/SGDBackend- Nex2/blob/master/docs/webservice.MD. For querying data in other model organisms, the Alliance of Genome Resources also has a JSON API which integrates data about disease associations and homology from multiple model organisms. Further, the Alliance offers a Neo4j graph database which users can query to see connections between genes, diseases, and GO terms.

308 Evolving yeast to resist an antifungal compound in a long-term project integrated into a high school biology class. R. Skophammer1, M.B. Taylor2, M. Dunham2 1) Science Department, Westridge School, Pasadena, CA; 2) Genome Sciences Department, University of Washington, Seattle, WA. Inquiry-based learning in the natural sciences leads to positive gains in educational outcomes including scientific literacy, 110 critical thinking, and attitudes toward science. Instruction in experimental design, hypothesis testing, data analysis, and communication models the behavior of working scientists and provides an effective model of inquiry. The recently revised AP Biology curriculum emphasizes science practice and provides flexibility in the choice of laboratory investigations. A series of connected labs that can answer questions in multiple biological domains over the course of a year could therefore substitute for a traditional list of independent projects. Here we present the results of an ongoing research project in which fourteen teams of high school students evolved differently-colored strains of Saccharomyces cerevisiae to resist the antifungal activity of FungiCure, an over-the-counter treatment for nail fungus (active ingredient: clotrimazole). Students passaged yeast in culture tubes by serial transfer each day of class. Initially, they each maintained three replicate cultures at a 1:12,000 dilution of the antifungal agent. During the experiment, students decided when and by how much to increase the dosage of the antifungal based on their observations. After six months of evolution most student groups maintained cultures at ~1:100 dilution of the antifungal, with one group maintaining a culture at a 1:1 dilution. At the two-month time point, whole-genome sequencing of samples from each yeast population revealed mutations in transcription factors that regulate drug response and genes involved in synthesis of cell wall components. Students searched databases and read primary literature to write a short paper explaining the evidence for these mutations causing the resistance phenotype. Students are learning to measure the relative fitness of evolved strains in fixed concentrations of the antifungal through competition experiments. The effects of this project on student learning and attitudes toward science will be studied and the project will be developed for dissemination and implementation to additional high schools.

309 SGD Migration to AWS Architecture. Ajay Shrivatsav Vichanthangal Prathivadhib, Travis K. Sheppard, Gail Binkley, Felix Gondwe, Kalpana Karra, Shuai Weng, Pedro H.R. de Assis, J. Michael Cherry, The SGD Project Genetics, Stanford University, 94304, CA. Over its twenty-five years of existence, the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/) has become an indispensable service for the yeast community. During that time, SGD has also experienced a proliferation of services, data types, and web pages which has made it more expensive to maintain. In order to make a more sustainable service for its users, SGD was recently re-architected for Amazon Web Services (AWS) cloud infrastructure to be consistent with industry best practices. Migrating SGD to AWS had many benefits including flexibility to provision and decommission servers as needed, while maintaining high reliability and robustness in service. The architecture is multi-layered. User requests are handled by the load balancer that reroutes them to one of the productions web servers, which in turn query the database slave servers for data replicated from the master database. These architecture changes have left SGD a leaner, more sustainable service consistent with industry best practices.

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