Characterizing the Assembly and Molecular Interactions of the Fission Yeast Atg1 Autophagy Regulatory Complex

Characterizing the assembly and molecular interactions of the fission yeast Atg1 autophagy regulatory complex

Tamiza Nanji B.Sc., M.Sc. (McMaster University) 2012, 2014

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY in The Faculty of Graduate and Postdoctoral Studies (Biochemistry and Molecular Biology)

THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)

April 2019

©Tamiza Nanji, 2019

The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled:

Characterizing the assembly and molecular interactions of the fission yeast Atg1 autophagy regulatory complex

submitted by Tamiza Nanji in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology

Examining Committee:

Calvin Yip, Biochemistry and Molecular Biology

Supervisor

Filip Van Petegem, Biochemistry and Molecular Biology

Supervisory Committee Member

Michel Roberge, Biochemistry and Molecular Biology

Supervisory Committee Member

Elizabeth Conibear, Medical Genetics

University Examiner

Chris Loewen, Cell and Developmental Biology

University Examiner

Abstract

Macroautophagy, often referred to as autophagy, is a non-selective degradation mechanism used by eukaryotic cells to recycle cytoplasmic material and maintain homeostasis. Upregulated under starvation to generate molecular building blocks for ongoing cellular processes, this pathway requires the coordinated action of six multi-protein complexes, the Atg1/ULK1 complex being the first. Although, the

Atg1 complex has been extensively studied in Saccharomyces cerevisiae, far less is known about the biochemical and structural properties of its mammalian counterpart, the ULK1 complex. Unlike the S. cerevisiae Atg1 complex which contains five subunits (Atg1, Atg13, Atg17, Atg29, and Atg31), the ULK1 complex consists of four proteins (ULK1, FIP200/RB1CC1, ATG13, and ATG101) that are technically more challenging to study. In this thesis, I characterized the Atg1 complex from fission yeast,

Schizosaccharomyces pombe, as the composition of proteins resembles the mammalian ULK1 complex but is more amenable to biochemical analyses. The Atg1 complex in S. pombe is composed of Atg1 (ULK1 counterpart), Atg13, Atg17 (FIP200 counterpart) and Atg101. We found that the interactions between

Atg1, Atg17, and Atg13 are conserved while Atg101 does not replace Atg29 and Atg31. Instead, Atg101 binds to Atg1 and the HORMA domain of Atg13. Furthermore, Atg101 was previously shown to contain a conserved loop, termed the WF finger, postulated to bind and recruit downstream autophagy-related proteins and effectors. Using affinity purification mass spectrometry, we further investigated the potential interacting partners of S. pombe Atg101 under autophagy-inducing and non-inducing conditions. We obtained 625 proteins that co-purified with Atg101-GFP from cells grown in defined media. We used in vitro pairwise studies to confirm the interaction between Atg101 and prey proteins. 9 of the 16 proteins tested were confirmed including Fkh1, an FKBP-type peptidyl-prolyl cis-trans isomerase. We further explored the interaction interface between Atg101 and Fkh1 and found that the WF finger is required for the interaction in vitro. Although the S. cerevisiae Fkh1 homologue, FKBP12, interacts with rapamycin;

Fkh1 it is not thought to be directly involved in autophagy. Collectively, our results give new insights into

iii an Atg101-containing Atg1/ULK1 complex and reveals that Atg101’s function may span beyond autophagy.

Lay Summary

All life forms consume nutrients and in turn produce waste. If not removed, waste accumulates leading to detrimental outcomes. Eukaryotic cells have evolved a mechanism termed autophagy (Greek for “self- eating”) to remove waste. Dysregulation in autophagy has been linked to devastating illnesses such as cancer. In this thesis, we explore the autophagy-related complex, Atg1, which is thought to help regulate autophagy initiation. Although the Atg1 complex has been extensively studied in budding yeast, the human Atg1 complex counterpart, the ULK1 complex, differs in subunit composition. We explored the

Atg1 complex from fission yeast as it resembles the human ULK1 complex. We found that many of the interactions in the Atg1 complex are conserved between budding and fission yeast; however, the unique

Atg101 protein forms interactions with Atg13HORMA and Atg1 within the Atg1 complex. We further explored potential Atg101 interactions. Our work suggests that Atg101’s function may extend beyond autophagy.

Preface

This thesis contains contributions made by other scientists from around the world and includes a version of a published paper. These contributions are outlined below.

A version of Chapter 2 was published as Tamiza Nanji, Xu Liu, Leon H. Chew, Franco K. Li, Maitree

Biswas, ZhongQiu Yu, Shan Lu, Meng-Qiu Dong, Li-Lin Du, Daniel J. Klionsky & Calvin K. Yip (2017)

Conserved and unique features of the fission yeast core Atg1 complex, Autophagy, 13:12, 2018-2027, DOI:

10.1080/15548627.2017.1382782. I took over this project from previous students, as such many of the constructs used for the pull down assays in Figure 2.1 were made by other students including Leon Chew,

Katarina Priecelova, Tianlei Sun and Franco Li. I generated the remaining constructs needed and conducted all pull down assays and associated western blots to devise the S. pombe Atg1 complex interaction network (Figure 2.1, 2.2, 2.4). Electron microscopy (EM) studies of S. pombe Atg17 (Figure 2.3) were conducted by Leon Chew and Calvin Yip. Autophagy assays in the budding yeast were conducted by

Xu Liu and Daniel J. Klionsky (University of Michigan). Autophagy assays in the fission yeast were conducted by ZhongQiu Yu and Li-Lin Du (National Institute of Biological Sciences, Beijing). I devised the study to compare the Atg1 complexes between fission and budding yeast with respect to Atg29 and Atg31.

I generated the cells necessary and completed pull down assays and associated western blots for this section. With assistance from Dr. Calvin Yip, I purified the S. pombe Atg17-S. cerevisiae Atg31-Atg29 chimera complex and completed negative-stain EM analysis (Figure 2.5). Crosslinking experiments were completed by me, with limited assistance from Maitree Biswas (University of British Columbia). Shan Lu and Meng-Qiu Dong (National Institute of Biological Sciences, Beijing) analyzed our crosslinked samples using crosslinking mass spectrometry (CXMS) (Figure 2.6).

Chapter 3 is an ongoing study. I generated the fission yeast Atg101-GFP construct in the S. pombe

ARC039 strain and grew these cells for proteomic studies. Samples were prepared and analyzed using

vi mass spectrometry by Aoki Hiroyuki and proteomic analysis was conducted by Sadhna Phanse from Dr.

Mohan Babu’s group (University of Regina). Sadhna Phanse generated Figure 3.2C. I generated the constructs required for the in vitro pull down experiments to confirm interactions between Atg101 and our prey proteins. Atg101 mutants and subsequent cloning into expression vectors used to assess the interaction surface of Atg101 with Atg13HORMA and Fkh1 were prepared with the assistance of Diana

Vasyliuk (Mitacs Globalink). I conducted the final pull down assays and associated western blots.

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Table of Contents

Abstract ...... iii Lay Summary ...... v Preface ...... vi Table of Contents ...... viii List of Tables ...... xi List of Figures ...... xii List of Abbreviations and Symbols ...... xiii Acknowledgements ...... xvi CHAPTER 1: Introduction ...... 1 1.1 Protein turnover helps preserve cellular homeostasis ...... 1 1.2 Two distinct mechanisms of cellular degradation: The ubiquitin-proteasome and lysosome- autophagy systems ...... 2 1.2.1 The ubiquitin-proteasome system (UPS) ...... 2 1.2.2 The lysosome-autophagy system ...... 5 1.3 Historical milestones of lysosomal/vacuolar degradation ...... 8 1.3.1 Early evidence of autophagy ...... 8 1.3.2 Early autophagy studies conducted in the budding yeast ...... 13 1.4 Macroautophagy ...... 17 1.4.1 The progression of the autophagy field ...... 18 1.4.2 Core autophagy machinery in the budding yeast ...... 21 1.4.2.1 The Atg1 kinase complex ...... 21 1.4.2.2 The Atg9 transmembrane protein ...... 24 1.4.2.3 The autophagy-specific class III phosphatidylinositol 3-kinase complex I (PI3K) ...... 25 1.4.2.4 Atg18-Atg2 complex...... 26 1.4.2.5 The Atg8 conjugation system ...... 27 1.4.2.6 The Atg12 conjugation system ...... 28 1.4.3 Diversity of autophagy machinery and its regulation in yeast and mammals ...... 28 1.4.4 Methods used in this study to measure autophagic flux ...... 33 1.5 Using fission yeast to study autophagy ...... 35 1.6 Overview of thesis ...... 37 CHAPTER 2: Conserved and unique features of the fission yeast core Atg1 complex ...... 38

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2.1 Introduction ...... 38 2.2 Materials and Methods ...... 39 2.2.1 Molecular cloning ...... 39 2.2.2 Co-precipitation experiments ...... 40 2.2.3 Protein purification ...... 40 2.2.4 Negative stain electron microscopy and image processing ...... 42 2.2.5 S. cerevisiae yeast strains and Pho8∆60 activity assay ...... 42 2.2.6 CFP-Atg8 cleavage assays...... 43 2.2.7 Melting temperature determination by differential scanning fluorimetry (DSF) ...... 43 2.2.8 Chemical crosslinking and crosslinking coupled to mass spectrometry (CXMS) ...... 44 2.3 Results ...... 44 2.3.1 Subunit interactions of the core S. pombe Atg1 complex ...... 44 2.3.2 S. pombe Atg17 is dimeric and adopts a rod-like architecture ...... 49 2.3.3 S. pombe Atg17 can interact with S. cerevisiae Atg29 and Atg31 in vitro ...... 51 2.3.4 S. pombe Atg17 cannot functionally complement S. cerevisiae Atg17 in vivo ...... 55 2.3.5 Atg101 binding to Atg13HORMA provides enhanced stability ...... 56 2.4 Discussion ...... 59 CHAPTER 3: Exploring the Atg101 interactome ...... 61 3.1 Introduction ...... 61 3.2 Methods ...... 65 3.2.1 S. pombe atg101-GFP yeast strain construction and growth ...... 65 3.2.2 Liquid chromatography-mass spectrometry (LC-MS) sample preparation ...... 66 3.2.3 Proteomic analysis ...... 68 3.2.4 In vitro co-precipitation experiments using E. coli cells ...... 68 3.3 Results ...... 71 3.3.1 Incorporation of a C-terminal GFP tag on S. pombe Atg101 ...... 71 3.3.2 LC-MS/MS analysis reveals that Atg101 potentially interacts with a variety of proteins ...... 72 3.3.3 In vitro co-precipitation experiments validate that Atg101 interacts with prey genes identified from LC-MS/MS studies ...... 77 3.3.4 Mapping the Fkh1-interacting interface of S. pombe Atg101 ...... 82 3.3.5 Atg101 is unable to simultaneously interact with Fkh1 and Atg13HORMA in vitro ...... 84 3.4 Discussion ...... 85 Chapter 4: Discussion and Future Directions ...... 93

4.1 Conserved and unique features of the yeast Atg1 complex ...... 93 4.1.1 Interactions and architecture of the Atg1 complex ...... 93 4.1.2 Autophagy initiation in Atg1 complexes with and without the Atg101 subunit ...... 96 4.2 AP-MS to identify novel Atg101 interacting partners ...... 100 4.2.1 Implication of the interaction between Atg101 and Fkh1 in fission yeast ...... 103 BIBLIOGRAPHY ...... 106 Appendices ...... 122

List of Tables

Table 1.1. Autophagy-related genes in yeast and mammals ...... 29 Table 2.1. List of S. cerevisiae strains used...... 43 Table 2.2. Intermolecular and intramolecular crosslinks observed from CXMS analysis...... 58 Table 3.1. Primers used to clone S. pombe genes ...... 69 Table 3.2. S. pombe proteins from proteomic analysis for follow up studies ...... 76 Table A1. Prey genes identified from proteomic mass spectrometry analysis of S. pombe cells expressing Atg101-GFP grown in YES media...... 122 Table A2. Prey genes identified from proteomic mass spectrometry analysis of S. pombe cells expressing Atg101-GFP in EMM and EMM-N ...... 151 Table A3. Prey genes identified from cells grown in EMM and EMM-N with associated descriptions .... 177 Table A4. Difference in prey genes identified in EMM and YES ...... 192

List of Figures

Figure 1.1. The ubiquitin-proteasome system ...... 4 Figure 1.2. Three branches of autophagy ...... 11 Figure 1.3. The six functional groups that make up the budding yeast core macroautophagy machinery...... 20 Figure 1.4. Structural properties of the budding yeast Atg1 complex ...... 24 Figure 1.5. Pho8∆60 activity assay to assess for autophagic flux in budding yeast ...... 34 Figure 1.6. Atg8 cleavage assay to assess autophagic flux ...... 35 Figure 2.1. Intersubunit interactions of the S. pombe Atg1 core complex ...... 46 Figure 2.2. The interaction between S. pombe Atg13CTD and Atg1CTD ...... 48 Figure 2.3. Negative stain EM analysis of S. pombe His-MBP-Atg17 ...... 50 Figure 2.4. S. pombe Atg17 binds S. cerevisiae Atg29 and Atg31 but cannot complement S. cerevisiae Atg17 function ...... 52 Figure 2.5. Interaction between S. pombe Atg17 and S. cerevisiae Atg31 ...... 54 Figure 2.6. The interaction between Atg101 and Atg13HORMA ...... 57 Figure 3.1. Integration of the gfp-kanMX tag and cassette into S. pombe strain ARC039 ...... 72 Figure 3.2. LC-MS/MS analysis of S. pombe Atg101 ...... 74 Figure 3.3. Co-precipitation assays with His-Atg101 to confirm protein-protein interactions from proteomic mass spectrometry analysis, in vitro ...... 80 Figure 3.4. Co-precipitation assays of E. coli cells expressing S. pombe Atg101 mutants with S. pombe Atg13HORMA and S. pombe Fkh1 ...... 83 Figure 3.5. Co-precipitation assay of S. pombe proteins, His-Atg101 co-expressed in E. coli with GST- Atg13HORMA supplemented with His-Fkh1 ...... 85 Figure 4.1. Model of autophagy initiation ...... 99 Figure A1.. . Co-immunoprecipitation assays with His-Atg101 and GST-Rpn6 to confirm protein-protein interactions from proteomic mass spec in vitro...... 198

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List of Abbreviations and Symbols

% Percent ∆ Delta °C degree Celsius 17BR Atg17 binding region 17LR Atg17 linking region 2D 2-dimensional Å Ångström A, Ala Alanine ABM Applied Biological Materials AFM atomic force microscopy ALS amyotrophic lateral sclerosis AP-MS affinity purification-mass spectrometry Atg autophagy-related C, Cys Cysteine C9ORF72 chromosome 9 open reading frame 72 CBB Coomassie Brilliant Blue CCD charged-coupled device CID collision-induced dissociation CMA chaperon-mediated autophagy pathway cryo-EM cryogenic electron microscopy CTD C-terminal domain Cvt cytoplasm-to vacuole targeting CXMS crosslinking coupled to mass spectrometry D, Asp aspartic acid DNA deoxyribonucleic acid DSF differential scanning fluorimetry DSS disuccinimidyl suberate DTT Dithiothreitol E, Glu glutamic acid EAT early autophagy targeting/tethering ECL enhanced chemiluminescence EEA1 Early Endosome Antigen 1 EM electron microscopy ER endoplasmic reticulum ESCRT endosomal sorting complex required for transport ESI electrospray ionization F, Phe Phenylalanine FEI Field Electron and Ion (Company) FIP200 focal adhesion family interaction protein of 200 kDa FKBP12 FK506-binding protein of 12 kDa FL full-length

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FRB FKBP12-rapamycin binding domain FRET fluorescence resonance energy transfer FTD frontotemporal dementia G, Gly Glycine GDP-GTP guanosine diphosphate–guanosine 5′-triphosphate GE General Electric (Company) GEF GDP-GTP exchange factor GFP green fluorescent protein GOI gene(s) of interest GST glutathione S-transferase GTP guanosine triphosphate h Hour H,His Histidine His-MBP histidine-maltose binding protein HORMA fold named after Hop1, Rev7, Mad2 HPLC high-performance liquid chromatography HRP horseradish peroxidase Hsc70 heat shock cognate 70 hygML hygromycin resistance cassette I, Ile Isoleucine IDR intrinsically disordered region IPTG isopropyl beta-D-1-thiogalactopyranoside ITC isothermal titration calorimetry K, Lys Lysine kDa kilo Dalton KO MEFs knockout mouse embryonic fibroblasts L, Leu Leucine L.E. long exposure LAMP-2A lysosomal-associated membrane protein-2A LC-MS liquid chromatography-mass spectrometry M Molar M, Met Methionine MDa Megadalton MEF mouse embryonic fibroblasts MIM MIT-interacting motif min Minutes MIT microtubule interacting and transport MS mass spectrometry MST microscale thermophoresis mTORC1 mammalian target of rapamycin complex 1 mug66 meiotically upregulated gene 66 MVB multivesicular body N Nitrogen N, Asn Asparagine

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NEB New England Biolabs NMR nuclear magnetic resonance P, Pro Proline PA S. cerevisiae protein A PAS phagophore assembly site PCR polymerase chain reaction PE phosphatidylethanolamine PI3K phosphatidylinositol 3-kinase PI3P phosphatidylinositol 3-phosphate PTMs post-translational modifications Q, Gln Glutamine qPCR quantitative polymerase chain reaction R, Arg Arginine RB1CC1 retinoblastoma 1-inducible coiled-coil 1 RNase Ribonuclease S, Ser Serine S.E. short exposure SAXS small angle X-ray scattering Sc S. cerevisiae SD standard deviation SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis s Second SMCR8 Smith-Magenis syndrome chromosomal region candidate gene 8 Sp S. pombe SPR surface plasmon resonance T, Thr Threonine TFA trifluoroacetic acid TOR target of rapamycin TORC1 target of rapamycin complex 1 ULK1 UNC-51-like kinase 1 ULK2 UNC-51-like kinase 2 UPS ubiquitin-proteasome system Ura Uracil V,Val Valine VMP1 vacuolar membrane protein 1 W, Trp Tryptophan WDR41 WD repeat-containing protein 41 WT wild-type X Times Y, Tyr Tyrosine YFP yellow fluorescent protein

Acknowledgements

I would like to thank everyone who supported me leading up to, and during the preparation of this work.

I would like to acknowledge my supervisor, Dr. Calvin Yip, who guided me through this project and allowed me to explore various biological questions in his laboratory. I would like to thank my committee members

Dr. Michel Roberge and Dr. Filip Van Petegem for their invaluable support and feedback. I am also indebted to my former mentors Dr. Alba Guarné, and Dr. Marie Elliot who helped me develop critical thinking skills, among many others, that were integral for the completion of this work. I would further like to acknowledge my fiancé Safir Kassam, and our families who have supported me throughout this endeavour. Lastly, most of the experimental portion of this work was conducted at UBC Point Grey

Campus on the traditional, ancestral territory of the Musqueam people, and supported by scholarships provided by UBC and the Natural Sciences and Engineering Research Council (NSERC), I would like to acknowledge their efforts to the advancement of education in Western Canada.

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CHAPTER 1: Introduction

1.1 Protein turnover helps preserve cellular homeostasis

The dynamic nature of biological systems allows them to cope with their changing environments. Healthy cells acquire nutrients for ongoing processes and produce waste products that must be properly recycled.

New molecular machinery is constantly being generated, it is estimated that 1x1015 hemoglobin molecules are synthesized in the average human body in one second (Ohsumi, 2016). Molecular machinery is made from resources acquired from outside of the cell as well as from recycled products within the cell.

Furthermore, older cellular material deteriorates in function. To maintain cellular homeostasis, cells balance the synthesis and degradation processes (Ohsumi, 2014). If this is not achieved, cells perform sub- optimally, and disease phenotypes arise (Choi, Ryter and Levine, 2013).

The degradation process is central to maintaining cellular homeostasis. However, the notion that cells degrade intracellular content was a foreign concept until the late 1930s when German biochemist

Rudolph Schoenheimer illustrated “protein turnover” using isotopes to track metabolic processes

(Schoenheimer, Ratner and Rittenberg, 1939; Schoenheimer and Clarke, 1942). The concept of protein turnover was further studied by assessing the rate of protein degradation, using pulse-chase experiments, after protein was injected into a cell (Ganschow and Schimke, 1969; Kalish, Chovick and Dice, 1979).

Collectively, studies on protein turnover illustrated that every protein in vivo has a distinct half-life broadly ranging from a few minutes to several months (Ganschow and Schimke, 1969; Kalish, Chovick and Dice,

1979; Ohsumi, 2016) demonstrating that cells have the capacity to degrade protein.

Synthesis and degradation of cellular material go hand in hand. If an organism is unable to obtain nutrients from outside sources, it goes through the process of starvation. To overcome this potentially harmful threat which affects homeostasis, cells recycle cellular content to generate new macromolecules.

The importance of cellular recycling has become more apparent in recent studies, it is now known that

1 most of the amino acids incorporated in proteins are from recycled products (Ohsumi, 2014). Proteins can be recycled by proteases which are enzymes that degrade proteins and peptides. The necessity of protein degradation is emphasized by the fact that all organisms without exception contain proteases (Ohsumi,

2014; Clark and Pazdernik, Nanette, 2016). Proteases help to maintain cellular homeostasis as they produce new molecular building blocks from pre-existing cellular material. However, as one can imagine, the degradation processes must be regulated to ensure degradation only occurs at the time and place needed. Cells regulate the degradation process through two distinct mechanisms discussed below.

1.2 Two distinct mechanisms of cellular degradation: The ubiquitin-proteasome and lysosome-autophagy systems

The maintenance of cellular homeostasis requires cellular material to be recycled by proteases and other hydrolytic enzymes. Detrimental outcomes may occur if this process is not controlled. One way in which cells regulate the degradation process is by compartmentalizing enzymes. Two forms of protein degradation, that segregate enzymes from the rest of the cytoplasm, exist in eukaryotic cells. They include: (1) the ubiquitin-proteasome system (UPS) which confines proteases to a barrel-like structure and threads individual proteins through for degradation, and (2) the lysosome-autophagy system which employs the acidic nature of this organelle and proteases within to degrade and recycle substrates. I will expand on these two pathways below.

1.2.1 The ubiquitin-proteasome system (UPS)

As the name suggests, the protein ubiquitin plays a significant role in this pathway as it tags specific proteins for degradation. The ubiquitin protein, composed of 76 amino acids, forms a covalent bond to the target protein using an elaborate mechanism (Figure 1.1A). First. the activating enzyme, E1, uses ATP to form a ubiquitin-AMP intermediate and ubiquitin-E1 thiol ester product. This product is recognized by

E2 enzymes. Ubiquitin is then transferred to the E2 enzyme forming an E2 thiol ester product. E2 enzymes

2 contain a conserved core that aids in forming the E2-E3 bond. E3 enzymes select and transfer ubiquitin to the target protein for degradation (Nandi et al., 2016). Comparative genome analysis in mice shows that few genes encode E1 and E2 enzymes, while hundreds of genes code for E3 ligases. This illustrates that there is some specificity of ubiquitin-protein conjugates in UPS (Semple, 2003). Polyubiquitin chains are used to link ubiquitin to the target protein by forming covalent linkages using the carboxy-terminal glycine of the added ubiquitin, and usually the epsilon-amino group lysine of the target protein. For those targets lacking lysine, the amino-terminal residue may be used (Pickart, 2001; Weissman, 2001; Ciechanover and

Iwai, 2004). Furthermore, the position of the lysine residue on ubiquitin used to form the polyubiquitin chain is important for appropriate delivery of the tagged protein. For example, polyubiquitin chains made using ubiquitin residue K48 or K29 act as a signal for proteasome-mediated degradation, while polyubiquitin chains made though linkages at K63 have been shown to act as a signal for DNA repair

(Weissman, 2001).

Proteins tagged as targets for degradation by ubiquitin are delivered to the 26S proteasome, a 2.5

MDa multi-catalytic molecular degradation machine. The 26S proteasome is made up of 2 subassemblies: the 20S “core particle” which is the central component of the proteasome, and the 19S “regulatory particle” which consists of the lid and base (Figure 1.1B). Collectively, these subassemblies form stacks of ring-shaped heptamers which adopt a barrel-like architecture with a lid and base at each end to keep the contents contained and segregated from the rest of the cytoplasm. The 19S regulatory particle recognizes the ubiquitinated substrate and produces a deubiquitylated product for further degradation. The regulatory particle is thought to help unfold and translocate the protein to the interior of the proteasome

(Tanaka, 2009; Nandi et al., 2016). The interior of the proteasome (made from the 20S subassembly) contains proteases that break down the targeted protein into oligopeptides which can be further broken down into individual amino acids. These products are recycled for ongoing cellular processes (Nandi et al.,

2016).

Figure 1.1. The ubiquitin-proteasome system. (A) Conjugation of ubiquitin to the target substrate using E1, E2, and E3 conjugation enzymes. Ubiquitin is depicted by the purple circle; the green star represents the substrate. (B) The ubiquitinated substrate is targeted to the 26S proteasome for degradation. The protein is transported through the lid of the 19S subassembly and degraded by proteases within the 20S barrel.

The 2004 Nobel Prize in Chemistry was awarded to Aaron Ciechanover, Avram Hershko, and Irwin

Rose for their discovery of the UPS (Giles, 2004; Ciechanover, 2009). As the UPS and other degradation mechanisms were being characterized, it became apparent that cellular degradation and recycling is critical for cellular processes including regulation of the cell cycle and division, DNA transcription and repair, neural and muscular degeneration, responses to stress and extracellular modulators, ribosome biogenesis, and viral infections (Nandi et al., 2016). The UPS is essential for cells to function optimally; however, on its own, the UPS cannot fulfill the degradation requirements of all living organisms. Protein targets are tagged by ubiquitin and then delivered to the proteasome, where they are first unfolded and

4 then threaded through the proteasome pore to the catalytic chamber where they are degraded. It is important to note that the targets degraded by the UPS are limited to the size of the proteasome; substrates must fit through the ~20 Å pore of the proteasome (Rabl et al., 2008; Bohn et al., 2010); hence this pathway only has the capacity to degrade protein substrates and cannot recycle other cellular content such as organelles. Furthermore, proteasomes themselves lose function over time and must be turned over (Bohn et al., 2010). A common thought is that the UPS is used primarily for short-lived or abnormally folded proteins that fit within the proteasome pore, while the lysosome-autophagy system (discussed below) targets long-lived macromolecular complexes, larger cargo, and organelles (Li, Li and Bao, 2012).

Like the UPS, the degradation machinery for the lysosome-autophagy system is sequestered in a compartment. However, unlike UPS, the lysosome is a dynamic organelle that can change morphology based on the content it degrades (Cooper, 2000). The next section will explore the role of the lysosome in degradation and briefly describe autophagy-lysosome pathways.

1.2.2 The lysosome-autophagy system

The mammalian lysosome and analogous plant/yeast vacuole (used interchangeably in this section) are membrane-enclosed organelles that contain an array of degradative enzymes to hydrolyze proteins, DNA,

RNA, polysaccharides, and lipids. The lysosome was initially isolated from rat liver cells by Christian de

Duve (de Duve et al., 1955), a discovery that led him to become a co-recipient of the 1974 Nobel Prize in

Physiology or Medicine (Winawer, 2015). This organelle has been shown to be a central component of the degradation process; hence, it is commonly referred to as the “garbage dump,”(Ohsumi, 2016) or more accurately, the “recycling facility” of the cell.

The lysosome membrane is sufficiently flexible to allow the incorporation of variously sized cargo

(Cooper, 2000). It has been reported that the budding yeast vacuole increases in size under nutrient- limiting conditions to meet the degradation demands of the cell, illustrating the dynamic nature of this organelle (Chan and Marshall, 2010). Moreover, lysosome biogenesis has been linked to cellular

5 environment conditions (Sardiello et al., 2009; Chan and Marshall, 2010) and the tissues in which they are found. Early EM studies showed that liver tissue had more lysosomes than other cell types and that the morphology of lysosomes is heterogeneous in various cells (Novikoff, Beaufay and De Duve, 1956). For example, animal cells contain many lysosomes (Ashford and Porter, 1962) observed as small round structures that can form tubular networks (Titorenko, Chan and Rachubinski, 2000), while budding yeast vacuoles generally exist as a single structure (Wiemken, Matile and Moor, 1970; Baba et al., 1994). The plant vacuole is very large, often taking up more than half the entire cell. This helps plants maintain turgor pressure and provides structural support (Eisenach et al., 2015). Furthermore, lysosomes have been shown to be mobile due to their interaction with microtubules (Wubbolts et al., 1999), which lets them sample cellular content (Chan and Marshall, 2010). This sampling mechanism may be useful in larger cells for the efficient breakdown of waste, aiding in the maintenance of cellular homeostasis. Lastly, lysosomal fusion events have been shown to affect lysosomal morphology (Wickner and Haas, 2000) further illustrating the dynamic nature of this organelle and its ability to fuse with other vesicles.

Although the term lysosome is often used to refer to both the lysosome and vacuole, there are many differences between the two organelles, a key difference being size. The yeast vacuole is a “gigantic”

(Klionsky and Eskelinen, 2014) organelle as it is approximately 2-3 µm in diameter (Matile and Wiemken,

1967; Indge, 1968; Pratt, Bryce and Stewart, 2007), while a budding yeast cell is 5-10 µm in diameter

(Klionsky and Eskelinen, 2014). Hence, the vacuole can encompass more than one-third of the cell, which is also true for plant vacuoles (see above). On the other hand, mammalian lysosomes are smaller at approximately 0.5 µm in diameter (Klionsky and Eskelinen, 2014). In addition, yeast typically contain one large vacuole while mammalian cells customarily contain multiple smaller lysosomes (Hoffman, Wood and

Fantes, 2015), illustrating the diversity of both lysosomes and vacuoles across various species.

Regardless of the difference between lysosomes and vacuoles, they both contain hydrolytic enzymes capable of processing intact macromolecules into reusable building blocks. The lumen of the

6 vacuole is maintained at an acidic pH of ~5 by the vacuolar ATPase, which pumps protons into the lumen

(Appelqvist et al., 2013). The enzymes within the organelle are active at acidic pH, but not at the neutral pH (~7.2) found in the cytoplasm, ensuring that they will not cause damage to cells if accidentally released.

The lysosome/vacuole is associated with multiple degradation pathways including phagocytosis, endocytosis, and autophagy pathways (Saftig and Klumperman, 2009; Jaishy and Abel, 2016). I will briefly describe phagocytosis and endocytosis in this section and describe autophagy in greater detail in section

1.4 as it is the focus of my thesis.

During phagocytosis, phagocytic cells including various white blood cells (neutrophils, macrophages, and monocytes) are used to clear pathogenic material such as bacteria and fungi (Gray et al., 2016). Endocytosis can be used by cells to sense their environment and take up extracellular nutrients.

This pathway begins at the plasma membrane, where vesicles are formed by clathrin-dependent or - independent mechanisms and bud off into the cell (Doherty and McMahon, 2009). The vesicles, including their membrane and contents, are delivered to early endosomes which contain marker proteins, the Ras- related protein Rab5, and Early Endosome Antigen 1 (EEA1) (Stenmark et al., 1995; Riezman et al., 1997).

Early endosomes mature from Rab5- to Rab7-positive endosomes leading to the dispersal of these vesicles back to the plasma membrane for sorting and transport to the Golgi complex for further sequestration into multivesicular bodies (MVBs). The sorting of vesicles is mediated by the endosomal sorting complex required for transport (ESCRT) machinery (Hanson and Cashikar, 2012). Details on the ESCRT machinery and its relation to autophagy were recently reviewed (Lefebvre, Legouis and Culetto, 2017). More recently the ESCRT machinery has been shown to directly mediate autophagosome closure (Takahashi et al., 2018).

Late endosomes and MVBs may mature into lysosomes or fuse with pre-existing lysosomes to recycle their contents (Saftig and Klumperman, 2009; Tooze, Abada and Elazar, 2014). The endocytic pathway overlaps with other lysosome-mediated degradation pathways including autophagy (Tooze, Abada and Elazar,

2014).

1.3 Historical milestones of lysosomal/vacuolar degradation

Lysosome-mediated degradation pathways are generally highly conserved and are essential in maintaining cellular homeostasis. The discovery of the lysosome was the first of many discoveries leading to the birth of the autophagy field. I will briefly outline noteworthy findings below.

1.3.1 Early evidence of autophagy

Forms of lysosome-mediated degradation were studied even prior to the discovery of the lysosome. As early as the late 1800s Metschnikoff observed phagocytosis, a pathway that degrades foreign material and is linked to immune health (Metschnikoff, 1891). One of the first indications that the lysosome played a role in protein/organelle degradation occurred in the mid-1950s using light microscopy. Novikoff et al. observed an organelle, now known to be the lysosome, containing structures resembling the mitochondria, endoplasmic reticulum (ER), and ribosomes (Novikoff, Beaufay and De Duve, 1956).

Microscopy was also used to observe the delivery of extracellular material to the lysosome (Novikoff,

Beaufay and De Duve, 1956) (now known as endocytosis, described above). Furthermore, when electron microscopy (EM) was used to study kidney tubule cells in mice, irregularly shaped lysosomes containing amorphous material was observed (Clark, 1957).

As lysosome-related studies progressed, lysosome morphology was observed to be associated with nutrient availability and cell type. Ashford and Porter reported an increase in lysosomes containing cytoplasmic components after treatment with the hormone glucagon (Ashford and Porter, 1962). Shortly after, De Duve coined the term “autophagy” (Greek for self-eating) as a pathway that delivers intracellular material to the lysosome (De Duve, 1963). These experiments suggested that a unique degradation pathway exists that responds to the concentration of glucagon. Further studies using EM, autoradiography, and cytochemical staining showed that cell types that take up material from outside the cell through endocytic events, such as macrophages and leukocytes, contain relatively more lysosomes

8 than other cell types (De Duve, Christian and Wattiaux, 1966). The relationship between nutrient availability and autophagy was strengthened with the observation that glucagon induces cellular autophagy while insulin suppresses autophagy in rat liver (Deter, Baudhuin and De Duve, 1967). These findings corroborate with older studies where 48 h of starvation resulted in the degradation of 30-40% of liver proteins in rats and insulin inhibited autophagy in rat kidney cells (Pfeifer and Warmuth-metz, 1983).

Furthermore, feeding and fasting between meals was shown to inhibit and promote autophagy respectively, reinforcing the link between nutrient availability and autophagy (Pfeifer and Warmuth-metz,

1983). Although it was known that nutrient levels affect autophagic flux, the mechanism by which autophagy progresses was not well characterized.

It is now known that the lysosome-autophagy degradation pathway consists of three branches, macroautophagy, chaperone-mediated autophagy (CMA), and microautophagy, described below (Figure

1.2). All branches employ features of the lysosome including its acidic interior and ability to recycle cellular content. Non-selective starvation-induced macroautophagy, the focus of my work, progresses as follows: a cup-shaped double membrane structure forms in the cytoplasm near the vacuole and engulfs cytoplasmic material. The structure (the phagophore in yeast, or isolation membrane in mammals) matures into a double membrane vesicle (the autophagosome). Ultimately, the outer membrane of the autophagosome fuses with the membrane of the lysosome/vacuole while the inner membrane and its contents are degraded by hydrolytic enzymes.

The general mechanism of autophagosome formation was not discovered until the mid-1970s.

Arstila and Trump used cell fractionation by centrifugation, microscopy, and histochemistry to determine that a de novo compartment, termed the autophagosome, is generated (Arstila and Trump, 1973). These studies identified that a double membrane-bound structure containing cytoplasmic components and organelles, but not hydrolytic enzymes, is present in the cytoplasm. Moreover, they observed the autolysosome, the organelle resulting from the fusion event between the autophagosome and lysosome.

The autolysosome was observed to contain mitochondria at various stages of degradation (Arstila and

Trump, 1973). Furthermore, the connection between amino acid availability and autophagic flux was explored by examining the release of amino acids from proteins in perfused rat liver. An increase in amino acid release was observed if the perfusion solution contained less amino acids, while an abundance of amino acids resulted in degradation at one-third the basal rate (Mortimore and Ward, 1976). Specific amino acids seemed to affect autophagy more significantly than others; Leu, Tyr, Phe, Gln, Pro, His, Trp, and Met seemed to suppress autophagy the most (Mortimore and Ward, 1976). Hepatocyte cultures also revealed similar findings, where Leu most strongly inhibited autophagy (Seglen, Gordon and Poli, 1980).

These experiments helped to establish the mechanism and regulation of what is now known as macroautophagy (Figure 1.2A).

The field of autophagy expanded and was studied in a wide range of eukaryotic organisms such as its discovery in plants (Matile, 1978). Moreover, the molecular mechanisms behind the pathway were being explored by multiple research groups. In the early 1980s, Mortimore and his team continued their work with liver perfusions in rats using biochemical and physiological methods (Mortimore and Ward,

1976; Mortimore, Hutson and Surmacz, 1983). Seglen and his team studied the effects of nutrient starvation on autophagy and autophagy regulation in cultured cells (Seglen, Gordon and Poli, 1980; Seglen and Gordon, 1982).

Figure 1.2. Three branches of autophagy. Black lightning bolts represent hydrolytic enzymes. (A) In macroautophagy, a cup-shaped double membrane structure forms in the cytoplasm near the vacuole and engulfs cytoplasmic material. The structure (the phagophore in yeast, or isolation membrane in mammals) matures into a double membrane vesicle (the autophagosome). Subsequently, the outer membrane of the autophagosome fuses with the vacuole while the inner membrane and its contents are degraded by hydrolytic enzymes. (B) In chaperone-mediated autophagy (CMA), target proteins containing the KFERQ degradation sequence are recognized by Hsc70 and brought to the LAMP-2A lysosomal transmembrane protein for transport into the lysosome and subsequent degradation. (C) Microautophagy functions non- selectively to “ingest” cytoplasmic content. The invaginations form an autophagic tube that bud at the ends and releases a vesicle into the lysosome for degradation by hydrolytic enzymes.

During this time, an additional form of self-eating was discovered which uses the chaperone- mediated autophagy (CMA) pathway (Neff et al., 1981). CMA is characterized as a specific form of selective

11 autophagy that targets single proteins for degradation (Cuervo, 2011). J. Fred Dice observed CMA using

RNase A as a substrate and discovered that the first 20 residues of target proteins were necessary for degradation. Further systematic analysis of the required residues identified a degradation signal made from the amino-acid sequence KFERQ (Figure 1.2B) (Dice et al., 1978; Cuervo, 2011). It was confirmed that this sequence was sufficient for CMA-targeted degradation (Cuervo, 2011). The chaperone heat shock cognate 70 (hsc70) recognizes proteins with a KFERQ or a KFERQ-like motif. The hsc70-protein conjugate then binds the lysosomal-associated membrane protein-2A (LAMP-2A), which transfers both the chaperone complex and the targeted protein to the lysosomal lumen for further degradation (Figure 1.2B)

(Neff et al., 1981; Li, Li and Bao, 2012).

In addition to macroautophagy and CMA, there is a third branch of autophagy known as microautophagy. In fact, De Duve and Wattiaux observed both microautophagy and macroautophagy back in 1966 (De Duve, Christian and Wattiaux, 1966) but the term microautophagy was not defined until

1983 (Mortimore, Hutson and Surmacz, 1983; Mijaljica, Prescott and Devenish, 2011). All three autophagy pathways (microautophagy, macroautophagy, and CMA; depicted in Figure 1.2) use the lysosome/vacuole to degrade cellular content, but the mechanism of transporting material to the vacuole is different in each case. In its truest form, microautophagy is a non-selective, lysosome-mediated degradation pathway but forms of selective microauophagy also exist (micropexophagy, piecemeal microautophagy of the nucleus, and micromitophagy) (Li, Li and Bao, 2012). Microautophagy involves the direct engulfment of cytoplasmic material by the lysosome/vacuole through invagination of the lysosomal membrane. The invagination of the lysosomal/vacuolar membrane occurs due to membrane bulges on its surface from the lateral segregation of lipids and the localization of large transmembrane proteins. Certain lipids and lipid-modifying proteins drive and maintain the formation of a spontaneous pit which has a propensity to cave in. The dynamin-related GTPase, Vps1p, regulates microautophagic invaginations to form differentiated autophagic tubes for capturing portions of the cytosol (Uttenweiler, Schwarz and Mayer,

2005). The tip of the tube forms vesicles that bud off into the lumen of the lysosome for degradation

(Figure 1.2C). The selective form of microautophagy involves the lysosomal membrane forming an arm- like protrusion that engulfs the target material and brings it into the lumen of the lysosome for degradation (Li, Li and Bao, 2012). Overall, microautophagy is thought to degrade less content compared to macroautophagy (Marzella, Ahlberg and Glaumann, 1981).

Early microautophagy research primarily used EM to examine sections of rat liver cells after subjecting them to short and long periods of starvation (Ahlberg, Marzella and Glaumann, 1982;

Mortimore, Lardeux and Adams, 1988). Yeast were later used to determine the vacuolar components and conditions required for microautophagy. Systematic screening of yeast mutants identified genes involved in microautophagy (Kunzt, Schwarz and Mayer, 2004). These genes were named “apg” and “Aut” at the time. Fluorescence microscopy was used to stain the various “apg” or “Aut” mutants to understand the function of specific genes and the state of microautophagy in these mutant strains (Bellu et al., 2001).

Although these early studies led to the identification of autophagy, the molecular mechanisms behind autophagy were difficult to study in mammalian cells due to the limited visibility and quantification of this dynamic process. It was not until Ohsumi’s pivotal genetic screen using the budding yeast model organism that the proteins involved in this pathway, and its regulation were better understood

(Zimmermann et al., 2016). In the next sections, I will focus on the invaluable discoveries made in budding yeast that lead to the characterization of the macroautophagy pathway, henceforth referred to as

“autophagy”.

1.3.2 Early autophagy studies conducted in the budding yeast

Yoshinori Ohsumi initiated the use of the budding yeast, Saccharomyces cerevisiae, as a model organism to study autophagy which lead him to becoming the 2016 Nobel Prize recipient for Physiology or Medicine.

Yeast serve as a good model organism to study autophagy for several reasons. Firstly, yeast cells are

13 unicellular making them amenable to biochemical analyses. Secondly, under glucose starvation yeast cells contain one large vacuole compared to multiple smaller vacuoles found in mammals (Mukaiyama et al.,

2009; Klionsky and Eskelinen, 2014; Hoffman, Wood and Fantes, 2015). These features enable the yeast vacuole to be readily detected by light microscopy. Ohsumi’s early studies in the budding yeast started with the intent to purify nuclei using density gradient centrifugation. During these experiments, Ohsumi noticed a white layer at the top of the tube which was later identified, through microscopy, as a fraction highly enriched in vacuoles (Ohsumi, 2016). This not only sparked his curiosity towards the yeast vacuole but also allowed him to delineate procedures for isolating vacuoles and making vacuolar membrane vesicles. Ohsumi further showed active transport of amino acids and calcium ions over vacuolar membranes (Ohsumi and Anraku, 1981, 1983), illustrating that cytoplasmic material was able to be transported across the membrane.

Ohsumi’s next goal was to visualize the analogous autophagy pathway that was previously discovered in mammalian cells. Ohsumi hypothesized that massive cellular remodeling, which would occur during sporulation, would require new macromolecules to be made through the yeast autophagy pathway (Ohsumi, 2016). Hence, he believed he should see degradation products within the vacuole upon autophagy induction during sporulation. Preliminary results were negative, and he reasoned that the products were being degraded too fast to be observed. Ohsumi next attempted his studies using yeast proteinase mutant strains from Elizabeth Jones (UC Berkeley Yeast Genetic Stock Center). These yeast cells lack the ability to degrade content delivered to the vacuole. After growing these cells in nitrogen- depleted media he observed spherical structures, which he termed autophagic bodies, accumulating inside the vacuole within 30 min and almost completely filled the vacuole within 3-4 hours (Kitamoto et al., 1988; Ohsumi, 2016). Although Ohsumi used sporulating cells, it was later found that autophagy is not specific to the process of sporulation but is a general cellular response to various nutrient starvation conditions (Takeshige et al., 1992).

The large budding yeast vacuole allowed Misuzu Baba and Masako Osumi to more easily visualize

Ohsumi’s findings using electron microscopic analysis (Baba et al., 1994). Furthermore, freeze-fracture techniques were able to capture images of fusion events between the autophagosome and vacuolar membrane in yeast (Baba, Osumi and Ohsumi, 1995). Detailed analysis of the autophagosomal membrane by EM illustrated that the membranes are thin compared to other organelles and that the composition of the outer membrane is different from that of the inner membrane. The outer membrane was found to contain additional particles (proposed to be the machinery for targeting to and fusion with the vacuole) compared to the inner membrane (Baba, Osumi and Ohsumi, 1995). Nevertheless, these EM studies suggest that yeast and mammalian autophagy use similar topological processes. Notably, the three main hallmarks of autophagy are conserved. First, a membrane sac appears and engulfs cytoplasmic material to form the double-membrane autophagosome. Second, the autophagosome fuses with the lysosome or vacuole. Lastly, the inner membrane of the autophagosome and its contents are degraded within the lysosome or vacuole (Figure 1.2A) (Baba et al., 1994).

Once the yeast autophagy pathway was successfully visualized, Ohsumi’s goal was to identify the genetic factors that orchestrate this pathway. The powerful genetic tools available for budding yeast enabled Ohsumi and his team to design the first genetic screen to isolate autophagy-related genes. Using the vacuolar proteinase-deficient mutant as the background, this genetic screen identified “apg”

(subsequently renamed “atg”) mutants that lost the ability to accumulate autophagic bodies after nitrogen starvation. When characterizing the first mutant (apg1), they observed that the apg1 homozygote diploids failed to sporulate (Tsukada and Ohsumi, 1993). Although the apg1 mutant strain failed to induce protein degradation in starvation conditions, under nutrient-rich conditions there was no difference in fitness observed between the wild-type (WT) and apg1 mutant strain, nor differences in vacuolar function or secretion. However, apg1 mutants lost viability faster after prolonged nitrogen starvation compared to WT cells (Tsukada and Ohsumi, 1993). Ohsumi and his team designed a secondary

15 genetic screen using this loss of viability phenotype (post nitrogen starvation) as an indication of defective autophagy, enabling them to identify more apg mutant genes (Tsukada and Ohsumi, 1993). The gene sequence of the first autophagy genetic factor, apg1, obtained through classical cloning by complementation approaches, revealed that it likely encodes a Serine/Threonine protein kinase

(Matsuura et al., 1997). The completion of the yeast genome sequencing project, shortly after, greatly facilitated the identification of the genetic sequence of other apg alleles. However, the apg genes identified did not share sequence homology with other known genes, this lead to a void in the field

(Ohsumi, 2016).

At around the same time, other research groups were independently working on pathways related to autophagy and identified similar or identical genetic factors involved in autophagy using yeast-based genetic screens (Nakatogawa et al., 2009). Notably, Daniel Klionsky and his team applied yeast genetics to identify genes involved in the cytoplasm-to-vacuole targeting (Cvt) pathway, a fungal-specific intracellular transport pathway found in Saccharomyces cerevisiae that constitutively and selectively transports vacuolar hydrolases to the vacuole (Baba et al., 1997; Lynch-Day and Klionsky, 2010).

Subsequent EM-based studies revealed that the membrane dynamics of the Cvt pathway are similar to macroautophagy, with the exceptions that “Cvt vesicles” exclude cytoplasmic proteins and selectively deliver the Cvt complex (α-aminopeptidase 1 complex and Ty1 virus-like particles) to the vacuole (Baba et al., 1997; Scott et al., 1997), and Cvt vesicles are much smaller than autophagosomes (150 nm versus 500 nm in diameter) (Suzuki et al., 2011). The genetic screen aimed to identify Cvt-specific genes also identified autophagy-related gene (Atg) products such as Atg19 (Cvt19) and Atg34 (Atg19-B) which were found to be receptor proteins of selective cargos α-aminopeptidase 1 and α-mannosidase 1, respectively

(Baba et al., 1997; Lynch-Day and Klionsky, 2010; Suzuki et al., 2010). Furthermore, the scaffold protein required to bridge the core Atg proteins and the target of selective autophagy, Atg11 (Cvt9/Cvt3), was

16 identified through studies exploring the Cvt pathway (Shintani and Klionsky, 2004; Yorimitsu and Klionsky,

2005).

Michael Thumm and his group also used a yeast genetic screen to search for autophagy-related proteins. They uncovered similar gene products as Ohsumi and Klionsky: Atg15 (Aut5/Cvt17), Atg1

(Aut3/Apg1/Cvt10), Atg4 (Aut2p/Apg4), and Atg8 (Aut7p, Cvt5, Apg8) (Thumm et al., 1994; Krick et al.,

2008). As many of the genes overlap between various vesicle trafficking/degradation pathways, the genes identified by the three groups were given multiples names with different prefixes (eg. APG, AUT, CVT). To avoid confusion the field agreed on a unified nomenclature, ATG (autophagy-related) to denote genes related to the autophagy pathway (Klionsky et al., 2003).

Although “atg” genes participate in the three branches of autophagy, macroautophagy is the focus of my thesis. The next section will explore the mechanisms involved in budding yeast macroautophagy.

1.4 Macroautophagy

Macroautophagy, henceforth referred to as autophagy, is an evolutionarily conserved degradation pathway responsible for the bulk degradation and recycling of cytoplasmic proteins, organelles, and harmful aggregates, as well as intracellular pathogens (Mizushima, Yoshimori and Ohsumi, 2011; Ragusa,

Stanley and Hurley, 2012; Reggiori and Klionsky, 2013). Autophagy is operated at a low, basal level during normal, nutrient-rich conditions and upregulated upon starvation and other cellular stresses. This enables cells to mobilize intracellular stores to ensure essential processes are not disrupted, aiding in the maintenance of cellular homeostasis (Shintani and Klionsky, 2004). In yeast, autophagic degradation begins with the formation of an initial sequestering compartment, known as the phagophore, which assembles at the phagophore assembly site (PAS) located next to the vacuole. The phagophore matures and expands promoting the engulfment of cytoplasmic material. This leads to the formation of a double- membrane vesicle, containing cytoplasmic content, termed the autophagosome. The outer membrane of

17 the autophagosome fuses with the vacuole where the content is degraded by hydrolytic enzymes, the resulting macromolecules are recycled by permeases that release them back into the cytosol (Figure 1.2A).

1.4.1 The progression of the autophagy field

Ohsumi’s pioneering work on autophagy in the budding yeast led to rapid growth in the field. Once specific genes were identified scientists were able to assess the function of individual gene products and their role on autophagy and its regulation. One of the first autophagy-related genes discovered was apg1/atg1 whose gene product was shown to have protein kinase activity, as mentioned above. Although protein kinase inhibitors were used to show the involvement of phosphorylation in autophagy (Holen, Gordon and Seglen, 1992, 1993), the targets of the kinases were unknown at the time.

Seglen and Gordon found one of the first inhibitors of autophagy, 3-methyladenine (Seglen and

Gordon, 1982), which was later discovered to inhibit the function of phosphatidylinositol 3-kinase (PI3K) essential for autophagosome formation (described below) (Blommaart et al., 1997). The effects of 3- methyladenine and another commonly used PI3K inhibitor, wortmannin have been explored (Wu et al.,

2010). Additional inhibitors of autophagy were discovered such as leupeptin, a cathepsin inhibitor, which blocks degradation in the lysosome (Furuya et al., 2001). It is now known that autophagy is regulated through phosphorylation events involving protein kinases. A key kinase in autophagy regulation—Tor, was first identified as a kinase that responds to nutrient availability (Thomas and Hall, 1997) and its function was thought to be conserved. Tor was further shown to be inhibited by the immunosuppressant rapamycin which induces characteristic phenotypes of starved cells (Hardwick et al., 1999). In 1998,

Ohsumi’s group found that the drug rapamycin induces autophagy in yeast irrespective of nutrient status

(Noda and Ohsumi, 1998). In 2000, Ohsumi and his team discovered a connection between the Tor pathway and the first protein they identified, Apg1/Atg1 (described below) (Kamada et al., 2000).

Although inadequate sequence homology between Atg genes identified from the yeast genetic screens described above and proteins with known function limited the ability to speculate the roles of Atg proteins in autophagy, Ohsumi and his group continued to characterize Atg proteins using various methods. They showed through immunoelectron microscopy that Atg8 localizes to the isolation membrane and autophagosome (Kirisako et al., 1999; Ohsumi, 2016). They hypothesized that this would make Atg8 a good marker to visualize membrane formation during autophagy. They later discovered the

Atg8 conjugation system (Ichimura et al., 2000) (further discussed below). Furthermore, the yeast phagophore assembly site was identified in 2001 as the site of Atg8 localization next to the vacuole (Suzuki et al., 2001). Ichimura et al. were able to reconstitute the Atg8 conjugation system in vitro (Ichimura et al., 2004) which allowed them to delineate the function of Atg8 (Nakatogawa, Ichimura and Ohsumi,

2007).

Suzuki et al. continued to study the localization of Atg proteins using fluorescent tagging. They initially assessed 16 green fluorescent protein (GFP)-tagged autophagy-related genes (atg1, atg2, atg3, atg4, atg5, atg7, atg8, atg9, atg10, atg12, atg13, atg14, atg16, atg17, atg18, and vps30/atg6) and found that these proteins at least partially localized to the PAS under starvation conditions (Suzuki et al., 2007).

Suzuki et al. then looked at the relationship between the Atg proteins and the PAS by assessing the localization of Atg proteins in strains lacking individual atg genes (Suzuki et al., 2007). They found that atg genes are recruited to the PAS in a hierarchical fashion and often operate in the context of multiprotein assemblies (Suzuki et al., 2007). Additional autophagy-related genes were studied including budding yeast

Atg29 and Atg31. Collectively the core autophagy-related proteins have been classified into six functional groups: (1) the Atg1 complex, (2) the Atg9 protein and its cycling system, (3) the autophagy-specific phosphatidylinositol 3-kinase (PI3K) complex, (4) the Atg18-Atg2 complex, (5) the Atg8 conjugation system, and (6) the Atg12 conjugation system (Feng et al., 2014). These functional groups are summarized in Figure 1.3. I will describe the discovery and roles of the budding yeast autophagy machinery below.

Figure 1.3. The six functional groups that make up the budding yeast core macroautophagy machinery. (A) The budding yeast Atg1 complex is composed of an elongated Atg17 dimer (orange) that interacts with the Atg31-Atg29 dimer (red) as well as the Atg1-Atg13 dimer (grey). (B) Atg9 (light green) is composed of six helices, 5 loops, and its N- and C-terminal tails face the cytosol. (C) The PI3K complex (blue) generates PI3P from PI using its catalytic subunit Vps34. PI3P is required to recruit (D) the Atg18-Atg2 complex (purple/fuchsia) to the PAS. (E) Atg8 conjugation system (green). Atg8 is first processed by Atg4 and then modified by E1- and E2-like enzymes, Atg7, and Atg3 respectively. Atg8 becomes conjugated to PE and incorporated into the inner and outer membrane of the growing autophagosome. (F) Atg12 conjugation system (pink/dark red). Atg12 is modified by E1-, and E2-like enzymes, Atg7 and Atg10, for Atg12-Atg5 complex formation. Atg16, which forms a homodimer, binds Atg5 bridging the complex together.

1.4.2 Core autophagy machinery in the budding yeast

1.4.2.1 The Atg1 kinase complex

The Atg1 complex is the first complex recruited to the PAS in budding yeast, and is thought to orchestrate early events in autophagy (Suzuki et al., 2001, 2007; Kawamata et al., 2007; Cheong et al., 2008; Noda and

Fujioka, 2015; Puente, Hendrickson and Jiang, 2016). The formation of the Atg1 complex in budding yeast regulates autophagy initiation. If the complex is unable to form, autophagy is not upregulated (Mizushima,

2010; Ragusa, Stanley and Hurley, 2012); hence, it is an essential complex in the autophagy pathway and is often called the initiation complex.

Structural and biochemical studies have shed light into the subunit organization and overall architecture of the budding yeast Saccharomyces cerevisiae Atg1 complex. This complex is composed of the Atg1 protein kinase (101.7 kDa), the regulatory protein Atg13 (83.3 kDa), and the Atg17-Atg31-Atg29 regulatory subassembly made up of Atg17 (48.6 kDa), Atg29 (24.7 kDa), and Atg31 (22.2 kDa) (Figure 1.3A,

Figure 1.4A). Crystallographic and small angle X-ray scattering analyses of Atg17-Atg31-Atg29 from the closely related yeast, Lachancea thermotolerans, revealed that this subassembly is heterohexameric and adopts an elongated double-crescent architecture. This architecture results from the dimerization of two highly helical Atg17 monomers at their C-termini, (Ragusa, Stanley and Hurley, 2012) and two globular

Atg31-Atg29 assemblies anchored to the concave surfaces of Atg17 by a direct interaction through Atg31

(Figure 1.4A) (Ragusa, Stanley and Hurley, 2012). Subsequent negative stain EM studies confirmed these structural features and further demonstrated that the Atg31-Atg29 subassembly stabilizes the unique curvature of Atg17 (Chew et al., 2013). Jim Hurley’s group observed that the curvature of Atg17 resembles the high curvature of the small Atg9-containing vesicles which are thought to be the precursors of the phagophore. This leads them to speculate that Atg17-Atg31-Atg29 mediates tethering of early vesicles in autophagy (Ragusa, Stanley and Hurley, 2012). This model was further supported by Tom Wollert’s group using in vitro reconstitution studies (Rao et al., 2016).

Additional structural studies using crystallographic analysis on the C-terminal region of Atg1 in complex with a C-terminal region of Atg13 showed that Atg1 contains two microtubule interacting and transport (MIT) domains at its C-terminus (Figure 1.4B) (Fujioka et al., 2014). The Atg1 MIT domains bind through hydrophobic interactions to 2 cognate MIT-interacting motifs (MIM) on Atg13 (Figure 1.4B). This interaction allows Atg13 to bridge the Atg17 central scaffold to Atg1. The intersubunit interactions in the latter two structures were validated by biochemical analysis and more recently by crosslinking coupled to mass spectrometry (CXMS) analysis (Chew et al., 2015). Furthermore, the crystal structure of the L. thermotolerans Atg13 N-terminal region revealed that it adopts a HORMA (Hop1, Rev7, Mad2) domain fold and might contain a phosphate sensor motif (Jao et al., 2013). Furthermore, the crystal structure of a small fragment of the C-terminal domain of Atg13 (residues 424-436) bound to the Atg17-Atg31-Atg29 hexamer shows that Atg13 makes at least one direct contact with the convex side of Atg17 (Figure 1.4C,

Atg13-Atg17BR) (Jao et al., 2013; Fujioka et al., 2014).

Atg1 complex assembly in S. cerevisiae is regulated, in part, by phosphorylation events directed by the Tor kinase (Loewith and Hall, 2011). Noda and Ohsumi first identified that the Tor kinase affected autophagic flux when they observed that rapamycin, a specific inhibitor of Tor, induced autophagy in budding yeast even under nutrient-rich conditions. This was a key finding that suggested that the Tor pathway likely regulates autophagy (Noda and Ohsumi, 1998). It was later shown that Tor regulates one of the two major intersubunit interactions essential to Atg1 complex assembly; the interaction between

Atg13 and Atg1 (Kamada et al., 2000; Fujioka et al., 2014). Under nutrient-rich conditions the Tor kinase heavily phosphorylates Atg13, blocking interaction sites, preventing the Atg1 complex from forming. As nutrients become diminished, Tor is inactivated resulting in Atg13 hypophosphorylation and promotion of Atg1 complex formation (Figure 1.3A, 1.4) (Kamada et al., 2000; Kabeya et al., 2005). This is a similar phenotype to that seen through rapamycin induction (Noda and Ohsumi, 1998). The interaction between

Atg1 and Atg13 was further shown to be mediated by dephosphorylation of serine residues in Atg13 in

22 response to starvation or Tor inactivation (Fujioka et al., 2014). Upon Atg1 complex formation, Atg1 plays roles in both the kinase-dependent phosphorylation of autophagy genes and the kinase-independent function of recruiting downstream Atg proteins to the PAS. In this way, Atg1 complex formation regulates autophagy.

Yamamoto et al. recently discovered a domain, in addition to the Atg17 binding region (17BR), on

Atg13 that interacts with Atg17. This domain is within the intrinsically disordered region (IDR) of Atg13, corresponding to residues 359-389 in budding yeast and has been termed the Atg17-Atg13 linking region

(Atg1317LR) (Figure 1.4C) (Yamamoto et al., 2016). The regulation of this interaction is also controlled by phosphorylation; dephosphorylation of Atg13 at position Ser379 was shown to mediate the interaction between the Atg1317LR and Atg17 (Yamamoto et al., 2016). It has been suggested that that the flexibility of the IDR of Atg13 allows for one Atg13 monomer to bind to two separate Atg17 dimers through the

Atg1317LR and the Atg1317BR. One Atg13 monomer could bind at either end of the Atg17 dimer allowing multimerization of linked Atg1 complexes (Yamamoto et al., 2016), causing a nucleation event leading to autophagosome formation.

The concept of Atg1 complex nucleation to promote autophagy is not foreign. Atg1 has also been shown to self-associate, which is promoted by Atg13 but does not require Atg17. Self-association of Atg1 has been shown to promote autophagy, while disruption results in decreased autophagy and Atg1 kinase activity. Dimerization of Atg1 in vitro and in vivo using a heterologous dimerization domain led to an increase in kinase activity (Yeh, Shah and Herman, 2011). Interestingly, activation of Atg1 though autophosphorylation at Thr226 seems to require Atg1 self-association (Yeh, Shah and Herman, 2011;

Yamamoto et al., 2016), suggesting that Atg1 may be phosphorylated by neighbouring Atg1 molecules and that autophagy initiation may require Atg1 complex nucleation.

Figure 1.4. Structural properties of the budding yeast Atg1 complex. (A) A schematic of the S. cerevisiae Atg1 complex subunits: Atg1 (gray) highlighting the tandem MIT domain, Atg13 (magenta) highlighting the LR (magenta) and BR (blue) that interact with Atg17, and the MIM domain that interacts with Atg1, Atg17 (yellow), Atg29 (orange), and Atg31(red). (B) Crystal structure of Atg1tMIT with Atg13MIM (PDB: 4P1N). Atg1 is shown in grey, Atg13 in magenta. (C) Crystal structure of Atg13(17BR)-Atg13(17LR)-Atg17- Atg31-Atg29 complex (PDB: 5JHF). Atg17 is shown in yellow, Atg29 in orange, Atg31 in red. The linking region (LR) of Atg13 is shown in magenta and the binding region (BR) of Atg13 is shown in blue.

Atg1 complex assembly has been shown to trigger downstream autophagic events and is thought to be responsible for autophagy initiation; hence, it is an important complex to study and is the focus of my research. I will briefly describe the other 5 autophagy-related groups below before discussing Atg1 complexes from other organisms and their role in autophagy regulation.

1.4.2.2 The Atg9 transmembrane protein

One of the next Atg proteins to be recruited to the PAS, Atg9, is the only conserved transmembrane protein amongst the 18 core Atg proteins essential for starvation-induced autophagy in budding yeast.

Atg9 (115 kDa) is predicted to contain six transmembrane helices linked by 5 loops, with its N- and C- terminal tail regions projecting into the cytosol (Figure 1.3B) (Young and Sharon A. Tooze, 2006).

Fluorescence microscopy studies showed that while Atg9 localizes mostly to small membrane vesicles arising from the Golgi apparatus, they also freely move throughout the cytoplasm (Yamamoto et al.,

2012). These cytoplasmic dot-like structures were described in 2010 (Mari et al., 2010). Upon starvation,

Atg9 accumulates at the PAS and is incorporated into the outer membrane of the autophagosome. Further studies revealed that only a few Atg9 vesicles are needed to make the “seed” that nucleates the formation of the autophagosome (Yamamoto et al., 2012). Due to these findings, Atg9 is thought to traffic between the PAS and peripheral sites to direct membrane needed for phagophore biogenesis and growth.

Localization of Atg9 to the PAS was initially shown to require both Atg17 and Atg1. Atg17 physically interacts with Atg9 in the presence of rapamycin in an Atg1-dependent manner. Interestingly, the kinase activity of Atg1 is not necessary for the interaction to occur or for the localization of Atg9 to the PAS

(Sekito et al., 2009). More recently, Ohsumi’s group showed that the HORMA domain of Atg13 is sufficient to recruit Atg9 to the PAS during starvation-induced autophagy (S. W. Suzuki et al., 2015). Furthermore, self-association of Atg9 is required for its localization to the PAS and for both selective and non-selective autophagy, suggesting that Atg9 multimerization facilitates autophagosome biogenesis and growth (He et al., 2008). Moreover, Atg9 is thought to be “recycled” as this protein can translocate from the PAS to cytoplasmic compartments dependent on Atg1 and Atg13, as well as the PI3K and Atg2-Atg18 complexes

(Yamamoto et al., 2012; Rao et al., 2016) described below.

1.4.2.3 The autophagy-specific class III phosphatidylinositol 3-kinase complex I (PI3K)

Two PI3K complexes are present in budding yeast. PI3K complex I is an autophagy-specific class III phosphatidylinositol 3-kinase complex (PI3K) that is recruited to the PAS after the Atg1 complex and plays a pivotal role in autophagy initiation (Figure 1.3C). Complex I is composed of Vps30/Atg6 (63.2 kDa), Atg14

(40.5 kDa), Atg38 (26.0 kDa), Vps15 (166.4 kDa), and the catalytic subunit Vps34 (101.0 kDa), all of which are essential for starvation-induced autophagy (Kihara et al., 2001). Vps34 is the catalytic subunit that generates phosphatidylinositol 3-phosphate (PI3P) required to recruit PI3P-binding proteins such as Atg18 to the PAS (Feng et al., 2014). Atg38 is required for the incorporation of PI3P into the isolation membrane

(Araki et al., 2013). Complex II, on the other hand, consists of Vps34, Atg6/Vps30, Vps15 and Vps38 (50.9 kDa) (in place of Atg14), and is involved in retrograde endosome-to-Golgi complex trafficking (Kihara et al., 2001). Hence, Atg14 is the autophagy-specific factor that directs the complex to the PAS (Obara, Sekito and Ohsumi, 2006). It has been suggested that Atg14 functions to direct the class III PI3K complex to the

PAS allowing for further recruitment of autophagy genes (Yang and Klionsky, 2010).

1.4.2.4 Atg18-Atg2 complex

In the early 2000s Atg18 (55.1 kDa) was discovered by the Thumm research group and was shown to be required for both Cvt and autophagy pathways (Barth et al., 2001). Atg18 is predicted to contain multi-

WD-repeats and an atypical PI3P binding region called a Phe-Arg-Arg-Gly (FRRG) motif. Furthermore,

Atg18 has been shown to be a PI3P effector (Dove et al., 2004; Strømhaug et al., 2004) and is essential for autophagy and regulation of vacuolar morphology (Dove et al., 2004). In Suzuki’s experiments to delineate the hierarchy of Atg proteins, Atg18 was shown to function after the recruitment of the Atg1 and PI3K complexes, but separate from the Atg8 and Atg12 conjugation systems (Suzuki et al., 2007). These results corroborate the thought that Atg18 functions downstream of the PI3K complex, with the Vps34 subunit generating the PI3P required for Atg18-Atg2 recruitment to the PAS (Obara et al., 2008; Feng et al., 2014).

Atg18 forms a heterodimeric complex with Atg2 (178.4 kDa) irrespective of PI3P binding. However,

Atg18 mutants unable to interact with PI3P display autophagy defects (Obara et al., 2008; Nair et al., 2010) and impaired localization of downstream proteins Atg8 and Atg16 (Petiot et al., 2000). This further suggests that PI3P promotes recruitment of the Atg18-Atg2 complex through Atg18 (Figure 1.3D) (Suzuki

26 et al., 2007). In addition, the Atg18-Atg2 complex may play a role in recruiting Atg16 and Atg8- phosphatidylethanolamine (PE) to the PAS and may further protect Atg8 from premature cleavage by Atg4

(described in section 1.4.2.5) (Nair et al., 2010). Atg21, an Atg18 homologue, that also contains WD-40 repeats has been shown to compensate for some roles of Atg18 (Nair et al., 2010) suggesting that WD-40 repeats contribute to Atg18 function. Collectively, these findings show that the Atg18-Atg2 complex is recruited to the PAS by the PI3K complex and is responsible for recruiting components of the Atg8 and

Atg12 conjugations systems.

1.4.2.5 The Atg8 conjugation system

The final functional groups required for phagophore assembly are the Atg8 and Atg12 conjugation systems that are thought to aid in phagophore membrane expansion (Noda and Inagaki, 2015). Atg8 (13.6 kDa), which is localized to the PAS and incorporated into the growing phagophore membrane during autophagy, is first processed by the cysteine protease Atg4 to remove the C-terminal arginine residue resulting in a mature form of Atg8 (Figure 1.3E; 1.6A, B) (Kirisako et al., 2000). Both Atg12 and Atg8 use the same E1 enzyme, Atg7, but the Atg8 conjugation system employs Atg3 in place of Atg10 as an E2 enzyme. The

Atg12 system was shown to play a role in forming the final Atg8-PE conjugated product (Hanada et al.,

2007). Atg8-PE deconjugation by Atg4 for Atg8 recycling is also important for autophagosome biogenesis

(Nair et al., 2012).

Most of Atg8 in a cell is conjugated to PE, recruited to the PAS, and incorporated into the inner and outer membrane of the autophagosome. It played an instrumental role in the identification of the yeast

PAS and is often used as a marker for the PAS/phagophore in fluorescence microscopy; furthermore, it can be used to measure autophagic flux (discussed in section 1.4.4). Atg8 has also been shown to be involved in membrane tethering and hemifusion events that support the expansion of the isolation

27 membrane (Nakatogawa, Ichimura and Ohsumi, 2007) further supporting that Atg8 plays integral functions for autophagosome biogenesis.

1.4.2.6 The Atg12 conjugation system

The Atg12 conjugation system is the second essential conjugation system required for autophagosome formation. The ability for Atg12 to form a conjugated product with Atg5 and Atg16 was first discovered by Noboru Mizushima who unexpectedly detected two bands through immunoblot analysis that corresponded to Atg12 (21.1 kDa); one band was much larger than anticipated and disappeared in the absence of Atg5 (33.6 kDa), Atg7 (71.4 kDa) or Atg10 (19.7 kDa) (Mizushima et al., 1998). It was later determined that these proteins make up a unique ubiquitin-like conjugation system. Atg12 is significantly larger than ubiquitin and contains a glycine residue at its C-terminus. Atg12 becomes activated by the E1 enzyme Atg7, resulting in a thioester intermediate that is transferred to the E2 enzyme Atg10. It then forms an isopeptide bond at a lysine residue that is located at the center of Atg5. The Atg12-Atg5 conjugate then binds a dimeric form of Atg16 (17.22 kDa) to form a dimer of Atg12-Atg5-Atg16 (Figure

1.3F) (Fujita et al., 2008; Fujioka et al., 2010; Ohsumi, 2016). This complex is formed constitutively irrespective of nutrient conditions and is localized to the PAS but not the completed autophagosome

(Suzuki et al., 2001). The Atg12 conjugation system is required for Atg8-PE formation (Hanada et al., 2007).

Like the Atg8 conjugation system, the Atg12 conjugation system is also required for autophagosome membrane expansion (Sakoh-Nakatogawa et al., 2013).

1.4.3 Diversity of autophagy machinery and its regulation in yeast and mammals

Although the process of autophagy is well conserved from yeast to mammals, autophagy-related proteins involved within the six functional complexes differ in composition among various organisms (Table 1.1).

The most significant differences are observed between the Atg1/ULK1 complex. The mammalian ULK1 complex possesses two functionally redundant Atg1 homologues, ULK1 (UNC-51-like kinase 1) and ULK2

(Kundu et al., 2008). In place of Atg17, mammals contain FIP200 (focal adhesion family interaction protein of 200 kDa )/RBCC1 (retinoblastoma 1-inducible coiled-coil 1) (referred to FIP200 from now on) which is almost four times larger than Atg17, and does not share significant sequence homology with Atg17 (Hara et al., 2008). Interestingly, the C-terminal region of FIP200 also shares limited sequence homology with the budding yeast selective autophagy-specific scaffold protein Atg11. That said, FIP200 has been shown to form a complex with ULK1 and mammalian ATG13 (Hara et al., 2008; Alers et al., 2011). Furthermore,

FIP200 is predicted to be predominantly helical in structure, similar to budding yeast Atg17 (Hara et al.,

2008; Ragusa, Stanley and Hurley, 2012). For these reasons, FIP200 is speculated to be functionally homologous to both yeast autophagy-related scaffolding proteins Atg11 and Atg17 (Noda and Inagaki,

2015).

Table 1.1. Autophagy-related genes in yeast and mammals

Functional Budding Yeast Fission Yeast Mammals Feature Complex Atg1/ULK1 Atg1 Atg1 ULK1/2 Ser/Thr kinase complex Atg13 Atg13 ATG13 Phosphorylated by (m)TORC1 Atg17 Atg17 FIP200/RB1CC1* Scaffold protein for Atg1/ULK1 complex Atg11 Atg11 * Scaffold protein for selective macroautophagy Atg29 - - Forms a ternary complex with Atg17 and Atg31 Atg31 - - Forms a ternary complex with Atg17 and Atg29 - Atg101/Mug66 ATG101 Interacts with Atg13 Class III PI3K Vps34 Pik3 Vps34 PI3K complex Vps15 Ppk19 Vps15 Ser/Thr kinase Vps30/Atg6 Atg6 Beclin 1 Beclin family protein Atg14 Atg14 Atg14(L)/Barkor Autophagy-specific subunit AMBRA1 Interacts with Beclin 1 Atg9 Atg9 Atg9 Atg9L1/2 Transmembrane protein

Functional Budding Yeast Fission Yeast Mammals Feature Complex Atg18-Atg2 Atg2 Atg2 Atg2A/B Forms a complex with Atg18 complex Atg18 Atg18 WIPI1/2/3/4 PI3P-binding protein DFCP1 PI3P-binding protein VMP1 Transmembrane protein Atg12 conjugation Atg12 Atg12 Atg12 Ubiquitin-like enzyme system Atg7 Atg7 Atg7 E1-like enzyme Atg10 Atg10 Atg10 E2-like enzyme Atg5 Atg5/Mug77 Atg5 Component of conjugation system Atg16 Atg16 Atg16L1/2 Forms a homodimer and interacts with Atg5 Atg8/LC3 Atg8 Atg8 LC3A/B/C, Ubiquitin-like protein, conjugation system GABARAP, conjugates to PE GABARAPL1/2/3 Atg4 Atg4 Atg4A-D Deconjugation enzyme Atg7 Atg7 Atg7 E1-like enzyme Atg3 Atg3 Atg3 E2-like enzyme *Note: FIP200 and RB1CC1 refer to the same gene product. The mammalian Atg1 complex contains one scaffold protein (FIP200/RB1CC1) that is thought to encompass roles of the 2 scaffold proteins in yeast (Atg17 and Atg11).

Another notable difference with respect to subunit composition between budding yeast and mammalian autophagy systems is that mammals lack counterparts to budding yeast Atg29 and Atg31 but contain a unique factor, ATG101 (Hosokawa et al., 2009; Sun et al., 2013; H. Suzuki et al., 2015b; Qi et al.,

2015). When commencing this thesis work, it was unclear if the ATG101 protein was the functional counterpart of the budding yeast Atg31-Atg29 complex. A short while after the X-ray crystal structures of

Atg101/ATG101 in complex with the HORMA domain of Atg13/ATG13 in both fission yeast and humans illustrated that unlike budding yeast Atg31-Atg29, which is anchored to Atg17, Atg101/ATG101 primarily interacts with the N-terminal HORMA domain of Atg13/ATG13 (H. Suzuki et al., 2015b; Michel et al., 2015;

Qi et al., 2015). This illustrates that Atg101/ATG101 likely does not function in the same way as the Atg31-

Atg29 complex. Budding yeast and mammals in part regulate autophagy through the Atg1/ULK1 complex;

30 however, budding yeast use the formation of the Atg1 kinase complex to signal autophagy initiation while the mammalian ULK1 complex is constitutively formed (Mizushima, Yoshimori and Ohsumi, 2011).

Not only do differences exist within the budding yeast and mammalian autophagy systems, but they are also apparent within fungal species. Both fission and budding yeasts use Tor proteins to aid in autophagic flux regulation; however Tor seems to play opposite roles in sexual development in the two organisms (Cafferkey et al., 1993; Kunz et al., 1993; Barbet et al., 1996; Zheng and Schreiber, 1997;

Schmidt et al., 1998; Zaragoza et al., 1998; Hardwick et al., 1999; Weisman and Choder, 2001). Both fission and budding yeast contain two Tor homologues, S. cerevisiae Tor homologues have only been shown to play a role in stress induced by starvation, while S. pombe homologues function under various stress conditions. Moreover, S. cerevisiae Tor (target of rapamycin) homologues overlap in function while S. pombe Tor homologues have distinct roles for growth and overcoming stress conditions. Interestingly, rapamycin initially did not seem to affect the functions of S. pombe Tor proteins (Weisman and Choder,

2001); however, further studies using S. pombe leucine auxotrophs illustrated that fission yeast cells with this auxotrophy are sensitive to rapamycin. Deletion of the S.pombe FKBP12 protein, Fkh1, suppresses this sensitivity suggesting that S. pombe Tor forms a complex with Fkh1 (Weisman et al., 2005). Although the Tor proteins are highly conserved, their N-terminal portions are less conserved and may account for the differences in Tor sensitivity to rapamycin (Weisman and Choder, 2001) as well as other differences observed between fission and budding yeast Tor proteins.

Further differences between budding yeast and mammalian autophagy systems exist. Mammals contain an additional transmembrane protein, the vacuolar membrane protein 1 (VMP1), in addition to

Atg9. This protein participates in the mammalian Atg18-Atg2 complex (Table 1.1). The localization of

VMP1 is controversial (Yang and Klionsky, 2010), it has been observed at the plasma membrane and colocalizes with LC3 and Beclin 1 (Ropolo et al., 2007), but has also been observed to localize to the ER

(Calvo-Garrido et al., 2008). If overexpressed, VMP1 induces autophagy by forming an interaction with

Beclin 1 (Ropolo et al., 2007). It has been suggested that VMP1 recruits Beclin 1, and possibly other PI3K complex components to the phagophore (Nowak et al., 2009) aiding in phagophore formation.

Yeast contain one PI3K, Vps34, which is essential to generate PI3P at the PAS for the recruitment of

other Atg proteins (Feng et al., 2014). Mammals contain a similar class III PI3K complex (see Table 1.1)

with Atg14 being the autophagy-specific component. Recent work on mammalian Atg14L shows that

Atg14L localizes to the ER under fed conditions and localizes to LC3- (mammalian Atg8 homologue) and

Atg16L-positive structures under starvation. Furthermore, reduced levels of Atg14L lead to reduced

autophagic flux as observed by a reduction in LC3 puncta formation, commonly used as an indicator of

autophagosome formation (Itakura et al., 2008; Sun et al., 2008). The function of the PI3K complex in

mammals and yeast seems to be conserved.

The two ubiquitin-like proteins and their conjugation systems, Atg12 and Atg8/LC3 are very similar in yeast and mammals (Figure 1.3D, E). Atg12 is conjugated to Atg5, using Atg7 and Atg10. The Atg12-Atg5 conjugate then interacts with Atg16 noncovalently to form a larger complex (Yang and Klionsky, 2010).

Although mammalian Atg8 homologues have a conserved function to the yeast Atg8 protein, mammals have evolved several Atg8 homologues including LC3A, LC3B, LC3C, GABARAP, and GABARAPL1/2/3 that have not been well characterized. Not all Atg8 homologues function similarly and it is thought that various

LC3/GABARAP proteins are used for various vesicle trafficking events that include targeting proteins to the autophagosome as well as fusion with the lysosome or endosome (Lee and Lee, 2016); hence, Atg8 homologues may act at different stages of autophagy. LC3 and Atg8 are incorporated into the autophagosome, and as discussed above, played a key role in the identification of the PAS and understanding of the development of the autophagosome. Atg8 homologues are also extensively used to measure autophagic flux, discussed in the next section.

Lastly, it is important to note that mammals and fission yeast generally contain many sites where autophagosomes can form to produce many small autophagosomes, while budding yeast contain one site,

32 the PAS, where autophagy-related proteins accumulate to form one large autophagosome. Due to the differences in autophagy machinery and the variability in number and size of autophagosomes across species, autophagy research in many organisms has been necessary.

1.4.4 Methods used in this study to measure autophagic flux

Takeshi Noda, who worked with Nobel Prize winner Yoshinori Ohsumi, made significant contributions to further our understanding of the autophagy pathway. One contribution he made was developing the standard method to quantitatively measure autophagic flux in the budding yeast. To do this he constructed a yeast strain containing a mutation in the precursor for the vacuolar alkaline phosphate,

Pho8. This mutant yeast strain lacked the N-terminal vacuolar targeting sequence of Pho8 (Pho8∆60); meaning, the expressed protein would be localized to the cytosol and taken to the vacuole only under autophagy-inducing conditions through non-selective uptake. This precursor can then be processed in the vacuole and perform its enzymatic activity; thus, acting as a quantitative measurement of autophagic flux

(Noda and Ohsumi, 1998; Noda and Klionsky, 2008). Figure 1.5, adapted from a review by Claudine Kraft’s group (Torggler, Papinski and Kraft, 2017) illustrates how this assay works. Pho8 without its localization sequence is found in the cytoplasm. Upon autophagy induction, the Pho8∆60 precursor is incorporated into the autophagosome and delivered to the vacuole through bulk autophagy. Pho8∆60 is activated within the lumen of the vacuole allowing for its activity to act as a quantitative measurement of autophagic flux.

Figure 1.5. Pho8∆60 activity assay to assess for autophagic flux in budding yeast. Pho8∆60 (yellow octagon) is a cytosolic protein as its localization sequence (the first 60 residues) are deleted. Upon autophagy induction, cytosolic Pho8∆60 is incorporated into the expanding phagophore and is transported to the vacuole where it is activated (blue octagon). The activity of Pho8∆60 is monitored as an indication of autophagic flux. Red and green shapes represent miscellaneous cytosolic proteins.

Another method to measure autophagic flux is the Atg8 processing assay. Broadly used in mammalian cells, this assay is based on monitoring the processing of Atg8 (or the Atg8 homologue LC3 in mammalian cells) (Figure 1.6). As described above, Atg8 which is normally processed by Atg4 and subsequently conjugation to PE, is incorporated into the inner and outer membranes of the autophagosome (Figure 1.6B). Atg8 molecules present on the outer autophagosome membrane are typically recycled by Atg4 but those integrated into the inner membrane are targeted to the lumen of the vacuole for degradation. Exploiting this unique feature, the Atg8 processing assay involves expressing an

N-terminal fluorescent-tag fusion of Atg8 and monitoring the fate of the inner autophagosome membrane pool of fluorescently-tagged Atg8 (Figure 1.6C). The fluorescent protein portion of the fusion protein is much more stable than Atg8/LC3 and persists in the vacuole/lysosome lumen, enabling detection of the fluorescent tag by western blotting (Delorme-axford et al., 2016).

Figure 1.6. Atg8 cleavage assay to assess autophagic flux. (A) Atg8 is processed and covalently linked to PE. (B) Atg8-PE is incorporated into the inner and outer membranes of the autophagosome which then fuses with the vacuole membrane. Atg8-PE on the outer membrane is recycled by Atg4. (C) To assess autophagy flux, a fluorescent tag (yellow star) is engineered at the N-terminus of Atg8. Upon autophagosome-lysosome fusion, the fluorescent tag is separated from Atg8 by vacuolar proteases (scissors). (D) Read-out of Atg8 cleavage assay through western blot analysis. Under non-autophagic conditions, Atg8 remains intact with the fluorescent tag (star), but under autophagy-inducing conditions the fluorescent tag is cleaved allowing for the detection of the tag separated from Atg8.

1.5 Using fission yeast to study autophagy

Many of the fundamental discoveries in autophagy were made using budding yeast. However, differences between the core autophagy machinery of S. cerevisiae and humans makes the transfer of knowledge difficult. Particularly intriguing is the difference in subunit composition of the Atg1/ULK1 complex, with the extensively studied budding yeast complex consisting of five proteins (Atg1, Atg13, Atg17, Atg29 and,

Atg31) opposed to the mammalian ULK1 complex which is composed of four proteins (ULK1, ATG13,

FIP200, and ATG101). Furthermore, a bioinformatic analysis showed that mammals and higher eukaryotes

35 do not harbor Atg29 or Atg31 homologues but contain the autophagy factor ATG101 (Table 1.1). In fact, this analysis showed that organisms containing Atg101 do not contain Atg29 or Atg31 homologues; hence, their presence in a genome is mutually exclusive (Noda and Mizushima, 2016). Interestingly, the absence of Atg29 and Atg31 homologues and presence of Atg101, initially identified as meiotically upregulated gene 66 (mug66), is observed in a fungal species—the fission yeast, S. pombe. S. pombe Atg101 has been shown to interact with a region of Atg13 that adopts a HORMA domain (Hop1, Rev7, Mad2). This interaction has been structurally characterized using the fission yeast and human Atg101-Atg13HORMA proteins. Notably, they uncovered that Atg101, which is also structurally defined as a HORMA domain, contains a conserved loop thought to be necessary for the recruitment of downstream autophagy-related proteins (H. Suzuki et al., 2015b; Qi et al., 2015). Due to the conservation of the Atg101 protein between humans and fission yeast, researchers have been turning to the fission yeast for a more relevant understanding of the autophagy pathway and the proteins involved.

One of the first documented autophagy studies in fission yeast was conducted by Kohda et al. where they observed autophagy induction under nitrogen starvation. They proposed that the function of autophagy in the fission yeast is to provide a nitrogen source for cellular maintenance (Kohda et al., 2007).

Another notable autophagy-related study analyzed the mating efficiencies of fission yeast deletion strains to reveal new fission yeast autophagy factors (Sun et al., 2013). Traditional techniques to measure autophagy were adapted to the fission yeast showing that this organism is amenable to autophagy research. Fission yeast seem to be more similar to mammals than budding yeast in that they contain multiple vacuoles, described as small fragmented vacuoles (Mukaiyama et al., 2010), instead of one large central vacuole; furthermore, the composition of proteins within the Atg1/ULK1 complexes are more comparable between humans and fission yeast (Klionsky and Eskelinen, 2014; Li, 2014). Although fission yeast cells have been used to study autophagy; the molecular organization of the fission yeast Atg1

36 complex had yet to be determined. Furthermore, the function of the Atg101 protein remains elusive.

These aspects of the Atg1 complex are addressed in my thesis.

1.6 Overview of thesis

As detailed above, the “replacement” of Atg29 and Atg31 with ATG101 is a key composition difference between the budding yeast Atg1 complex and the orthologous mammalian ULK1 complex. Despite recent advances in structural and biochemical characterization of the budding yeast Atg1 complex, how the aforementioned subunit composition difference affects the overall architecture, subunit organization, and molecular interactions of the mammalian ULK1 complex is not fully understood. Fission yeast S. pombe offers a genetically trackable and biochemically less challenging system to investigate this biological question as this unicellular eukaryotic model organism possesses an Atg1 complex that seemingly shares identical composition to the mammalian ULK1 complex. The major goal of my thesis research is to gain new knowledge on the structural and biochemical properties of the S. pombe Atg1 complex by completing the following three aims. 1) Develop an interaction network of the S. pombe Atg1 complex to further compare the budding and fission yeast Atg1 complexes; 2) gain insights into the function of Atg101 using proteomic mass spectrometry; and 3) characterize the interaction between

Atg101 and Fkh1 in S. pombe. Chapter 2 will focus on my first aim, Chapter 3 will focus on my last 2 aims, and Chapter 4 will provide a summary and discussion of the results found in my thesis and will further outline future experiments that could be conducted to further this work.

CHAPTER 2: Conserved and unique features of the fission yeast core Atg1 complex

2.1 Introduction

The mammalian ortholog of the budding yeast Atg1 complex, the ULK1 complex, consists of 4 subunits:

ULK1, FIP200, and ATG13 (the orthologs of Atg1, Atg17, and Atg13, respectively), and a unique subunit known as ATG101 in place of S. cerevisiae Atg29 and Atg31. Interestingly, Atg29 and Atg31 are essential to autophagy in budding yeast and function to stabilize the S-shape architecture of Atg17 (Kawamata et al., 2005; Kabeya et al., 2009; Chew et al., 2013). What allows FIP200, which is 4 times larger in size and lacks discernable sequence homology to yeast Atg17, to bypass the requirement of the Atg31-Atg29 complex is not known. Recent crystallographic and biochemical studies show that ATG101, unlike Atg31-

Atg29, is a HORMA-domain-containing protein that interacts with the N-terminal HORMA domain of

ATG13 (Hegedus et al. 2014; Qi et al. 2015; Michel et al. 2015). However, whether ATG101 is capable of binding Atg17 and replacing the function of Atg29 and Atg31 has not been investigated. It is also unclear how the difference in composition between the S. cerevisiae Atg1 complex and the mammalian ULK1 complex affects subunit organization and overall architecture of these complexes.

The fission yeast, S. pombe, Atg1 complex provides a simplified model to investigate these questions. Despite having Atg1, Atg13, and Atg17 subunits that share similar size and secondary structure elements as their counterparts in the budding yeast, S. cerevisiae, Atg1 complex, the S. pombe Atg1 complex is devoid of Atg29 and Atg31 but contains an Atg101 protein that adopts an identical structure to human ATG101 (Hegedus et al., 2014; H. Suzuki et al., 2015a; Michel et al., 2015; Qi et al., 2015). Using biochemical and structural approaches, we show that the core subunit interactions observed in the S. cerevisiae Atg1 complex are preserved in the S. pombe Atg1 complex, with Atg17 serving as a central scaffold to anchor Atg13 and Atg1. However, we found that S. pombe Atg101 does not bind Atg17, and instead of forming an S-shape architecture as in the case of budding yeast, fission yeast Atg17 adopts an

38 overall rod-shape architecture. Furthermore, we show that S. pombe atg17 cannot rescue autophagy in an S. cerevisiae atg17∆ mutant strain even if S. pombe atg101 is incorporated. Reverse complementation experiments show that budding yeast atg17 cannot function in place of fission yeast atg17. Finally, we demonstrated that the heterodimerization of Atg101 with the HORMA domain of Atg13 enhances the stability of both proteins. Collectively, our studies provide insight into the conserved and unique properties of an Atg101-containing Atg1/ULK1 complex and establish the framework to further dissect the functions of the ULK1 complex in higher eukaryotes.

2.2 Materials and Methods

2.2.1 Molecular cloning

The coding regions of S. pombe Atg1CTD (589–830), Atg13HORMA (1–269), Atg13MIM (476–545), Atg17, and

Atg101 were amplified by PCR from genomic DNA with primers designed to incorporate N-terminal BamHI

(Thermo Fisher Scientific, FD0054) and C-terminal NotI (Thermo Fisher Scientific, FD595) restriction sites.

The Atg13CTD gene was synthesized (GenScript, Piscataway, NJ, USA) to remove internal restriction sites.

This construct was used for subsequent cloning of peptides within the CTD of Atg13. For co-expression constructs used in co-precipitation experiments, the pQLINK system of expression vectors was used

(Scheich et al., 2007). In particular, each gene was cloned into either the pQLinkG2 vector (Addgene,

13671; deposited by Konrad Buessow) encoding the GST-tag or the pQLinkH vector (Addgene, 13667; deposited by Konrad Buessow) encoding the hexahistidine-tag, and subsequently linked by ligase- independent cloning (Chew et al., 2015). To express His-MBP-tagged S. pombe Atg17, the gene was cloned into a modified version of the pET28a vector (Novagen, 69864) encoding an N-terminal His-MBP tag.

The CUP1 promoter and CYC1 terminator fragments were ligated into the SacI and SpeI sites, or

XhoI and KpnI sites of pRS416, respectively, to construct pCu(416). Three HA tandem repeats or a GFP fragment was ligated into the SpeI and XmaI sites of pCu(416) to construct pCu-HA(416) or pCu-GFP(416).

39 pCu(414) and pCu-HA(414) were constructed similarly from pRS414. The SpATG17 ORF fragment was ligated into the XmaI and XhoI sites of pCu(416), pCu-HA(416) and pCu-GFP(416) vectors to construct pCu-

SpAtg17(416), pCu-HASpAtg17(416) and pCu-GFP-SpAtg17(416). The SpATG101 ORF fragment was ligated into the XmaI and XhoI sites of the pCu-HA(414) vector to construct pCu-HA-SpAtg101(414).

2.2.2 Co-precipitation experiments

Different co-expression constructs were transformed in T7 Express E. coli cells (NEB, C2566I). Cells were grown in 2xYT media at 37°C to an OD600 of ~0.6. Expression was induced with 0.5 mM IPTG (Gold

Biotechnology, I2481) and cells were grown at 16°C for 18 h. Cells were lysed by sonication and cleared by centrifugation (30,966 x g for 30 min). Cell lysates were incubated with glutathione resin (GenScript,

L00206) for 1 h. Glutathione beads were pelleted and washed with wash solution (50 mM Tris, pH 8.0,

150 mM NaCl, 0.1% Triton X100 [Sigma, T8787], 5% glycerol, 0.5 mM DTT) 3 times. SDS loading dye was added to the beads, followed by boiling for 10 min. Samples were loaded onto two 12% SDS-PAGE gels.

One gel was stained with Coomassie Brilliant Blue, and the second gel was first transferred to a nitrocellulose membrane, subsequently probed with the anti-His antibody (mouse monoclonal; ABM,

G020) followed by detection using HRP-conjugated anti-mouse-IgG (Sigma, A4416). Chemiluminescence signal was detected using Amersham ECL Prime Western Blot Detection Reagent (GE Healthcare) and visualized using a Konica Medical Film Processor (Model SRX-101A) on Mandel 8x10 film (MED_CLE810).

2.2.3 Protein purification

For the purification of the Atg101-Atg13HORMA complex, His-tagged Atg101, and untagged Atg13HORMA were co-expressed in T7 Express E. coli cells (NEB, C2566I). Cells were grown in 2xYT media and lysed in buffer

A (50 mM Tris, pH 8.0, 150 mM NaCl, 10 mM PMSF) as described above. His-Atg101 as well as the His-

Atg101-Atg13HORMA complex were purified using nickel resin (ThermoScientific, 88222). The resin was washed with buffer B (50 mM Tris, pH 8.0, 150 mM NaCl) followed by buffer B supplemented with 50 mM

40 imidazole. The protein was eluted with buffer B supplemented with 100 mM imidazole, concentrated, and further purified using the Superdex 200 Increase gel filtration column (GE Healthcare Life Sciences,

28990944) in buffer B. For the purification of untagged Atg13HORMA, the protein was expressed in the

LOBSTR E. coli Expression strain (Kerafast, EC1002). Cells were lysed in buffer B, cleared by centrifugation

(as described above) and loaded onto a column containing Q Sepharose Fast Flow resin (GE Healthcare

Life Sciences, 7051001). The flow-through was loaded onto a HiPrepTM SP HP 16/10 (GE Healthcare Life

Sciences, 29-0181-83) column. The protein was eluted using a gradient from 0.15 M NaCl to 1.50 M NaCl.

His-MBP-Atg17 was expressed in T7 Express E. coli cells and grown as stated above. For the purification of His-MBP-Atg17, this protein was expressed in the BL21 (DE3) E. coli strain (NEB, C2527I). Cells were lysed in buffer B, and the protein was purified using nickel resin and eluted with 50 mM Tris, pH 8.0, 150 mM NaCl, and 250 mM imidazole. Fractions containing His-MBP-Atg17 were further purified using amylose resin (NEB, E8021S) and a Superpose 6 (10/300) gel filtration chromatography column (GE

Healthcare Life Sciences, 17517201).

To reconstitute the S. pombe His-MBP-Atg17 S. cerevisiae Atg31-Atg29 complex (SpAtg17-ScAtg31-

29), the Atg31-Atg29 complex was first purified. Atg31 and Atg29 were co-expressed in T7 Express E. coli cells using the pCOLADuet-1 vector (Novagen, 71406) and grown in 2xYT media. Cells were lysed in buffer

A, and the proteins were purified using HisPur nickel-NTA resin (ThermoScientific, CAP188222). The resin was washed with buffer B followed by buffer B supplemented with 50 mM imidazole. The protein was eluted with buffer B supplemented with 100 mM imidazole. The concentrated protein was further purified using the Superdex 75 10/300 GL (GE Healthcare Life Sciences, 17517401). His-MBP-Atg17 was purified as described above; however, purified Atg31-Atg29 was added to His-MBP-Atg17 during the amylose purification. The SpAtg17-ScAtg31-29 complex was separated using the Superose 6 (10/300) gel filtration chromatography column (GE Healthcare Life Sciences, 17517201) in 50 mM Tris, pH 8, 150 mM NaCl and used for negative stain EM studies.

2.2.4 Negative stain electron microscopy and image processing

Negative stain specimens were prepared as previously described (Ohi et al., 2004). Images of His-MBP- tagged S. pombe Atg17 or reconstituted S. pombe Atg17 in complex with S. cerevisiae Atg31-Atg29 were recorded at a nominal magnification of 49,000 and a defocus value of -1.0 µm using a 4K x 4K Eagle charged-coupled device (CCD) camera (FEI) mounted on a Tecnai Spirit transmission electron microscope

(FEI) operated at an accelerating voltage of 120 kV. For image processing of the S. pombe Atg17 dataset,

5,382 particle images were interactively selected using Boxer (Ludtke, Baldwin and Chiu, 1999), the selected particles were then translationally aligned and subjected to 10 cycles of multireference alignment using SPIDER (Frank et al., 1996). Each round of alignment was followed by K-means classification specifying 50 classes, and an averaged image was calculated from the particle images making up each of the 50 classes. End-to-end distances of Atg17 were measured using Jweb (Shaikh et al., 2009). For image processing of the SpAtg17-ScAtg31-29 dataset, 4,313 particle images were interactively selected using

Boxer, with 2D classification performed using RELION (version 1.4) (Scheres, 2012), specifying 50 classes.

2.2.5 S. cerevisiae yeast strains and Pho8∆60 activity assay

S. cerevisiae yeast strains used in this study are listed in Table 2.1. For the Pho8∆60 activity assay, WLY176

S. cerevisiae cells were transformed with an empty vector, while the S. cerevisiae atg17∆ strain was transformed with either an empty vector or vectors harboring a gene encoding ScAtg17, SpAtg17, HA-

SpAtg17 or GFP-SpAtg17. These cells were cultured to mid-log phase in SMD-Ura (0.67% yeast nitrogen base without amino acids, 2% D-glucose, and appropriate amino acids and nucleic acid bases to satisfy any auxotrophies) before they were shifted to nitrogen starvation medium (SD-N; 0.17% yeast nitrogen base without amino acids and ammonium sulfate, 2% D-glucose, and appropriate amino acids and nucleic acid bases to satisfy any auxotrophies) for 4 h. Samples were collected and the Pho8∆60 assay was performed as described (Noda and Klionsky, 2008).

Table 2.1. List of S. cerevisiae strains used

Name Genotype Reference

MATα leu2-3,112 ura3-52 his3-Δ200 trp1-Δ901 suc2-Δ9 (Tomotake Kanki et al., WLY176 lys2-801 GAL pho13∆ pho8∆60 2009)

XLY134 WLY176 atg17∆::KANMX6 (Liu et al., 2016)

XLY159 WLY176 atg17∆::KANMX6 ATG31-PA This study

XLY160 WLY176 atg29∆::HIS3 atg31∆::KANMX6 This study

2.2.6 CFP-Atg8 cleavage assays

An S. pombe atg17∆ strain expressing CFP-Atg8 was transformed with a plasmid expressing mCherry, mCherry-tagged ScAtg17, or mCherry-tagged SpAtg17. Along with a WT S. pombe strain expressing CFP-

Atg8, strains were cultured to mid-log phase in EMM growth media (14.7 mM potassium hydrogen phthalate, 15.5 mM Na2PO4, 93.5 mM NH4Cl, 2% glucose, plus salts, vitamins, and minerals mixture) before they were shifted to EMM-N, nitrogen-free media to induce autophagy. Cells were harvested after

2 h and 4 h. Lysates were prepared from approximately 3 OD600 units of cells using a TCA lysis method

(Ulrich and Davies, 2009). Samples were separated on 10% SDS-PAGE and then immunoblotted with an anti-GFP mouse monoclonal antibody (Abmart, ACAM20004). Coomassie Brilliant Blue R-250 staining of

PVDF membrane after immunodetection was used as a control for protein loading and blotting efficiency.

2.2.7 Melting temperature determination by differential scanning fluorimetry (DSF)

To perform differential scanning fluorimetry (DSF) analysis (Vedadi et al., 2010), we combined protein samples at ~1 mg/mL (in 50 mM HEPES, pH 8, 150 mM NaCl) with SYPRO® Orange Protein Gel Stain

(ThermoFisher, S6650) to a final concentration of 5x in 96-well plates. The plates were placed in a

StepOnePlus Real Time PCR System (Applied Biosystems), used in conjunction with MicroAmp® Fast

Optical 96-well reaction plates (ThermoFisher Scientific, 4346906) and optical adhesive covers

(ThermoFisher Scientific, 4306311). Samples were heated from 25°C to 95°C with a change in temperature

43 of 1°C per minute. The fluorescence of the specimens was monitored using the StepOne software 2.1 at every 0.5°C interval to determine the melting temperature. All graphs and calculations were generated using Microsoft Excel.

2.2.8 Chemical crosslinking and crosslinking coupled to mass spectrometry (CXMS)

For determining their stoichiometry, His-Atg101, Atg13HORMA, and the His-Atg101-Atg13HORMA complex in

150 mM NaCl, 50 mM HEPES, pH 8 were subjected to chemical crosslinking using DSS (0–640 mM;

ThermoFisher Scientific, P121555) for 30 min at room temperature. Samples were resolved by 15% SDS-

PAGE and complex formation was assessed via Coomassie Brilliant Blue staining. For CXMS analysis, bands of crosslinked products were excised from the gel and digested with trypsin (Sigma, T6567). Digestion products were analyzed by liquid chromatography-mass spectrometry (LC-MS) as described previously

(Chew et al., 2015). Crosslinked peptides were identified using the pLink search engine (Yang et al., 2012).

2.3 Results

2.3.1 Subunit interactions of the core S. pombe Atg1 complex

The major difference between the subunit composition of the Atg1 complex in S. cerevisiae and more complex eukaryotes is the replacement of Atg29 and Atg31 with Atg101 (Hosokawa et al., 2009). To determine how the absence of Atg29 and Atg31 and the presence of Atg101 affect the overall organization of the S. pombe Atg1 complex, we first applied a co-precipitation approach to systematically probe the subunit interactions among the 4 core subunits of this complex. This established approach involves co- expressing a GST-tagged subunit with a hexahistidine-tagged subunit in the same E. coli host and assessing for an interaction by GST co-precipitation and western blotting (Chew et al., 2015). We could express full- length S. pombe Atg17 as well as Atg101. However, due to technical difficulty in expressing full-length

Atg1 and Atg13 subunits, we focused our analyses on the core domains of Atg1 and Atg13, specifically the

C-terminal domain (CTD) of Atg1 (Atg1CTD, residues 589–830), and the N-terminal HORMA domain and

CTD of Atg13 (Atg13HORMA, residues 1–269; Atg13CTD, residues 392–758) (Figure 2.1A). These proteins/domains form the core S. cerevisiae Atg1 complex that our lab and others have previously reconstituted and characterized (Ragusa, Stanley and Hurley, 2012; Chew et al., 2015).

Our analyses show that S. pombe Atg17, whose counterpart was demonstrated to be the central scaffolding subunit in the S. cerevisiae Atg1 complex (G Stjepanovic et al., 2014; Chew et al., 2015), interacts with S. pombe Atg13CTD but not Atg1CTD or Atg101 (Figure 2.1B). The Atg17-Atg13CTD interaction was observed despite the presence of very low levels of GST-Atg13CTD bait. Notably, we need to use 10 times the amount of lysate to precipitate enough protein to visualize GST-Atg13CTD on our Coomassie Blue- stained gel. We also found that Atg13CTD interacts strongly with Atg1CTD (Figure 2.1C and D), suggesting that similar to its ortholog in S. cerevisiae, S. pombe Atg13 anchors Atg1 to Atg17. The co-expression of

GST-Atg13CTD with Atg1CTD also appears to enhance the stability of Atg13CTD as demonstrated by a substantially higher amount of this protein recovered from the co-precipitation (compare Figure 2.1B to

2.1D).

It was previously shown that the S. cerevisiae Atg13 MIM domain interacts with the C-terminal MIT domain of Atg1 (Fujioka et al., 2014). However, we found that the putative MIM domain of S. pombe

Atg13 (residues 475–545; sequence alignment shown in Figure 2.2C) is not capable of, or sufficient for, binding to S. pombe Atg1CTD (Figure 2.1D). To map the Atg1 binding region of Atg13, we generated a series of Atg13CTD fragments (Figure 2.2A) and tested their abilities to interact with Atg1CTD. We found that the fragment containing a C-terminal extension to the putative MIM domain (residues 476–640) interacted weakly with Atg1CTD. Furthermore, the addition of a fragment of Atg13 to include an N-terminal extension

(residues 392–640) to the putative MIM domain substantially enhanced binding (Figure 2.2B). Although we could not test binding of a fragment containing only the N-terminal extension (residues 392–545) due to its instability, our results show that additional elements, both N- and C-terminal to the putative MIM domain, are required for a stable interaction between Atg13 and Atg1 (Figure 2.2).

Figure 2.1. Intersubunit interactions of the S. pombe Atg1 core complex. (A) A schematic of the S. pombe Atg1 complex subunits: the CTD of Atg1 (gray), the HORMA domain (pink), and CTD of Atg13 (pink), full- length Atg17 (yellow), and Atg101 (green). Panels (B to E) show results of co-precipitation experiments performed by co-expressing one of the proteins of interest with a histidine tag and another protein with a GST tag or the GST tag alone. The Coomassie Brilliant Blue-stained gel (top panel) shows the GST

46 construct inputs (cell lysate) as well as the samples precipitated by glutathione beads (affinity isolate [pull down]). The GST-tagged baits are indicated by an asterisk. We loaded equal amounts of GST-tagged protein except in the case of GST-Atg13CTD co-expressing with His-Atg17, in which we could only detect a faint band of GST-Atg13CTD even after 10 times the amount of cells were used. For our western blot analyses, we loaded equal amounts of cell lysate and twice as much of the affinity isolate, except for GST- Atg1CTD, His-Atg13CTD (bottom panel C) and GST-Atg13CTD, His-Atg1CTD (bottom panel D) where half the volume of the cell lysate was loaded due to saturation of western blot signals. (B) His-Atg17 was co- expressed either with GST, GST-Atg1CTD, GST-Atg13CTD, or GST-Atg101. (C) His-Atg13CTD was co-expressed with GST, GST-Atg1CTD, or GST-Atg101. (D) His-Atg1CTD was co-expressed either with GST, GST-Atg13(476- 545), Atg13CTD, or GST-Atg101. (E) His-Atg101 was co-expressed either with GST, GST-Atg13HORMA (Atg13H), or GST-Atg1CTD. (F) Schematic comparing the subunit interactions between the fission (left panel) and budding (right panel) yeasts Atg1 complex.

Recent biochemical and structural studies revealed that the N-terminal HORMA domain of Atg13

(Atg13HORMA) from both fission yeast and humans binds Atg101, another HORMA-fold protein, in their respective organisms (H. Suzuki et al., 2015b; Qi et al., 2015). Our co-precipitation data not only confirmed this finding but also uncovered that Atg101 can interact with Atg1CTD, suggesting that this novel subunit makes additional contacts with other components of the Atg1 complex (Figure 2.1D and E). Collectively, our data suggest that the intersubunit interactions among the core subunits of the S. cerevisiae Atg1 complex (Atg1, Atg13, Atg17) are largely conserved in the S. pombe Atg1 complex, despite a difference in overall subunit composition. Furthermore, the finding that Atg101 does not interact with Atg17 suggests that this protein serves a function distinct from Atg29 and Atg31. A schematic summarizing the observed subunit interactions of the S. pombe Atg1 complex and its comparison to the orthologous S. cerevisiae

Atg1 complex is shown in Figure. 2.1F.

Figure 2.2. The interaction between S. pombe Atg13CTD and Atg1CTD. (A) Schematic of Atg1 and Atg13 constructs. (B) Co-precipitation experiments co-expressing His-Atg1CTD with GST or various constructs of

GST-Atg13. The Coomassie Brilliant Blue-stained gel (top panel) shows the GST construct inputs (cell lysate) as well as the samples precipitated by glutathione beads (affinity isolate [pull down]). The asterisks to the right of each band indicate the GST-tagged proteins. Glutathione resin was used for precipitation; 25% of each affinity isolate was loaded. The western blot (bottom panels) was probed using anti-His antibody where 25% of the affinity isolate was loaded. Both short exposures (S.E.) and long exposures (L.E.) were imaged. (C) Sequence alignment of S. cerevisiae Atg13 (GenBank: AJW27260.1) and S. pombe Atg13 (GenBank: CAB11710.1) using Clustal Omega from EMBL-EBI.

2.3.2 S. pombe Atg17 is dimeric and adopts a rod-like architecture

The high degree of conservation observed between the intersubunit interactions underlying the S. pombe and S. cerevisiae Atg1 complexes implies that its Atg17 component, despite having limited sequence identity (22%) to S. cerevisiae Atg17, likely serves an analogous scaffolding function as its budding yeast ortholog. Previous negative stain single-particle EM studies revealed that although S. cerevisiae Atg17, in complex with Atg29 and Atg31, forms a highly extended dimeric assembly with a double-crescent shape essential for mediating early vesicle tethering in autophagy, S. cerevisiae Atg17 cannot stably adopt an overall S-shaped conformation on its own (Chew et al., 2013) (Figure 2.3A). We; therefore, hypothesized that S. pombe Atg17 and other Atg17 orthologs in more complex eukaryotes (which lack Atg29 and Atg31) would have evolved the ability to dimerize into a structure with a curvature consistent with that of S. cerevisiae Atg17-Atg31-Atg29. To test this hypothesis, we overexpressed and purified recombinant S. pombe Atg17 from E. coli and analyzed the structural properties of the purified protein using single- particle EM. Similar to S. cerevisiae Atg17, we needed to fuse an N-terminal histidine-maltose binding protein (His-MBP) tag to maintain the stability of the overexpressed protein. We subjected the purified protein to negative stain EM analysis and observed highly extended particles in the raw images. The class averages obtained from 2-dimensional (2D) classification of a dataset containing 5,382 particles subsequently showed that S. pombe Atg17, similar to S. cerevisiae Atg17, was an obligate dimer with the

N-termini of the 2 proteins projecting away from the tips of the dimer. However, contrary to our hypothesis, S. pombe Atg17 adopted an apparent “rod” shape architecture that lacks discernable curvature over its entire length (Figure 2.3B).

Figure 2.3. Negative stain EM analysis of S. pombe His-MBP-Atg17. (A) Representative class averages obtained from the classification of 5,922 negatively stained His-MBP-tagged S. cerevisiae Atg17 particles showed that S. cerevisiae Atg17 can adopt 3 major conformations: S-shaped, asymmetric, and extended. The side length of each panel is 64 nm. (B) Representative class averages obtained from the classification of 5,382 negatively-stained His-MBP-tagged S. pombe Atg17 particles showed that this protein adopts a rigid rod-shaped conformation. The side length of each panel is 64 nm. (C) Distribution of junction-to- junction lengths of S. pombe Atg17 showing that most particles have lengths consistent with S. cerevisiae Atg17-Atg31-Atg29 with two minor populations having shorter lengths.

By measuring the junction-to-junction distance of these 2D averages, we found that the majority

(~77%) of S. pombe Atg17 molecules have lengths ranging from 320 to 350 Å (average length of 335 Å)

(Figure 2.3C), a value consistent with what has been observed for the dimeric S. cerevisiae Atg17 and the

Atg17-Atg31-Atg29 complex (Chew et al., 2013). Furthermore, we also detected two minor subpopulations of particles with averaged lengths of ~245 Å and 290 Å, respectively (Figure 2.3C). These

50 observations suggest that although S. pombe Atg17 appears to be able to extend laterally, this protein possesses limited conformational flexibility compared to its S. cerevisiae counterpart.

2.3.3 S. pombe Atg17 can interact with S. cerevisiae Atg29 and Atg31 in vitro

Results previously conducted in our lab show that the S. cerevisiae Atg31-Atg29 subcomplex induces and stabilizes the unique S-shape overall architecture of S. cerevisiae Atg17 (Chew et al., 2013). To determine if the lack of inherent curvature observed for S. pombe Atg17 could be attributed to the absence of Atg29 and Atg31 orthologs, we first tested whether S. cerevisiae Atg29 and Atg31 can bind S. pombe Atg17. To our surprise, we found that GST-Atg29, as well as GST-Atg31, were both able to pull down histidine- tagged S. pombe Atg17 (Figure 2.4A), similar to what was observed for S. cerevisiae Atg17 (Chew et al.,

2015). Furthermore, Atg31 appears to have stabilizing effects on S. pombe Atg17 as shown by a substantial increase in Atg17 protein that was pulled down (16 times less of the affinity isolate was loaded compared to the negative control lane) (Figure 2.4A).

Figure 2.4. S. pombe Atg17 binds S. cerevisiae Atg29 and Atg31 but cannot complement S. cerevisiae Atg17 function. (A) Histidine-tagged S. pombe Atg17 was co-expressed either with GST or GST-tagged S. pombe Atg13CTD, S. cerevisiae Atg29, or S. cerevisiae Atg31. Coomassie Brilliant Blue-stained gel (top panel) shows the GST construct inputs. Glutathione resin was used for precipitation; 25% of each co-precipitation was loaded. Western blot (bottom panel) was probed using anti-His antibody; 10% of the affinity isolate (pull down) was loaded for both the GST construct alone and with S. cerevisiae Atg29, while 17% was loaded for the S. pombe Atg13CTD construct and only 0.6% loaded for the S. cerevisiae Atg31 construct. (B) S. pombe Atg17 complementation assay using Pho8∆60 activity to measure autophagy in S. cerevisiae. The WT strain was transformed with an empty vector, while the atg17∆ strain was transformed with either an empty vector or vectors harboring S. cerevisiae (Sc) Atg17, S. pombe (Sp) Atg17, HA-SpAtg17 or GFP- SpAtg17. Cells were grown in nutrient-rich medium (SMD-Ura) and then shifted to nitrogen starvation medium (SD-N) for 4 h. Phosphatase activity was measured and plotted. Experiments were performed in triplicate. (C) Histidine-tagged S. pombe Atg17 was co-expressed either with GST or GST-tagged S. pombe Atg13CTD or full-length S. cerevisiae Atg13 (ScAtg13FL). Coomassie Brilliant Blue-stained gel (top panel) shows the GST construct inputs. Glutathione resin was used for precipitation; 25% of each affinity isolate was loaded. Western blot (bottom panel) was probed using anti-His antibody where 10% of the affinity isolate was loaded. (D) S. cerevisiae Atg17 failed to complement the autophagy defect of S. pombe atg17∆. Autophagy in S. pombe was assayed by monitoring CFP-Atg8 cleavage. Coomassie Brilliant Blue staining of the PVDF membrane was used as a loading control. (E) The WT (WLY176) S. cerevisiae strain was transformed with an empty vector, while the S. cerevisiae atg29∆ atg31∆ (XLY160) strain was transformed with either an empty vector or vector harboring a gene encoding HA-SpAtg101. These cells were cultured to mid-log phase in SMD-Trp before they were shifted to nitrogen starvation medium (SD-N) for 3 h. Samples were collected and the Pho8∆60 assay was performed. The value of starved WT cells was set to 100% and other values were normalized. The error bars indicate the standard deviation (SD) of 3 independent experiments. (F) The S. cerevisiae atg17∆ (XLY134) or atg17∆ ATG31-PA (XLY159) strains were transformed with either pCu(416)-GFP-ScAtg17 or pCu(416)-GFP-SpAtg17. These cells were cultured to mid-log phase in SMD-Ura before they were shifted to nitrogen starvation medium (SD-N) for 3 h. Cell lysates were prepared and incubated with IgG-Sepharose for co-precipitation. The total lysates and eluted proteins were analyzed using SDS-PAGE and detected with monoclonal anti-YFP antibody and an antibody that binds to PA. S.E., short exposure and L.E., long exposures were acquired.

Interestingly, we observed altered electrophoretic mobility of S. pombe Atg17 in the co- precipitation involving S. cerevisiae Atg31 (Figure 2.4A). To determine if this observation was due to a conformational change to S. pombe Atg17 induced by S. cerevisiae Atg31, we attempted to reconstitute a complex containing S. pombe Atg17 and the S. cerevisiae Atg31-Atg29 complex (denoted SpAtg17-

ScAtg31-29) by mixing purified His-MBP-tagged S. pombe Atg17 and the S. cerevisiae Atg31-Atg29 subcomplex and further purifying the “full complex” by gel filtration chromatography. Although this reconstitution procedure was quite inefficient, with the “full complex” representing less than 1% of the peak fraction in our size exclusion purification, we detected a small population of S. pombe Atg17

53 particles containing an additional bound density by negative stain EM. Class averages obtained from 2D analysis of 4,313 SpAtg17-ScAtg31-29 particles containing the additional density showed that this density is located close to the tip of S. pombe Atg17 juxtaposed to the density corresponding to the His-MBP tag.

Furthermore, multiple class averages showed that S. pombe Atg17 displays a characteristic bend over its entire length (Figure 2.5A), suggesting that S. cerevisiae Atg31-Atg29 binding can indeed have an impact on the overall architecture of S. pombe Atg17.

Figure 2.5. Interaction between S. pombe Atg17 and S. cerevisiae Atg31. (A) Top 15 most populated class averages obtained from the 2D classification of 4,313 negatively stained SpAtg17-ScAtg31-29 particles using Relion (Scheres, 2012). The side length of each panel is 672 nm. Class averages with SpAtg17 showing characteristic bending are outlined in red. (B) The S. cerevisiae atg17∆ (XLY134) or atg17∆ ATG31-PA (XLY159) strains were transformed with either pCu(416)-GFP-ScAtg17 or pCu(416)-GFP-SpAtg17. These cells were cultured to mid-log phase in SMD-Ura before they were shifted to nitrogen starvation medium (SD-N) for 3 h. Cell lysates were prepared and incubated with IgG-Sepharose for co-precipitation. The total lysates and eluted proteins (IP) were analyzed using SDS-PAGE and detected with monoclonal anti-YFP antibody and an antibody that binds to PA. Both short and long exposures were taken (S.E. and L.E. respectively).

2.3.4 S. pombe Atg17 cannot functionally complement S. cerevisiae Atg17 in vivo

Given the fact that S. pombe Atg17 could bind S. cerevisiae Atg29 and Atg31 in vitro, we next tested if this protein could restore the function of its counterpart in S. cerevisiae in vivo. More specifically, we employed the Pho8∆60 activity assay (described in Chapter 1) to assess the level of autophagy upon nitrogen starvation in an S. cerevisiae atg17∆ deletion strain expressing S. pombe Atg17 by transforming with an expression vector encoding untagged S. pombe Atg17, HA-tagged S. pombe Atg17, or GFP tagged S. pombe

Atg17. Results from this assay show that S. pombe Atg17, despite being able to interact with Atg29 and

Atg31 in vitro, could not complement the function of S. cerevisiae Atg17 (Figure 2.5B). We also attempted to perform in vivo co-precipitation assays by co-expressing GFP-tagged S. pombe Atg17 with S. cerevisiae protein A (PA)-tagged Atg31 in an S. cerevisiae atg17∆ deletion strain. We were unable to detect an interaction between S. cerevisiae Atg31 and S. pombe Atg17 from these immunoprecipitation studies.

Furthermore, unlike S. cerevisiae Atg17, the presence of S. pombe Atg17 did not enhance the stability of

S. cerevisiae Atg31 in vivo, based on a visible increase of precipitated Atg31 in the presence of S. cerevisiae

Atg17 which is not observed when S. pombe Atg17 is used in place of S. cerevisiae Atg17 (Figure 2.5B).

Interestingly, using our bacterial based co-precipitation approach, we found that S. pombe Atg17 was capable of interacting with full-length S. cerevisiae Atg13 in vitro (Figure 2.4C). These results suggest that the lack of complementation could be due primarily to the inability of S. pombe Atg17 to effectively recruit and anchor the S. cerevisiae Atg31-Atg29 complex.

S. pombe does not encode Atg29 and Atg31 proteins nor are they required for starvation-induced autophagy. To determine if S. cerevisiae Atg17 can complement the function of S. pombe Atg17 in vivo, we performed the reverse complementation assay whereby we used the moderate strength P41nmt1 promoter to express, in the S. pombe atg17∆ mutant, mCherry-tagged S. cerevisiae Atg17, mCherry- tagged S. pombe Atg17, or mCherry alone. Using the CFP-Atg8 processing assay (described in Chapter 1),

55 whereby CFP is cleaved from Atg8 upon autophagic body degradation (Sun et al., 2013), we found that S. cerevisiae Atg17 was unable to rescue the autophagy defect of S. pombe atg17∆ cells (Figure 2.4D).

2.3.5 Atg101 binding to Atg13HORMA provides enhanced stability

The lack of interaction between Atg101 and Atg17 suggests that Atg101 plays a role distinct from Atg29 and Atg31. Indeed, we found that S. pombe Atg101 could not rescue autophagy defects in an S. cerevisiae atg29∆ atg31∆ strain. Moreover, co-expression of S. pombe Atg17 and S. pombe Atg101 was unable to rescue autophagy defects in an S. cerevisiae atg17∆ strain (Figure 2.4E and F). Recent biochemical studies and crystallographic analysis show that S. pombe Atg101 adopts a HORMA domain fold and forms an obligate heterodimeric complex with Atg13HORMA (H. Suzuki et al., 2015b). A conserved feature of HORMA domains is the ability to self-associate into dimers (Rosenberg and Corbett, 2015). We purified recombinant His-tagged S. pombe Atg101 and tagless S. pombe Atg13HORMA (residues 1 to 269) and analyzed them by chemical crosslinking in conjunction with SDS-PAGE. In the presence of the crosslinker disuccinimidyl suberate (DSS), neither Atg13HORMA nor Atg101 produced new higher molecular weight bands indicating that they do not self-associate (Figure 2.6A). Our findings validate results obtained from small-angle X-ray scattering (SAXS) and gel filtration chromatography analyses which show that Atg101 and Atg13HORMA cannot form homodimers (H. Suzuki et al., 2015b).

To gain further insights into the interaction between Atg101 and the HORMA domain of Atg13, we next analyzed the crosslinked His-Atg101-Atg13HORMA complex by crosslinking coupled to mass spectrometry (CXMS). This approach provides information on the proximity between different regions of the protein subunits within the complex (Yang et al., 2012). We detected a total of 6 intermolecular and

19 intramolecular crosslinks. Of these, 3 intermolecular and 7 intramolecular crosslinks occurred on lysine residues that can be mapped onto the published crystallographic structure of S. pombe Atg101-

Atg13HORMA (H. Suzuki et al., 2015b). We found that all except one of these subsets of mappable crosslinks occurred between lysine residues that are located within 30 Å, the maximum Calpha-Calpha distance

56 allowable by the DSS crosslinker (Table 2.2). Hence, results from the CXMS analysis suggest that the solution state of the Atg101-Atg13HORMA complex recapitulates the conformation of the previously reported crystal structure (Figure 2.6B).

Figure 2.6. The interaction between Atg101 and Atg13HORMA. (A) Atg13HORMA, His-Atg101, and the His- Atg101-Atg13HORMA complex were purified from E. coli cells and subjected to the DSS crosslinker (0-640 µM). The double asterisks indicate a higher molecular weight complex indicative of a heterodimer. (B) CXMS analysis of the His-Atg101-Atg13HORMA complex. Inter-(dashed lines) and intramolecular (solid lines) interactions observed between the two proteins (top panel). Intermolecular crosslinks observed mapped onto the S. pombe Atg101-Atg13HORMA crystal structure using PDB 4YK8 (bottom panels). (C) Differential scanning fluorimetry analyses of His-Atg101 (green), Atg13HORMA (purple) and the His-Atg101-Atg13HORMA complex (orange). The relative fluorescence signal is calculated and plotted as a function of the increase in temperature (left). The first derivative of the relative fluorescence is plotted as a function of the increase in temperature. The peak for each sample is used to estimate the melting temperature of the protein-protein complex (right).

Table 2.2. Intermolecular and intramolecular crosslinks observed from CXMS analysis

Intermolecular interactions Atg101 Atg13 Distance (Å) 14 199 24.3 14 196 20.3 91 172 23 Intramolecular interactions Atg13 Atg13 Distance (Å) 39 96 21.9 96 105 14.4 196 261 19.9 39 76 12.1 39 172 16.3 194 199 6.9 73 96 16.6 Atg101 Atg101 Distance (Å) 14 92 36.7

The best-characterized HORMA domain protein, Mad2, is an essential spindle assembly checkpoint mediator and can fold into two distinct conformations: a more stable O-Mad2 form and a less stable C-

Mad2 form. The crystal structures of the S. pombe Atg101-Atg13HORMA complex reveal that Atg101

58 resembles the O-Mad2 HORMA domain conformation, whereas Atg13HORMA resembles the C-Mad2

HORMA domain conformation (H. Suzuki et al., 2015b). This observation leads to the proposal that one of the functions of Atg101 is to stabilize its inherently less stable binding partner. To more systematically examine how heterodimerization affects the stability of these 2 binding partners, we used differential scanning fluorimetry (DSF) to analyze recombinant His-tagged S. pombe Atg101, Atg13HORMA, and the His-

Atg101-Atg13HORMA complex. By taking advantage of the property of the SYPRO® Orange dye to only fluoresce upon binding to hydrophobic surfaces, DSF can be used to monitor protein unfolding and estimate the melting temperature of a protein or protein complex (Vollrath et al., 2014). Our analyses show that the estimated melting temperatures for Atg13HORMA, His-Atg101, and the His-Atg101-Atg13HORMA complex are ~43°C, ~48°C, and ~63°C, respectively (Figure 2.6C). These results not only confirm that

Atg101 adopts a more stable HORMA conformation than Atg13HORMA but also reveals that heterodimerization enhances the overall stability of Atg101, as shown by an ~15°C increase in melting temperature.

2.4 Discussion

Our systematic subunit interaction analysis of the S. pombe Atg1 complex provides insight on the subunit organization of an Atg101-containing Atg1/ULK1 complex. Our data show that the core interactions between Atg17, Atg1, and Atg13 are conserved from S. cerevisiae to S. pombe. Furthermore, Atg101 does not appear to functionally replace the Atg31-Atg29 complex. Instead of stabilizing Atg17 to maintain an

S-shape conformation (Chew et al., 2013), Atg101 interacts with and stabilizes the N-terminal HORMA domain of Atg13, a finding that agrees with results from studies conducted by other research groups (H.

Suzuki et al., 2015b; Qi et al., 2015). Interestingly, we also found that Atg101 is capable of interacting with the CTD of Atg1, suggesting that this subunit might function beyond stabilizing Atg13HORMA. Future studies should focus on validating this interaction in vivo and elucidating the functional implication of this interaction.

Our finding that Atg101 cannot bind Atg17 led to the hypothesis that S. pombe Atg17 can readily adopt an architecture with distinct curvature. Our negative stain EM studies refuted this idea by showing that His-MBP-Atg17 adopts a rod-shape architecture. Although it remains to be seen if the mammalian

Atg17 ortholog, FIP200, shares similar structural properties with fission yeast Atg17, our finding suggests that the S-shape architecture observed for S. cerevisiae Atg17-Atg31-Atg29 may not be absolutely required for the biological function of Atg17 proteins, at least in non-Atg31-Atg29-containing Atg1 complexes. Our finding that S. cerevisiae Atg29 and Atg31 can bind, albeit inefficiently, and possibly induce a conformational change to S. pombe Atg17 in vitro, was unexpected due to the relatively low sequence identity between the two proteins. The observation that S. pombe Atg17 cannot rescue autophagy in atg17∆ strains suggests that this protein lacks the required elements to efficiently recruit S. cerevisiae

Atg29 and Atg31 essential for the function of the full budding yeast Atg1 complex. In agreement with this finding, our observation that the Atg1 binding domain of S. pombe Atg13 spans a region beyond the putative MIM domain suggests that there is some divergence in the mode of interaction among the core subunits in the S. pombe Atg1 complex. Further work on an Atg101-containing Atg1 complex is required to delineate the mechanisms involved in the mammalian autophagy pathway and to understand the functions of each protein involved.

CHAPTER 3: Exploring the Atg101 interactome

3.1 Introduction

The overarching process of autophagy is thought to be conserved from yeast to mammals; however, the composition of proteins within the pathway are not conserved (Table 1.1) The Atg1/ULK1 complex is a prime example of this phenomenon. The Atg1 complex in budding yeast is composed of 5 proteins (Atg1,

Atg13, Atg17, Atg29, and Atg31) while its mammalian counterpart, the ULK1 complex, is composed of 4 proteins (ULK1, ATG13, FIP200, and ATG101). Atg1, Atg13, and Atg17 in the budding yeast have homologues proteins to the mammalian ULK1 complex, ULK1, ATG13, and FIP200; while Atg29 and Atg31 do not have counterparts within the ULK1 complex. Instead, the ULK1 complex contains a different subunit, ATG101, that does not share sequence or structural similarity to Atg29 or Atg31. Furthermore, a recent bioinformatic analysis revealed that the presence of Atg29 and Atg31, or that of Atg101 is mutually exclusive (Noda and Mizushima, 2016). Atg29 and Atg31 were shown to play a role in stabilizing the S- shape of the Atg17 dimer in the budding yeast (Ragusa, Stanley and Hurley, 2012; Chew et al., 2013); however, the role of ATG101 has been less defined.

Both mammalian ATG101 and yeast Atg101 proteins were structurally characterized to adopt

HORMA folds (named after Hop1, Rev7, Mad2) and shown to form a heterodimer with the HORMA domain of ATG13/Atg13 (H. Suzuki et al., 2015b; Qi et al., 2015). These structures reveal that Atg101 and

Atg13 in both organisms form a heterodimer in a Mad2-like manner. Mad2, one of the first proteins determined to contain a HORMA fold forms a homodimer where one monomer adopts an open conformation (O-Mad2; considered the autoinhibited form), while the other adopts a topologically distinct closed conformation (C-Mad2; considered the active form). Although, Mad2 can flip between the open and closed states (Luo and Yu, 2008), Atg101 adopts a stable locked open conformation while

Atg13HORMA takes the less stable closed conformation and is stabilized through the interaction with Atg101

(Hegedus et al., 2014; H. Suzuki et al., 2015a, 2015b; Michel et al., 2015; Qi et al., 2015; Nanji et al., 2017).

The structure of Atg13HORMA alone has been solved in Lachancea thermotolerans where the Atg1 complex is similar in composition to the budding yeast S. cerevisiae complex, containing Atg29 and Atg31 but not

Atg101 (Ragusa, Stanley and Hurley, 2012; Kofinger et al., 2015). To compensate for not having stabilization through Atg101, L. thermotolerans Atg13 (which also adopts a C-Mad2-like conformation) contains an additional three-stranded beta-sheet that forms a “cap” to stabilize the protein (Ragusa,

Stanley and Hurley, 2012). These results provide evidence supporting that one of Atg101’s functions in autophagy is to stabilize Atg13.

The X-ray crystal structures of the Atg13HORMA-Atg101 complex from both S. pombe (PDB: 4YK8) and humans (PDB: 5C50, 5XUY) not only indicate that the two proteins adopt HORMA folds and form a heterodimer, but also provide evidence to suggest that Atg101 might play an additional function in autophagy recruitment of downstream proteins to the PAS. Notably, these structures revealed that

Atg101 contains a unique loop with conserved tryptophan (W) and phenylalanine (F) residues, termed the

WF finger (H. Suzuki et al., 2015b; Qi et al., 2015; Kim et al., 2018). Flow cytometry and localization assays in knockout mouse embryonic fibroblasts (KO MEFs) with mutant Atg101 show that the WF finger is important for adapting to starvation and hence for autophagy (H. Suzuki et al., 2015b).

Mammalian Atg101 also contains a WF finger that has a proline residue between the two residues of interest. The W110A, P111A, F112A mutation in MEFs has been shown to be dispensable for the interaction between Atg101 and the HORMA domain of Atg13 but is required for Atg101 to participate in the Atg1 complex, observed via co-precipitation assays. Furthermore, Atg101 mutant W110A, P111A,

F112A as well as mutations at the surface of Atg101 that mediate it’s interaction with the HORMA domain of Atg13 (L30A, H31A) in Atg101-KO MEFs were shown to negatively affect autophagy under starvation conditions using flow cytometry (H. Suzuki et al., 2015b). Although these mutants have been characterized in MEFs, their corresponding residues have not been explored in fission yeast.

Furthermore, Atg101 has been shown to form complexes with autophagy-related and unrelated proteins. Atg101 functions in autophagy-related complexes not only through stabilizing the HORMA domain of Atg13, but ATG101 has been recently shown to bridge the ULK1 and PI3K complexes in mammalian autophagy (Kim et al., 2018). In addition, mammalian ATG101 was shown to interact with the

CTD of the Patched1 (PTCH1) protein, a receptor of the Hedgehog pathway and a tumor suppressor that ultimately induces apoptosis (Chen, Morales-Alcala and Riobo-Del Galdo, 2018). ATG101 functions to bridge PTCH1 to the ULK1 complex blocking autophagic flux and causes autophagosomes with undegraded cargo to accumulate (Chen, Morales-Alcala and Riobo-Del Galdo, 2018). Moreover, ATG101 has been shown to form a complex with Smith-Magenis syndrome chromosomal region candidate gene 8

(SMCR8), chromosome 9 open reading frame 72 (C9ORF72), and WD repeat-containing protein 41

(WDR41). The C9ORF72 complex functions as a guanosine diphosphate–guanosine 5′-triphosphate (GDP-

GTP) exchange factor (GEF). Abnormalities in the C9ORF72 region of the chromosome has been linked to frontotemporal dementia (FTD) as well as amyotrophic lateral sclerosis (ALS), while the function of SMCR8 is unclear (Yang et al., 2016). SMCR8/C9ORF72 has been shown to affect the activity of ULK1; Smcr8 knockout and C9orf72 knockdown studies show that these two components modulate autophagy induction similarly by regulating ULK1 (Yang et al., 2016). The C9ORF72 protein was further shown to affect autophagy initiation as it forms a complex with Rab1 and ULK1 to modulate the Rab1a-dependent trafficking of ULK1. Although ATG101 containing complexes have mainly been characterized through autophagic flux to monitor their roles in autophagy, these complexes may function in other aspects of the cell. Whether or not the WF finger of ATG101 participates in these interactions have yet to be determined.

We hypothesize that Atg101 plays additional roles in fission yeast as well as in humans including forming key protein-protein interactions for both autophagy-related and non-autophagy-related processes. To test this hypothesis, we generated a GFP-tagged S. pombe strain for mass spectrometry- based proteomics to identify potential novel protein interacting partners of Atg101. S. pombe cells were

63 grown in media with and without nitrogen to determine the difference in the protein interaction profile of Atg101 under autophagy-inducing and non-inducing conditions. We found that Atg101 has a unique protein interaction profile under the two conditions tested, with some overlapping proteins identified.

We validated various protein candidates using pairwise interaction studies and were able to confirm more than 50% of the tested proteins to co-precipitate with His-Atg101 in vitro.

One protein that was confirmed to interact with Atg101 in vitro is Fkh1. The rapamycin-

Fkh1/FKBP12 complex is speculated to bind Tor1p in fission yeast. The function of Tor1p is modulated by amino acid uptake; furthermore, it has been determined that Tor1p function is sensitive to rapamycin-

Fkh1/FKBP12 inhibition (Weisman, Finkelstein and Choder, 2001; Weisman et al., 2005; Laplante and

Sabatin, 2013). Due to the relationship between the Tor proteins (described in Chapter 1) which contribute to autophagy regulation and cellular homeostasis (Eltschinger and Loewith, 2016; Pérez-Pérez,

Couso and Crespo, 2017) and the fission yeast Fkh1 protein, we decided to further explore the interaction between Atg101 and Fkh1 from the fission yeast S. pombe. We validated that Atg101 interacts with Fkh1 through the aforementioned WF finger, and we also explored the S. pombe Atg101-Atg13HORMA interaction interface using pairwise co-precipitation studies. Collectively, our results suggest that Atg101 plays an important role in maintaining cellular homeostasis under stress conditions. The protein interaction profile of Atg101 differs based on the conditions in which the cells are grown, suggesting that Atg101 functions in response to nutrient status. Furthermore, the interaction identified between Atg101 and Fkh1 may highlight roles of Atg101 outside of autophagy as Fkh1/FKBP12 has been shown to affect Tor function through the drug rapamycin (Pérez-Pérez, Couso and Crespo, 2017), which is produced by the soil dwelling actinomycete Streptomyces hygroscopicus (Kim et al., 2014) and not by budding or fission yeast. Our findings lead us to speculate that Atg101 has many potential protein interacting partners involved in more than one cellular pathway, suggesting that Atg101 may have cellular functions that extend beyond autophagy.

3.2 Methods

3.2.1 S. pombe atg101-GFP yeast strain construction and growth

Fission yeast strain ARC039 h− leu1-32 ura4-C190T (Mukaiyama et al., 2009) was engineered to incorporate a C-terminal GFP-tag using primers (IDT) TN013 (5’-GGG AAA TAC AAT TCC TTG GGA ACA ATG

GAT C-3’), TN014 (5’-GGG GAT CCG TCG ACC TGC AGC GTA CGA ATC ACC TCC ACT AAC GTT TTC-3’), TN015

(5’-GTT TAA ACG AGC TCG AAT TCA AAT CTC ACC AGG TTT GTA C-3’), and TN016 (5’-GAT ATA AAT GGC ACA

GCA TCC GTC CTA AAT-3’); primers were designed as previously described (Noguchi et al., 2008). Briefly,

PCR was carried out in 2 steps. First, one ~250 bp product was obtained using TN013 and TN014, and a second ~250 bp product was obtained using TN015 and TN016. These products were purified and used as templates for the second PCR step. A third template, a vector containing the sequence for the GFP-tag and kanMX resistance cassette (pFA6a-GFP(S65T)-kanMX6; Addgene, plasmid #39292), was used; TN014 and TN015 are engineered to contain overhangs which allow for the incorporation of the needed portion of the vector into the final PCR product. TN013 and TN016 were used to amplify this final product (~2500 bp). This product was separated on a 1% agarose gel, extracted and further gel purified. The purified DNA product was used for transformation into S. pombe ARC039 using the heat shock procedure (Suga and

Hatakeyama, 2005). Transformed cells were plated onto YES agar plates followed by replica plating onto

YES agar plates supplemented with 200 µg/mL of the G418 antibiotic (Gold Biotechnology, G-418-1).

Colonies that formed were confirmed to incorporate atg101-GFP by colony PCR using primers that flank the C-terminal integration site to yield a product only if the gfp-kanMX cassette is integrated. Atg101-GFP expression was further confirmed through western blot analysis. Cells confirmed to express Atg101-GFP were first grown in YES at 32°C and 220 rpm, cells were washed and transferred to 1.2 L of EMM or EMM-

N (Sunrise Science), both supplemented with 225 µg/mL leucine, and 225 µg/mL uracil (Bioshop) and grown for ~4 h at 32°C, 220 rpm. ~ 200 OD600 units of cells were harvested and separated into three tubes to be used in triplicate experiments for proteomic mass spectrometry analysis. Cells were verified to

65 express Atg101-GFP; samples were lysed for 5 min at 4°C in SDS-loading dye supplemented with 10 mM

PMSF with glass beads. The supernatant of the boiled, lysed samples were loaded onto a 12% SDS-PAGE gel. The gel was transferred to a nitrocellulose membrane and blotted to detect Atg101-GFP using the anti-GFP antibody (Roche, 11 814 460 001). Blots were subsequently probed using HRP-conjugated anti- mouse-IgG (Sigma, A4416). Chemiluminescence signal was detected using Amersham ECL Prime Western

Blot Detection Reagent (GE Healthcare) and visualized using a Konica Medical Film Processor (Model SRX-

101A) on Mandel 8x10 film (MED_CLE810).

3.2.2 Liquid chromatography-mass spectrometry (LC-MS) sample preparation

Harvested cells were centrifuged at 1,000 rpm for 5 min (Beckman Table-Top). The pelleted cells were resuspended in 2 mL RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Na-deoxycholate, 0.01%

SDS, 0.1% NP-40, and 1mM EDTA) with protease inhibitor cocktail (1 x Sigma P1724) and phosphatase inhibitor cocktail (Sigma P5726, P0044). After 30 min incubation on ice, the supernatant was collected by centrifugation at 11,000 rpm for 10 min at 4 oC. 100 µL of anti-GFP magnetic beads (Miltenyi Biotec No.

130-094-252) were added, samples were rotated for 3 h at 4oC. µMACS columns in the magnetic holder were equilibrated with 250 µL RIPA buffer with protease and phosphatase inhibitors as described above.

After loading 1 mL lysate to the column, the column was washed twice with 1 ml of 10% RIPA wash buffer

(50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% Na-deoxycholate, 0.01% SDS, 0.1% NP-40, and 1mM EDTA) and once with 1 ml no detergent RIPA buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, and 1mM EDTA).

For on-column Trypsin digestion, 25 µL of Trypsin digestion buffer (50mM Tris-HCl pH 7.5, 2 M urea,

1 mM DTT and 5 µg/mL Trypsin) was added to the column and incubated for 30 min at room temperature.

Tryptic samples were eluted with 100 µL of the elution buffer (50mM Tris-HCl pH 7.5, 2 M urea and 0.5mM

2-chloroacetamide). After incubation at 37oC with shaking for 16 hours, 1 µL of trifluoroacetic acid (TFA) was added to the samples to stop trypsin digestion. Digested samples were then desalted with Zip-Tip C-

18 cartridge (Millipore). ZipTip was wet with a wetting solution (65% acetonitrile, 1% acetic acid in water) and then equilibrated in equilibration solution (2% acetonitrile and 1% acetic acid in water). Peptide samples were loaded by pipetting up and down 10 times with a 10 µL pipettor in the solution. After washing with equilibration solution, peptide samples were eluted by 5 µL of elution solution (65% acetonitrile and 1% acetic acid in water). Eluted peptides were air-dried and dissolved in 20 µL of water with 0.1% formic acid.

All samples were analyzed by nanoLC coupled to the Orbitrap Elite mass spectrometer (Thermo

Fisher Scientific). Chromatographic separation of peptides was performed on a Pro Xeon EASY nLC 1000

System (Thermo Fisher Scientific) equipped with a Thermo Scientific™ Acclaim™ PepMap™ C18 column,

10 cm x 50 μm ID, 3 μm, 100 Å employing a water/acetonitrile/0.1% formic acid gradient. Samples were loaded onto the column for 100 min at a flow rate of 0.30 μl/min. Peptides were separated using 2% acetonitrile and increasing to 6% acetonitrile in the first 1 min, and then using a linear gradient from 6 to

24% of acetonitrile for 75 min, followed by a gradient from 24 to 100 % of acetonitrile for 14 min. Samples were then washed for 10 min at 100 % of acetonitrile. Eluted peptides were directly sprayed into the mass spectrometer using positive electrospray ionization (ESI) at an ion source temperature of 250°C and an ion spray voltage of 2.1 kV. Full-scan MS spectra (m/z 350–2000) were acquired in the Orbitrap elite at

60 000 (m/z 400) resolution. The automatic gain control settings were 1e6 for full FTMS scans and 5e4 for

MS/MS scans. Fragmentation was performed with collision-induced dissociation (CID) in the linear ion trap when ion intensity was >1500 counts. The 15 most intense ions were isolated for ion trap CID with charge states ≥2 and sequentially isolated for fragmentation using the normalized collision energy set at

35%, activation Q at 0.250, and an activation time of 10 ms. Ions selected for MS/MS were dynamically excluded for 30 s. The Orbitrap Elite mass spectrometer was operated with Thermo XCalibur software.

3.2.3 Proteomic analysis

Mass spectrometry cycle spectra were searched using SEQUEST (ver. 27 = rev.9) (Yates et al., 1995) against

S. pombe target and decoy protein sequences downloaded from UniProt (UP000002485) (Bateman et al.,

2017). Search parameters were set to allow for two missed cleavage sites, precursor mass tolerance ranged from -2 to 4 Da (daughter mass ion tolerance set to the default of 0), variable modification of methionine oxidation, protein N-terminal acetylation, and one fixed modification of cysteine carbamidomethylation. A stringent 1% false discovery rate threshold was used to filter candidate peptides for protein identification. The SEQUEST search results were evaluated by the STATQUEST probability algorithm (Kislinger et al., 2003) to assign a probability based confidence score to identified proteins.

3.2.4 In vitro co-precipitation experiments using E. coli cells

S. pombe genes were cloned from genomic DNA extracted from ARC039 (Mukaiyama et al., 2009) using primers outlined in Table 3.1 or genetically synthesized by GeneArt Gene Synthesis (Thermo Fisher

Scientific) to incorporate N-terminal BamHI and C-terminal NotI restriction sites for easy integration into the pQLinkG2 vector (Addgene, plasmid #13671). This vector contains a GST tag and is part of the pQLink expression vector family (Scheich et al., 2007), described in section 2.2.1, allowing for subsequent cloning into our plasmid pQLinkH-atg101.

Atg101 mutants were generated using site-directed mutagenesis. Top and bottom primers (see

Table 3.1) were used to mutate atg101 from our lab’s pQLinkH-atg101 WT construct, using PCR. WT vector used as the template was digested with Dpn1 (ThermoFisher Scientific) and subsequently transformed by heat shock into TOP10 chemically competent E. coli cells (ThermoFisher Scientific) and plated onto selective plates. Colonies that formed were verified to have the desired mutation by Sanger sequencing

(Genewiz). The pQLinkH-atg101 mutant vectors were subsequently cloned into pQLinkG2-fkh1 and

68 pQLinkG2-atg13HORMA to generate co-expression constructs using the pQLINK vector expression system

(Scheich et al., 2007) described in section 2.2.1.

Table 3.1. Primers used to clone S. pombe genes

Primer Sequence Function

TN033 5’-AAG GAT CCA TGG TTG CTG TCG GAT CTA CTT TGC C Synthesize pmp20 F*

TN034 5’-AAG CGG CCG CTT AAA GAG AGC TAA GGA CCT TGT CAG C Synthesize pmp20 R**

TN035 5’-AAG GAT CCA TGC CTG CCT CCA AAG AAC AAA CCG Synthesize cam2F 5’-AAG CGG CCG CCT ATT TTG CCA TGA TTC TCT GTA CAA TN036 AGT CAT AAT AGT C Synthesize cam2 R 5’-AAG GAT CCA TGA TTC GTC TTC AAA AGT TTG GTG AAA TN037 TTG TTG GG Synthesize pdb1 F 5’-AAG CGG CCG CTT ATT TAA TAT ACA AGC ATT TTT TAG TN038 CAG CAG CAA CAA C Synthesize pdb1 R

TN057 5’-AAG GAT CCA TGA ACT TCC TCC ATT TTT TAA CCA CTT CTC Synthesize gas5 F 5’-AAG CGG CCG CTT AAG CAA AAA CAA GTC CGC TAA TTG TN058 CCA GTA TAC Synthesize gas5 R 5’-AAG GAT CCA TGA GCC GTT TGG ATG GAA AAA CGA TTT TN059 TAA TC Synthesize spac521.03 F 5’-AAG CGG CCG CCT ACG CTT GCT TTC TGT ACA CAT GAT TN060 TGG Synthesize spac521.03 R 5’-AAG GAT CCA TGG CTG CAA TCA ACA TTG TCA AAA AGC TN061 GCA C Synthesize rpl3201 F 5’-AAG CGG CCG CTT ATT CTT GAG AAC GAA CTT TAG CGC TN062 CGG CAT TC Synthesize rpl3201 R

TN083 5’-AAG GAT CCA TGG CTT CAA GTA AGG AAA ATA ACT TG Synthesize spbc29a3.06 F

TN084 5’-AAG CGG CCG CTC AAT CAT AAT GAG CTA GTT TCC AAA G Synthesize spbc29a3.06 R

5’-GAA AGA TCG CCT AAA AAG TCT GCA GCT GGG AAG GGA TN132 AAT ACA ATT C Clone Atg101_WF94AA T#

5’-GAA TTG TAT TTC CCT TCC CAG CTG CAG ACT TTT TAG TN133 GCG ATC TTT C Clone Atg101_WF94AA B##

Primer Sequence Function

5’-GTT TTA GGT GTA ATT CTA GCC GCG AGA CAG TTT TCT TN134 ACT GTT CCT G Clone Atg101_FH29AA T

5’-CAG GAA CAG TAG AAA ACT GTC TCG CGG CTA GAA TTA TN135 CAC CTA AAA C Clone Atg101_FH29AA B

5’-GCA CAC TAC CAT TTT AGC AGC CGG TGA CTC TTA TCA TN138 AGA ATC TTC Clone Atg101_EE114AA T

5’-GAA GAT TCT TGA TAA GAG TCA CCG GCT GCT AAA ATG TN139 GTA GTG TGC Clone Atg101_EE114AA B *F- Forward primer, **R- Reverse primer, #T-Top primer, ##B- Bottom primer

Co-expression constructs were transformed into T7 Express E. coli cells (NEB, C2566I). Cells were grown in 2xYT media at 37°C to an OD600 of ~0.6. Expression was induced with 0.5 mM IPTG (Gold

L00206) for at least 30 min. Glutathione beads were pelleted and washed with wash solution (50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Triton X100 (Sigma, T8787), 5% glycerol, 0.5 mM DTT) 3 times. SDS loading dye was added to the beads, followed by boiling for 10 min. Samples were loaded onto two 12% SDS-

PAGE gels. One gel was stained with Coomassie Brilliant Blue, and the second gel was first transferred to a nitrocellulose membrane, and the blots were subsequently probed with an anti-His antibody (mouse monoclonal; ABM, G020) followed by detection using HRP-conjugated anti-mouse-IgG (Sigma, A4416).

Chemiluminescence signal was detected using Amersham ECL Prime Western Blot Detection Reagent (GE

Healthcare) and visualized using the Bio-Rad ChemiDoc™ MP Imaging System with a 2x2 binning, exposures were taken at optimal, as well as 30-900 sec exposure times for short and long exposure (S.E.,

L.E.) images.

3.3 Results

3.3.1 Incorporation of a C-terminal GFP tag on S. pombe Atg101

Our group decided to use an affinity chromatography mass-spectrometry (AP-MS)-based proteomics method to identify novel Atg101 interacting proteins. To study protein-protein interactions via methods such as X-ray crystallography, nuclear magnetic resonance (NMR), or EM which provide information on interaction interfaces, or isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and fluorescence resonance energy transfer (FRET) which delineate binding affinities and kinetics, a prior knowledge of the protein complex is necessary. LC-MS/MS allows us to generate a novel pool of potential proteins that interact with our protein of interest, Atg101. This technique has been demonstrated to have broad proteome coverage, good accuracy, and precision in quantification (Xie et al., 2011) which allows us to more accurately and quantitatively determine and compare Atg101 interacting proteins from S. pombe cells grown in vivo under various growth conditions (Meyer and Selbach, 2015). Hence, LC-MS/MS allows us to test our hypothesis and assess the role of Atg101 in both autophagy-related and non- autophagy-related cellular functions. This was achieved by growing S. pombe cells expressing a fusion protein of Atg101 with the green fluorescent protein (GFP) under autophagy-inducing and non-inducing conditions using nitrogen starvation. To integrate the gfp-kanMX tag and resistance cassette into the S. pombe genome downstream of the coding sequence for atg101, a 2-step PCR approach followed by homologous recombination using yeast transformations was used (Section 3.2.1). Colonies were confirmed to contain the gfp-kanMX cassette using PCR (Figure 3.1A) and further confirmed to express

Atg101-GFP in S. pombe cells grown in YES, EMM and EMM-N media through western blot analysis (Figure

3.1B-D).

Figure 3.1. Integration of the gfp-kanMX tag and cassette into S. pombe strain ARC039. (A) Colony PCR to amplify genomic DNA at the C-terminus of Atg101 and the start of the integration site, was used to confirm integration of the gfp-kanMX cassette (+ lane). A 1kb DNA ladder was used as a marker (M), the – lane represents a negative control; water was introduced instead of the PCR amplified DNA fragment during the yeast transformation. The colony used to generate the PCR product (+ lane), as well as a negative control (– lane), were used to assess for Atg101-GFP expression. S. pombe cells were grown in (B) YES, (C) EMM and (D) EMM-N. Cell lysates were cleared and subsequently probed for Atg101-GFP using western blot analysis using the anti-GFP antibody. S. pombe ARC039 cells were grown in place of S. pombe ARC039 cells expressing Atg101-GFP for the negative control (– lane) in our western blot analysis.

3.3.2 LC-MS/MS analysis reveals that Atg101 potentially interacts with a variety of proteins

Our overall experimental approach involved isolating a tagged version of Atg101 and its interacting partners by affinity purification using the GFP-tagged Atg101 S. pombe strain and anti-GFP magnetic beads. Isolated content is digested with trypsin and the digested peptides are separated using high- performance liquid chromatography (HPLC) prior to MS/MS analysis. Identified peptides were searched against a UniProt S. pombe target and decoy protein library (see Methods 3.2.3.). Untagged S. pombe cells were used as a negative control to generate a list of potential false positive hits.

As a first trial, we performed LC-MS/MS analysis on GFP-tagged Atg101 S. pombe cells grown in nutrient-rich undefined YES media, which contains yeast extract and glucose (Forsburg and Rhind, 2006).

From the peptides isolated, 635 genes were identified as prey genes (the total number of prey genes in

Table A1). Amongst these, we were able to recover our bait Atg101-GFP as peptides corresponding to atg101 (annotated as mug66) (Table A1) were isolated. In addition, we identified peptides corresponding to Atg13 and Atg1 (Table A1), subunits of the Atg1 complex which were previously shown to interact directly with Atg101 (Figure 2.1E). These findings provided confidence in our overall AP-MS/MS workflow.

Table A1 in the appendix provides a list of potential Atg101-interacting partners that were found in YES media (named based on gene ontology), the number of peptides found that correspond to that gene of interest, as well as the confidence score associated with the number of peptides found.

After our pilot experiments with S. pombe cells expressing Atg101-GFP grown in YES validated our technique, we next analyzed these cells under our desired experimental conditions, autophagy-inducing and non-inducing conditions. This was achieved by growing cells in defined media with and without nitrogen using EMM and EMM-N media (Forsburg and Rhind, 2006). Although peptides corresponding to the original bait, atg101 (mug66), were identified from samples prepared from cells grown in EMM and

EMM-N, no peptides were found that correspond to the Atg1 complex components including Atg13 or

Atg1 (Table A2). This shows that regardless of media type, we are able to recover our bait; however, the proteome of Atg101 may differ under the various conditions tested. Furthermore, we found differences in peptides isolated from cells grown under non-autophagy-inducing conditions (YES and EMM) in different media (Figure 3.2A). This phenomenon is further discussed in section 3.4. A comparison of the prey genes identified in YES and those identified in EMM was performed. Table A4 in the appendix lists the genes that were exclusively found in either YES or EMM when relating the two media types. A greater number of genes are found in YES compared to EMM when considering all hits as well as those exclusive to each media type (Figure 3.2A).

Figure 3.2. LC-MS/MS analysis of S. pombe Atg101. (A) The total number of prey genes found in EMM (green), EMM-N (pink) and YES (blue) as well as the number of overlapping hits (in brackets). (B) Proteins identified to interact with Atg101 under defined conditions were grouped based on their functional descriptions, results are displayed in a pie chart created using the Pivotchart function in Microsoft Excel. Descriptions that were enriched are identified on the pie chart. (C) Functional profiling using g:Profiler (Reimand et al., 2016) of identified proteins that were enriched in S. pombe cells grown in EMM, EMM-N, and proteins found in both media types. The data was displayed using the Cytoscape Enrichment Map Application (Shannon et al., 2003; Merico et al., 2010). The lines represent shared genes between connected GO Term descriptors (the circles or nodes). The width of the line represents the number of

74 shared genes; green represents genes identified from cells grown in EMM-N while blue represents genes identified from cells grown in EMM.

A combined number of 625 prey genes were identified from S. pombe cells expressing Atg101-GFP grown in EMM and EMM-N (total number of prey genes in Table A2 with their associated descriptions in

Table A3) which are classified as defined media. Peptides associated with proteins of various functions were identified illustrating that Atg101 may participate to some degree in multiple cellular pathways in vivo. However, it is unclear if the proteins pulled out from our affinity purification directly interact with

Atg101. In addition to co-eluting with Atg101 due to a direct interaction with Atg101, eluted peptides may co-purify with Atg101-GFP due to several reasons, for example, not having adequate washes, or if a protein is bridged to Atg101 through one or multiple proteins. To prevent us from identifying proteins that may interact with the resin used for GFP affinity purification, WT S. pombe cells were used as a control to eliminate contaminating hits, as mentioned above. Our final list of proteins identified from defined media is shown in the appendix, Table A2, while the total number of hits found in EMM, EMM-N, and YES as well as the number of overlapping hits are displayed in Figure 3.2A. Hit proteins identified in defined media (EMM and EMM-N media) were grouped based on function (Figure 3.2B). Functional groups that contained more hits compared to other groups include the eukaryotic translation initiation factor, the 60S ribosomal protein, the 40S ribosomal protein, and the 26S protease regulatory subunit. The interaction between Atg101 and a regulatory subunit of the proteasome further illustrates that there is crosstalk between the UPS (discussed in section 1.2.1). and the autophagy pathway, this paradigm was recently reviewed (Nam et al., 2017).

Proteins identified from defined media were further sorted to assess for the enrichment of various cellular functional groups, determined by the gene ontology (GO) term associated with the gene.

Functional profiling using g:Profiler (Reimand et al., 2016) was conducted and displayed using the

Cytoscape Enrichment Map Application (Figure 3.2C) (Shannon et al., 2003; Merico et al., 2010). Proteins

75 associated with various GO terms were identified and are illustrated as nodes (circles). Three interaction networks were identified for hits enriched in EMM-N (Figure 3.2C, nodes with red on the left half with green connectors, the width of the connector indicates the number of genes shared between the nodes), one interaction network was identified for hits enriched in both media types (Figure 3.2C, filled in circles; the green and blue lines represent shared genes that were identified from cells grown in EMM-N and

EMM respectively) while one interaction network was identified for hits enriched in EMM-N and both media types (Figure 3.2C; the green line illustrates that only hits from EMM-N overlap).

To determine if the hits indeed represent direct binding partners of Atg101, we selected a total of

16 candidate prey genes: 5 found exclusively from the EMM dataset, 5 found exclusively from the EMM-

N dataset, and 5 found in both datasets (Table 3.2). An additional gene that was identified from preliminary studies using S. pombe cells expressing Atg101-GFP grown in YES media, cam 2, was also tested. We selected candidate prey genes based on the number of technical replicates in which they were found and the associated confidence score, biasing our candidate genes to those found in multiple replicates tested and those having higher confidence scores. We also included technical considerations

(eg. size of proteins, ease of cloning/presence of introns) in selecting this first set of candidates for follow- up studies.

Table 3.2. S. pombe proteins from proteomic analysis for follow up studies

Media Length Weight Prey gene Description Length (bp) used (aa) (kDa) cam2 Myosin I light chain Cam2 YES 442 143 16.22 crn1 Coronin-like protein crn1 EMM 1803 601 67.02 Adenylate kinase isoenzyme 6 homolog fap7 (AK6) (EC 2.7.4.3) (Dual activity adenylate EMM 525 175 20.22 kinase/ATPase) (AK/ATPase) Peptidyl-prolyl cis-trans isomerase (PPIase) fkh1 EMM-N 336 112 12.04 (EC 5.2.1.8) (FK506-binding protein) (FKBP)

Media Length Weight Prey gene Description Length (bp) used (aa) (kDa) Probable UTP--glucose-1-phosphate fyu1 uridylyltransferase (EC 2.7.7.9) (UDP-glucose Both* 1518 506 56.43 pyrophosphorylase) (UDPGP) (UGPase) gas5 1,3-beta-glucanosyltransferase gas5 Both 1530 510 53.69 Pyruvate dehydrogenase E1 component pdb1 subunit beta, mitochondrial (PDHE1-B) (EC Both 1101 366 39.62 1.2.4.1) Putative peroxiredoxin pmp20 (EC 1.11.1.15) pmp20 (Peroxisomal membrane protein pmp20) Both 471 156 16.67 (Thioredoxin reductase) ATP-dependent 6-phosphofructokinase rpl3201 (ATP-PFK) (Phosphofructokinase) (EC EMM-N 381 127 14.44 2.7.1.11) (Phosphohexokinase) Probable 26S proteasome regulatory subunit rpn6 EMM 1263 421 47.34 rpn6

rps1001 40S ribosomal protein S10-A EMM-N 432 144 16.28 NADP-dependent 3-hydroxy acid spac521.03 dehydrogenase (L-allo-threonine EMM-N 777 259 28.15 dehydrogenase) (EC 1.1.1.381) spbp8b7.26 Uncharacterized protein P8B7.26 EMM 786 262 27.49 tef5 Elongation factor 1-beta (EF-1-beta) Both 642 214 23.48 urg1 Uracil-regulated protein 1 (EC 3.5.4.-) EMM-N 1317 439 49.03 Probable U3 small nucleolar RNA-associated utp18 protein 18 (U3 snoRNA-associated protein EMM 1665 555 62.47 18) *Both- EMM and EMM-N

3.3.3 In vitro co-precipitation experiments validate that Atg101 interacts with prey genes identified from LC-MS/MS studies

We first carried out pairwise interaction studies with the 16 chosen candidate proteins using E. coli-based in vitro co-precipitation experiments, similar to those conducted in Chapter 2. We overexpressed histidine-tagged S. pombe Atg101 with each of our proteins of interest fused to a GST tag using T7 Express

E. coli cells. The GST-tagged bait protein was pulled down using glutathione beads and the co-precipitated material was assessed by western blot analysis. We struggled to produce one of the cloning products required for the co-expression of a protein identified in EMM, Rpn6. For this reason, we expressed GST-

Rpn6 in one vector and His-Atg101 in another and then pooled the cell lysates for our pull down experiments. However, the poor expression of Rpn6 precluded us from making firm conclusions regarding the interaction between Atg101 and Rpn6 (pull down results between Atg101 and Rpn6 are shown in the appendix, Figure A1).

Results for the remainder of our co-precipitation studies are shown in Figure 3.3, with protein candidates grouped based on the growth conditions from which their related peptides were isolated.

Panel A contains proteins isolated from cells grown in both conditions (EMM and EMM-N). Panel B contains pull down results for proteins identified from cells grown in the absence of nitrogen to induce starvation-induced autophagy (EMM-N). Panel C contains results for proteins isolated from cells grown in autophagy non-inducing conditions from both defined minimal media containing nitrogen (EMM), as well as one hit isolated from YES media, Cam2.

Of the five selected candidates isolated in both EMM and EMM-N (Pmp20, Pdb1, Tef5, Fyu1, and

Gas5) we were able to confirm that three interact directly with His-Atg101 in vitro (Figure 3.3A). Pmp20, which is conserved in archaea, bacteria, eukaryotes, fungi, and vertebrates (Gould et al., 1990; Kim et al.,

2010), is a predicted thioredoxin-related chaperone protein that localizes to the cytosol and nucleus

(Matsuyama et al., 2006) to mediate protein refolding (Kim et al., 2010). On the other hand, Pdb1 is a cellular component of the mitochondrial pyruvate dehydrogenase complex (Matsuyama et al., 2006;

Beltrao et al., 2009) that affects the biological acetyl-CoA biosynthetic process (Cavan and MacDonald,

1994, 1995). It is important to note that Pdb1 visibly pulled down Atg101 despite not expressing at a high level in our E. coli co-expression strain, suggesting that it may have a high binding affinity to Atg101 (Figure

3.3A). Furthermore, this gene is conserved in eukaryotes and fungi (Vilella et al., 2009). Lastly, Tef5, which is conserved in archaea, eukaryotes, fungi, and metazoa is the translation elongation factor EF-1 beta subunit that plays a role in extending translation products (Matsuyama et al., 2006). GST-Tef5 is highly expressed in our co-expression strain yet is only able to isolate a small amount of His-Atg101 suggesting

78 that it has a weak affinity for His-Atg101 (Figure 3.3A). Of the proteins tested that were from both EMM and EMM-N growth conditions, Pmp20, Pdb1, and Tef5 have confirmed interaction in vitro based on our pairwise co-precipitation studies.

Amongst the five selected candidates from the EMM-N dataset (Rps1001, Urg1, Fkh1, Spac521.03, and Rpl3201), four were confirmed to interact directly with His-Atg101 in vitro (Figure 3.3B). Rps1001 is predicted to be the 40S ribosomal protein S10 (Matsuyama et al., 2006). GST-Rps1001 expresses well in our co-expression strain and pulls down a generous amount of His-Atg101 compared to the other proteins tested (Figure 3.3B). Urg1 is a predicted GTP cyclohydrolase II with no apparent S. cerevisiae ortholog

(Watt et al., 2008; Lock et al., 2018). Urg1 has been found in the cytosol as well as the nucleus (Matsuyama et al., 2006); based on the electronic annotation of Urg1, it has the capacity to bind metal, is predicted to play a role in riboflavin biosynthesis, and has GTP cyclohydrolase II activity (Watt et al., 2008). GST-Urg1 expresses well in our E. coli co-expression construct but only minimally pulls down His-Atg101 (Figure

3.3B). Fkh1 is an FKBP-type peptidyl-prolyl cis-trans isomerase (Tonthat et al., 2016) that has been shown to interact with Tor1 (Weisman et al., 2005). Furthermore, Fkh1 has been shown to be sensitive to rapamycin, based on cell growth assays (Ikai et al., 2011) and can be functionally complemented by S. cerevisiae FPR1 (Weisman, Finkelstein and Choder, 2001). GST-Fkh1 expressed well in our pairwise co- precipitation studies and we were able to detect His-Atg101 in our western blot confirming that Fkh1 interacts with Atg101 in vitro; however, this interaction may be weak due to the amount of His-Atg101 recovered in the pull down compared to the amount expressed. Lastly, GST-SPAC521.03 a predicted short chain dehydrogenase from the human DHRS7 family (28.15 kDa) SPAC521.03 has oxidoreductase activity and is found in the cytosol (Matsuyama et al., 2006).

Figure 3.3. Co-precipitation assays with His-Atg101 to confirm protein-protein interactions from proteomic mass spectrometry analysis, in vitro. Panels (A to C) show results of co-precipitation experiments performed by co-expressing His-Atg101 with the GST-tagged protein of interest or the GST tag (~26 kDa) alone. 2-50% of the pull down was loaded to allow for optimum visualization of the co- expressed constructs and co-precipitated samples. The Coomassie Brilliant Blue-stained gel (top panel) shows the GST construct inputs (cell lysate) as well as the samples precipitated by glutathione beads (affinity isolate [pull down]). The GST-tagged baits are indicated by an asterisk. Panels show hits isolated

80 from S. pombe cells grown (A) in both EMM and EMM-N, (B) those found exclusively in EMM-N, and (C) exclusively in EMM or YES media. (D) Motif-based sequence analysis results for hits that were confirmed to interact with Atg101 in vitro using the MEME suite (Bailey et al., 2009). The logo of the 10 unique sequences identified are displayed on the left with the number of hits that the site can be found in on the right.

Hits selected from non-starvation media contained Cam2 which was isolated from S. pombe cells expressing Atg101-GFP grown in YES media, as well as Fap7, SPBP8B7.26, Utp18, and Crn1 which were isolated from cells grown in minimal defined media containing nitrogen (EMM). Fap7 and Crn1 were confirmed to interact with His-Atg101 in our co-immunoprecipitation experiments (Figure 3.3C).

Conserved in eukaryotes, fungi, metazoan and vertebrates, Fap7 is a predicted nucleoside-triphosphatase involved in SSU-rRNA maturation (Hellmich et al., 2013). Based on data inferred from the electronic annotation and sequence model, Fap7 is suggested to have adenylate kinase activity and ATPase activity

(Loc’h et al., 2014). Fap7 is found in the cytosol and nucleus and has been shown to play a role in nucleoside monophosphate phosphorylation (Gaudet et al., 2011). Crn1, or coronin, is an actin-binding protein that is thought to be involved in actin filament bundle assembly as well as actin filament polymerization (Dodgson et al., 2010). Crn1 is conserved in eukaryotes, fungi, metazoan, and vertebrates, and has been shown to be a cellular component of the actin cortical patch and actomyosin contractile ring

(Pelham and Chang, 2001), as well as part of the growing pseudohyphal cell tip (Dodgson et al., 2010).

Collectively, nine proteins were confirmed to interact with His-Atg101 using in vitro co-precipitation studies (Figure 3.3).

We next attempted to identify similarities between the proteins that were confirmed to interact with Atg101 in vitro, to see if Atg101 potentially binds proteins with a common motif/fold. To assess if there is a common peptide sequence that Atg101 binds, sequences of identified proteins confirmed in our pairwise interaction studies were analyzed using the MEME suite, motif-based sequence analysis tool

(Bailey et al., 2009) (Figure 3.3D). This tool finds common peptide sequences among the protein sequences of interest. Ten logos, or peptide sequence, were identified from the nine confirmed protein

81 sequences searched; however, each logo was only found within two or three of the nine proteins analyzed

(Figure 3.3D, sites). Hence, no significant motifs were identified. Furthermore, Prosite, a database that can be used to find common protein families or domains (Sigrist et al., 2013) was able to find ten structured domains in five of the sequences. However, the identified domains were distinct in each protein and no overlap was seen. Hence, no significant commonalities were found amongst the nine hits confirmed to interact with His-Atg101 in vitro.

3.3.4 Mapping the Fkh1-interacting interface of S. pombe Atg101

We next wanted to further characterize the interaction between Atg101 and one of the confirmed hits.

Fkh1, one of the validated hits from the EMM-N dataset, is associated with the Tor pathway which directly regulates autophagy. Hence, Fkh1 was the protein candidate we chose to pursue. Based on the published

X-ray crystal structure of the S. pombe Atg101-Atg13HORMA complex (H. Suzuki et al., 2015b) we designed three sets of mutations targeting different solvent-accessible surfaces of Atg101. We next examined the impact of these mutations on the Atg101-Fkh1 interaction using in vitro co-precipitation assays (Figure

3.4). The first set of mutations target F29 and H30, two residues of Atg101 (referred to as Atg101FH29AA) that are directly involved in mediating the interaction with Atg13 (H. Suzuki et al., 2015b) (Figure 3.4A).

This mutant was also used as a control to asses the interaction between S. pombe Atg101 and Atg13HORMA.

The second set of mutations (Atg101WF94AA) target the “WF finger”, which is thought to mediate the recruitment of downstream autophagy factors (Figure 3.4B). The third set of mutations target glutamic acid residues at positions 114 and 115 (Atg101EE114AA) which form an exposed negatively charged surface and may provide a suitable interaction site for Atg101 (Figure 3.4C).

Figure 3.4. Co-precipitation assays of E. coli cells expressing S. pombe Atg101 mutants (A) WF94AA, (B) FH29AA, and (C) EE114AA with (D) S. pombe Atg13HORMA, and (E) S. pombe Fkh1. Gels were loaded to optimize visualization of the co-expressed constructs and pull down samples. Coomassie Brilliant Blue- stained gel (top panel) shows the GST construct inputs (cell lysate) as well as the samples precipitated by glutathione beads (pull down). The GST-tagged baits are indicated by the arrowhead on the left of the gel. Western blots detecting His-Atg101 obtained from short and long exposures (S.E., L.E.) are displayed (bottom panels) for both cell lysates and pull down samples.

Atg101 mutants did not all behave the same; expression of Atg101WF94AA is reduced compared to

Atg101WT and the other Atg101 mutants. Furthermore, the electrophoretic mobility of Atg101EE114AA seems

83 to be shifted. This may be due to a change in conformation of Atg101 which in turn changes the ability of

SDS to denature the protein; thus, altering its migration within the acrylamide gel matrix. Nonetheless, we observed a reduction in the interaction between S. pombe Atg101 and Atg13HORMA when F29 and H30 of Atg101 were mutated to alanine as expected while mutations made to the WF finger (Atg101WF94AA) or the glutamic acid rich surface (Atg101EE114AA) did not affect the Atg101-Atg13HORMA interaction (Figure

3.4D). In contrast, both His-Atg101FH29AA and His-Atg101WF94AA mutants had reduced binding affinity for

GST-Fkh1 compared to His-Atg101WT, with the most significant disruption in binding observed for the His-

Atg101WF94AA mutant (Figure 3.4E). This suggests that Fkh1 interacts with Atg101 through the WF finger, residues F29 and H30 may also contribute to the interaction as His-Atg101 is not recovered as efficiently when either WF94AA or FH29AA mutations are made to Atg101 (Figure 3.4E).

3.3.5 Atg101 is unable to simultaneously interact with Fkh1 and Atg13HORMA in vitro

We next tested if Atg101 could simultaneously bind Fkh1 and Atg13HORMA. We first mixed lysates from cells expressing His-Fkh1 and cells co-expressing His-Atg101 and GST-Atg13HORMA and then carried out GST co- precipitation assays. We used double the amount of cell lysate for His-Fkh1 due to the lower affinity between Fkh1 and Atg101 compared to Atg13HORMA and Atg101. This finding was determined by comparing amounts of pull down sample needed to detect His-Atg101 in studies conducted in Chapters 2 and 3. Although a significantly larger amount of His-Fkh1 is present compared to His-Atg101 (Figure 3.5), only Atg101 is sufficiently pulled down by GST-Atg13HORMA. This shows that Atg101 does not form a ternary complex with Atg13HORMA and Fkh1, or Fkh1 does not compete with Atg13HORMA for binding to Atg101.

Figure 3.5. Co-precipitation assay of S. pombe proteins, His-Atg101 co-expressed in E. coli with GST- Atg13HORMA supplemented with His-Fkh1. Coomassie Brilliant Blue-stained gel (top panel) shows the GST construct inputs (cell lysate) as well as the samples precipitated by glutathione beads (pull down). The GST-tagged baits are indicated by the arrowhead on the left of the gel. A western blot detecting His- Atg101 and His-Fkh1 are displayed (bottom panel) for both cell lysates and pull down samples.

3.4 Discussion

The original goal of our LC-MS/MS study was to identify novel binding partners of S. pombe Atg101 to assess autophagy-related and non-autophagy-related functions of Atg101. Recent studies suggested that

Atg101 possesses a unique WF-finger motif that might function to recruit downstream autophagy factors

(H. Suzuki et al., 2015b; Qi et al., 2015; Kim et al., 2018); however, this conserved surface may also function in non-autophagy-related complexes. Despite obtaining encouraging results from our pilot analysis on cells grown in YES media, we were unable to detect any autophagy-related factors from the 625 prey genes detected from cells grown in defined media (EMM and EMM-N). The most puzzling finding was that we did not observe peptides corresponding to Atg13 from cells grown in autophagy-inducing conditions.

This is perplexing as Atg101 has been shown to form a stable complex with Atg13HORMA; furthermore, deficient formation of the ATG13-ATG101 complex was shown to result in autophagy defects (Wallot-

Hieke et al., 2018). In addition, S. pombe cells were grown in non-autophagy-inducing media, EMM and

YES. These media showed variable interactomes for Atg101, illustrating that media type may affect the expression level of various proteins and/or downstream post-translational modifications (PTMs) required for a successful interaction to be detected. These aspects will be discussed in further detail later in this chapter.

The proteomic results for cells expressing Atg101-GFP grown in YES show that Atg101 not only pulled down Atg13, and Atg1, but also Atg4. Atg4 is a core autophagy-related protein involved in processing Atg8 for conjugation to PE (Figure 1.3D) and is required for phagophore expansion (Suzuki et al., 2017). YES media which is composed of yeast extract and glucose (Forsburg and Rhind, 2006) produces a different interactome for Atg101 than EMM, another non-autophagy-inducing condition. Why the interaction network for Atg101 differs between two non-autophagy-inducing conditions is perplexing. We hypothesize that although the interaction between S. pombe Atg101 and Atg13HORMA in vitro is strong, factors in the media may weaken the interaction between Atg101 and Atg13HORMA or change the expression of S. pombe genes in vivo altering the proteome of cells in various growth conditions; thus, producing variable Atg101 interactomes for the different media types tested. As the interactome for

Atg101 changes based on growth condition, and due to our inability to detect peptides corresponding to other autophagy-related proteins from cells grown in defined media, we speculate that the function of

Atg101 may extend beyond regulating autophagic flux.

Although common atg genes were not found in both EMM and YES media, 416 proteins were found to overlap between Atg101-GFP interactomes from cells grown in EMM and YES media types. It is interesting to note that more genes overlap between EMM and EMM-N conditions, which are autophagy non-inducing and inducing conditions respectively, compared to that between cells grown in EMM and

YES media types which are both non-autophagy-inducing conditions (Figure 3.2A); albeit, the difference in the number of genes found may not be significant. Nevertheless, this further highlights that factors in the media other than the presence of nitrogen may affect protein expression level as well as downstream protein modifications. Moreover, the S. pombe cells grown in EMM-N that were used for LC-MS/MS analysis were never explicitly confirmed to be undergoing autophagy. Although we starved our S. pombe cells for 4 h which was previously shown to be sufficient to induce autophagy, monitored by CFP-Atg8 degradation (Nanji et al., 2017), the cells used for our autophagy-inducing conditions were never specifically tested. Cells were washed in EMM-N media to remove any residual nitrogen from starter cultures grown in YES media prior to starvation; however, there may be residual nitrogen in the media that altered autophagic flux. To explicitly confirm that the cells used for LC-MS/MS both express Atg101-

GFP and undergo autophagy, an S. pombe strain expressing both CFP-Atg8 and Atg101-GFP should be engineered and assessed. Western blots that detect Atg101-GFP as well as cleavage of CFP from CFP-Atg8 would indicate that these cells not only express the tagged protein of interest but ensures that the cells used for LC-MS/MS analysis are undergoing autophagy. Alternatively, the localization of Atg101-GFP could be assessed in these cells to ensure that localization of Atg101-GFP is consistent with that of autophagy- inducing conditions described during the analysis of fission yeast genes (Sun et al., 2013).

Although Atg101-GFP did not pull out autophagy-related proteins from cells grown in EMM or

EMM-N, we did observe an enrichment of hit proteins associated with phosphatidylinositol-4,5- bisphosphate binding (Figure 3.2C). This protein description is related to membrane dynamics and plays an important role in lysosome reformation (Rong et al., 2012; Liu and Klionsky, 2018) illustrating that

Atg101 may play additional roles in autophagy-related functions through mediating membrane dynamics.

We decided to extend our search for Atg101 interacting proteins to include non-autophagy-related proteins. We first wanted to determine if proteins isolated from our LC-MS/MS studies interact with

Atg101 by recapitulating the interaction in an isolated environment, such as that in a pairwise pull down

87 experiment. Although this method does not allow us to assess interactions in a particular growth condition, it provides us with a convenient way to assess if two proteins can interact. We compiled a list of genes of interest (GOI) for follow up studies (Table 3.2). We confirmed nine of the 16 GOI (56%); three of the confirmed hits were identified from cells grown in both EMM and EMM-N, four of the confirmed hits were isolated from cells grown in autophagy-inducing conditions, and two hits were confirmed from cells grown in autophagy non-inducing conditions (Figure 3.3A-C). Hence, we were able to confirm that more than 50% of the genes identified illustrated positive interactions with Atg101 in vitro. However, we were unable to detect significant sequence or structural similarity amongst these nine genes, using bioinformatics (Figure 3.3D), suggesting that Atg101 does not bind to a group of proteins with a common motif/domain.

Of the seven proteins that we were unable to detect an interaction with His-Atg101 using in vitro pull down studies, three (Fyu1, Gas5, and Rpl3201) did not express well in our GST-tagged constructs.

Although these proteins may not directly interact with Atg101, their stability and expression levels hinder our ability to make conclusions about their interaction with His-Atg101. Further effort could be made to optimize expression conditions for these proteins. Aspects that could be altered and assessed are E. coli strains used, OD of cells at the point of IPTG induction, the concentration of IPTG used for induction, as well as growth conditions. We used T7 Express Competent E. coli cells (NEB); however, additional cell lines could be tested under various growth conditions. In some cases when cloning our genes of interest, gene sequences were optimized for expression in E. coli; however, in other cases, rare codons may be needed and call for bacterial expression strains that can supply the tRNAs for rare codons. In these cases, E. coli

Rosetta™(DE3) competent cells (Novagen) can be used. Furthermore, BL21 Star(DE3) competent cells

(Invitrogen) can also be assessed as they illustrate reduced mRNA degradation which may lead to increased protein expression. ArcticExpress competent cell lines (Agilent Technologies) can be used to overcome hurdles of protein insolubility. Induction conditions can also be altered to increase or decrease

IPTG concentration as well as the OD600 of cells prior to induction. Cells can be cold shocked and grown at various temperatures, shaking at various speeds to test which conditions lead to optimal protein expression. Hence further steps must be taken to confirm if proteins identified in our LC-MS/MS studies are unable to directly interact with His-Atg101 using in vitro pull down experiments.

We initially set out to determine the difference in the interactome of Atg101 under autophagy- inducing and non-inducting conditions, with the hypothesis that a downstream core autophagy-related protein may be identified, or we may discover novel functions of Atg101. Although additional Atg proteins were not isolated from cells grown in defined media, a wide variety of proteins were identified to interact with Atg101, illustrating that Atg101 may play other important cellular functions. It is important to note that S. pombe cells expressing Atg101-GFP grown in YES media did isolate peptides corresponding to Atg1,

Atg13, and Atg4. The interaction between Atg13 and Atg101 has been characterized, but further study of

Atg101’s interaction with Atg1 and Atg4 may be of interest.

We could also further explore interactions made by S. pombe Atg101. Atg101 may form biologically important complexes with other proteins that we did not pursue in our list of 16 GOI. Further work into each of the genes identified from our proteomic analysis can be conducted. This may help us further understand the function of Atg101. Furthermore, interactions should be confirmed in vivo under the nutrient conditions in which the GOIs were identified. This ensures that the proteins identified to interact with Atg101 exclusively in a specific growth condition are accurately identified. Although interactions between Atg101 and prey genes may have been identified exclusively in one growth condition, this may not mean that Atg101 cannot interact with that candidate protein in other conditions. Perhaps the level of expression is altered causing the interaction to be missed in a condition where it is expressed at a lower level. We could extend this study by assessing the level of expression of candidate genes, using quantitative PCR (qPCR) to measure mRNA levels. However, it biologically makes sense for the cell to adapt protein synthesis to generate genes that are necessary, and in the quantity required, for a given

89 stress condition. This further suggests that Atg101 may play a role in cellular processes that extend beyond autophagy as it is shown to interact with a variety of proteins under the various conditions tested. In addition to considering changes in protein expression amongst various growth conditions, PTMs on

Atg101 and its interacting partners likely change based on growth condition. PTMs made to Atg101 and candidate interacting proteins in the various conditions can be assessed using electrochemistry or mass spectrometry-based methods (Silva et al., 2013; V. Shumyantseva et al., 2014). For our purposes, we decided to follow up with the proteins confirmed to interact with Atg101 in vitro.

We next considered the functions of the nine GOI that were confirmed to interact with Atg101 in vitro. While most of the genes belonged to various cellular functions, one, in particular, Fkh1, had a connection to autophagy through its link with Tor, the S. cerevisiae Fkh1 homolog, FKBP12, was shown to bind rapamycin, restricting access to the kinase domain of Tor; thus, inhibiting Tor function (Loewith and

Hall, 2011). We found that Atg101 interacts with Fkh1 through our pairwise interaction studies and we further mapped the interaction site of Fkh1 to the WF finger of Atg101 (Figure 3.4E). Suzuki et al. identified the WF finger in the X-ray crystal structure of S. pombe Atg101 and further characterized the mutant in

KO MEFs (H. Suzuki et al., 2015b). They found that their WF finger mutant (W110A, P111A, F112A) did not affect the interaction with Atg13; however, it did show impairments in autophagy. They suggest that both the Atg13-binding surface and the WF finger of Atg101 are required for autophagy and to recruit WIPI1, the mammalian Atg18 functional homologue, to the PAS (determined using GFP-WIPI1 fluorescence microscopy). Furthermore, flow cytometry, assessing autophagic flux using GFP-LC3, illustrated that

Atg101 is essential in KO MEFs (H. Suzuki et al., 2015b). Collectively, their results suggest that Atg101 has one or more important role(s) in addition to the role of stabilizing Atg13 (H. Suzuki et al., 2015b). Our proteomic results also suggest that Atg101 may play an important biological role that extends from its effect on Atg1 complex formation.

We identified 625 prey genes, collectively, from the peptides isolated in our experiments using cells grown in defined conditions. For follow up studies, we attempted to select hits with high peptide counts and confidence scores in multiple replicates; however, we also wanted to select hits that were shared among both growth conditions as well as ones that were exclusive to either growth condition. We also had to consider technical aspects required for cloning the gene for downstream verification. For this reason, in some cases we selected hits with fewer peptide counts and confidence scores than we would have initially wanted; for example, in the case of Fkh1, only one replicate was able to identify two peptides with a confidence score of 99.58%. However, since LC-MS/MS is a sensitive approach and we were able to confirm some of the interactions in vivo, even for hits with limited peptide counts in few replicates. We feel confident that the peptides isolated are still good potential Atg101-interacting proteins and are valid targets for follow up studies.

Furthermore, the interaction between Atg13, Atg101 and Fkh1 was also assessed. Atg101 was previously shown to interact with Atg13HORMA through residues F29 and H30 while the WF finger is shown to protrude from the Atg101 structure away from the Atg13 binding site (H. Suzuki et al., 2015b; Qi et al.,

2015). We found that Atg101 cannot simultaneously bind Atg13HORMA and Fkh1. This could be due to steric hindrance; although both binding sites are available, proteins are in constant motion, surface residues on

Atg13 or Fkh1 may occupy the same space causing for a weaker interaction. In addition, our co- precipitation experiments were performed in vitro using N-terminally tagged proteins. The tags may also affect binding as they require additional space and thus could occlude potential binding interfaces. We could pursue this question further by switching the location of the tags. Alternatively, we could repeat LC-

MS/MS analysis using Atg13 and Fkh1 as bait proteins by engineering C-terminal GFP tag constructs for these proteins in separate S. pombe strains. Isolation of Atg101 and Atg13/Fkh1 from AP-MS using these cells may provide further insight to determine if a three-component complex is formed and if this complex is physiologically relevant.

Although we discovered a novel direct interaction between Atg101 and Fkh1 and have discussed a link to autophagy through the relationship between Fkh1 and the drug rapamycin, which in turn affects

Tor activity and thus affects autophagic flux, it is important to remember that rapamycin is not naturally produced by fission yeast. As rapamycin would unlikely be physiologically relevant, it is more likely that the interaction between Atg101 and Fkh1 functions outside of the autophagy pathway. In addition, unlike

S. cerevisiae, autophagy-related genes in humans and higher eukaryotes have been shown to function outside of the autophagy pathway. ULK1 has been shown to affect innate immunity, vesicle trafficking as well as endocytosis (Konno, Konno and Barber, 2013; Saleiro et al., 2015; Joo et al., 2016; Hwang et al.,

2018). We feel that Fkh1 is not a novel autophagy protein as it was not identified in the comprehensive fission yeast genetic screen conducted to identify autophagy-related genes (Sun et al., 2013; Li-Lin Du, personal communication). Furthermore, S. pombe cells that contain mutant atg genes share a meiosis/sporulation defective phenotype which is not shared with fkh1 mutants; fkh1 is required for mating but not for meiosis/sporulation (Weisman, Finkelstein and Choder, 2001). This suggests that Fkh1 is not likely a new autophagy factor and that Atg101 has additional uncharacterized roles in the cell that affect biological functions beyond autophagy. Perhaps fission yeast cells are more related to humans and higher eukaryotes in that regard. This is reinforced by the recent data showing that Atg101 forms complexes with proteins outside of the core autophagy machinery such as with PTCH1 (Chen, Morales-

Alcala and Riobo-Del Galdo, 2018), and with the SMCR8, C9orf72, WDR41 complex (Yang et al., 2016). This is further emphasized as the components of the Atg1 complex in fission yeast resemble the human ULK1 complex more than the homologous budding yeast complex. We found that Atg101 is able to co-purify with a variety of proteins and may play roles in various cellular functions. Hence there is an increasing need for continued studies on Atg101 and Atg101 containing Atg1/ULK1 complexes to further understand the detailed functions of autophagy-related proteins and the potential roles they have that extend beyond autophagy.

Chapter 4: Discussion and Future Directions

4.1 Conserved and unique features of the yeast Atg1 complex

4.1.1 Interactions and architecture of the Atg1 complex

One key objective of this thesis was to assess how the subunit organization and architecture of the fission yeast Atg1 complex differs from that of the budding yeast Atg1 complex. We found that the core interactions formed by Atg17, Atg13, and Atg1 are conserved between budding and fission yeast. Atg17 seems to serve a scaffolding function for both complexes as Atg17 forms an elongated dimer in both budding and fission yeasts. Using structural EM studies of the Atg17 dimer in budding and fission yeasts, it can be argued that the budding yeast Atg17 dimer is more flexible than its counterpart in fission yeast; in the absence of the S. cerevisiae specific Atg31-Atg29 complex, the S. cerevisiae Atg17 dimer displays a wide range of flexibility and is stabilized upon complex formation with the Atg31-Atg29 subassembly. On the other hand, the S. pombe Atg17 dimer exhibits limited flexibility with lateral changes in the length of the dimer. The variable flexibility of Atg17 may be specific to each organism allowing Atg17 to function in the desired conditions to make autophagosomes with the specific molecular machinery available. The flexibility of Atg17 can be further explored using single-molecule force microscopy with optical tweezers

(Neuman, Keir and Nagy, 2008). Although the shape and flexibility of the Atg17 dimer differs between the yeasts tested, the end to end distance of the majority of the images collected from our EM studies, of the

S. cerevisiae Atg17-Atg31-Atg29 hexamer and S. pombe Atg17 dimer were of similar size, 290-310 Å (Chew et al., 2013) and 320-340 Å (Figure 2.3C), respectively. As the length of the Atg17 dimers have more conservation than the shape, perhaps the length is more important than the shape of the dimer for autophagosome formation based on evolution.

We found that the S. pombe Atg17 dimer formed an elongated complex rather than an S-shape complex; however, SpAtg17, which shares ~22% identity to ScAtg17, was still able to interact with S. cerevisiae Atg29 and Atg31; however, SpAtg17 was unable to complement the function of ScAtg17 in an

S. cerevisiae atg17 KO strain. This suggests that the region on Atg17 that interact with Atg29 and Atg31 may be conserved, while other regions of Atg17 have been adapted for the S. pombe system. It is unclear why S. pombe would retain the Atg29 and Atg31 binding regions. Although SpAtg17 may interact with

Atg29 and Atg31 (Figure 2.4A) as well as with ScAtg13 (Figure 2.4C) in vitro, they may not in vivo or may not localize or recruit downstream proteins efficiently. The budding and fission yeast species diverged approximately 350 million years ago (Hoffman, Wood and Fantes, 2015); hence they have developed species-specific genomes through evolution. This may lead to an absence of additional components required to rescue autophagy; for example, the Atg1 complex in fission yeast contains Atg101. Atg101 was also added in our complementation studies using budding yeast to see if it was needed to rescue autophagy in atg17 KO cells; however, even in the presence of Atg101, SpAtg17 was unable to rescue autophagy in S. cerevisiae cells (Figure 2.4F).

When considering our work on Atg17 and its translation to a mammalian system, it is important to consider the differences in these proteins and the systems from where they have been characterized.

Fission yeast contain two scaffold proteins, Atg11 (106.77 kDa) and Atg17 (48.6 kDa) which seem to be required to perform the same function as mammalian FIP200 (183.1 kDa), a single scaffolding protein that resembles both Atg11 and Atg17. Human FIP200 shares 24.07% and 22.61% sequence identity with S. pombe Atg11 and Atg17, respectively. It has been suggested that both selective and non-selective autophagy (outlined in Chapter 1) in mammals uses FIP200 as a scaffolding protein and forms the ULK1 complex (Lin and Hurley, 2016), while budding yeast use Atg11 for selective autophagic events and Atg17 for non-selective events (Kamber, Shoemaker and Denic, 2015). In any case, both mammalian and fission yeast cells contain Atg101 in place of Atg29 and Atg31 allowing us to potentially gain new information on the human ULK1 complex through studies conducted on the fission yeast Atg1 complex.

Although the core interactions between the budding and fission yeast Atg1 complex components are conserved and Atg17 appears to serve as a scaffold, the Atg101 proteins seems to play a distinct role

94 than that of budding yeast Atg29 and Atg31. Atg101 does not form a complex with Atg17 but instead interacts with Atg13HORMA and in turn, has been suggested to recruit downstream autophagy factors. It has been reported that Atg13HORMA recruits Atg9 (S. W. Suzuki et al., 2015) as well as directly interacts with phospholipids and members of the Atg8 family (Wallot-Hieke et al., 2018) promoting PAS formation.

Furthermore, a disruption in the interaction between ATG101 and Atg13HORMA in mouse embryonic fibroblasts (MEFs) illustrated a stronger autophagy-inhibitory phenotype compared to a disruption in the binding sites between Atg13 and ULK1 or FIP200 proteins (Wallot-Hieke et al., 2018). Due to these findings, and the overwhelming data showing that autophagy impacts human health (Meijer and

Codogno, 2006; Shintani and Klionsky, 2009; Choi, Ryter and Levine, 2013; Shanware, Bray and Abraham,

2013), the interaction between Atg101 and Atg13HORMA has been suggested to be a promising drug target

(Wallot-Hieke et al., 2018). Small molecule screens can be used to find inhibitors of this interaction which may aid in downstream therapeutic applications.

Our studies focused on the assembly of the Atg1 complex in fission yeast as this process controls autophagy initiation in the budding yeast. However, the assembly of the Atg1 complex does not signal autophagy initiation in all organisms; for example, the mammalian ULK1 complex is constitutively formed and inhibited through mTORC1 (Park et al., 2016). It is unclear if the fission yeast Atg1 complex is constitutively assembled or if its assembly triggers autophagy initiation. Our AP-MS results suggest that the latter is the case as we did not detect many autophagy-related complexes from the genes that were isolated with Atg101-GFP. If the Atg1 complex in fission yeast was constitutively formed we should detect peptides corresponding to the complete Atg1 complex (Atg1, Atg13, Atg17 and Atg101) while we only detected Atg101 in all conditions. To further assess the conditions required for Atg1 complex assembly in fission yeast, AP-MS could be performed by tagging various Atg1 complex components and determining which Atg1 complex components are co-purified with each tagged protein under various conditions. This will provide us with more information on overall complex assembly as well as help us determine

95 physiologically relevant subassemblies made within the fission yeast Atg1 complex in a given growth condition. AP-MS can also be used to find new interacting partners for the other Atg1 complex components within fission yeast, similar to that conducted for Atg101 in Chapter 3. As AP-MS analysis on each Atg1 complex component would be taxing, the localization of the individual Atg1 components could also be visualized by fluorescence microscopy. In addition, FRET can be used to assess if two autophagy- related proteins change in proximity in response to a change in growth conditions. As the fission yeast vacuole is not as readily visible as the budding yeast vacuole, it may be more difficult to identify single phagophore initiation sites. Hence, identified interacting partners should be tested using in vitro and in vivo pairwise pull down studies to confirm the presence of an interaction as well as ensure the interaction is a direct interaction between the two proteins in question and not mediated by additional factors.

4.1.2 Autophagy initiation in Atg1 complexes with and without the Atg101 subunit

Although my thesis explored the Atg1 complex and its components which regulate autophagy initiation, we never assessed autophagy initiation itself. Furthermore, the function of each component and its role in autophagy initiation remains elusive. Autophagy initiation in the budding yeast has been assessed and the function of each module is slowly being characterized. Hurley and his group determined the crystal structure of a 2:2:2 complex of S. cerevisiae Atg17, Atg29, and Atg31, to a resolution of 3.05 Å. They found that Atg17 is crescent-shaped with a 10 nm radius of curvature. It was found that the dimerization of the

Atg17-Atg31-Atg29 complex is critical for both PAS formation and autophagy. Each dimer contains two separate and complete crescents which are suggested to tether Atg9 containing vesicles causing autophagy-related proteins to accumulate at the PAS, leading to autophagy initiation (Ragusa, Stanley and

Hurley, 2012). Furthermore, multimerization of the Atg1 complex through the binding of Atg1317LR and

Atg1317BR, within the intrinsically disordered domain of Atg13 in budding yeast, with Atg17 has been postulated to aid in phagophore nucleation (Yamamoto et al., 2016).

S. pombe Atg17 readily forms a dimer similar to budding yeast Atg17; however, this protein does not form the signature curved S-shape conformation adopted by the S. cerevisiae Atg17 dimer in complex with Atg31-Atg29 proposed to aid in vesicle tethering (Ragusa, Stanley and Hurley, 2012). Moreover, the

S. cerevisiae phagophore was shown to nucleate from a cluster of 20-30 nm diameter Atg9-containing vesicles (Ragusa, Stanley and Hurley, 2012). Although the budding yeast model suggested above relies on the curvature of Atg17 for Atg9 tethering, more recent evidence suggests that the Atg1 complex is able to recruit Atg9 containing vesicles through alternate mechanisms. For example, it has been shown that the HORMA domain of Atg13 can directly interact with Atg9 recruiting Atg9-containing proteoliposomes to the PAS (S. W. Suzuki et al., 2015). The HORMA domain of Atg13 seems to be conserved from fission yeast to humans (H. Suzuki et al., 2015b; Qi et al., 2015). Ohsumi and his team found that the HORMA domain of Atg13 was required for autophagy and the recruitment of the phosphatidylinositol PI3K subunit, Atg14 (S. W. Suzuki et al., 2015). Furthermore, Atg13HORMA contains a pair of conserved arginine residues. As these residues are found in the region of Mad2 where conformational switching occurs between the opened and closed Mad2 configurations, and these two residues are essential for autophagy, it has been suggested that Atg13HORMA could function as an activation switch. These arginine residues are situated away from the Atg101 binding site and thus, would not interfere with Atg1 complex assembly, but have been shown to affect downstream recruitment of autophagy-related genes (Jao et al., 2013).

Furthermore, mammalian Atg13HORMA has been shown to interact with the middle region of Atg14

(residues 201-395) (Park et al., 2016); thus, assuming that the HORMA domain of Atg13 in fission yeast can also interact with fission yeast Atg14 to recruit PI3K, it can be postulated that Atg1, Atg13, Atg17 and

Atg101 function together to form the initiation complex.

Furthermore, the length of Atg17 determines the spacing between the HORMA domains on the symmetrical complex, allowing the Atg1 complex to be correctly positioned when interacting with adjacent Atg9 molecules to form the phagophore assembly site. This would lead to the recruitment of the

PI3K complex through the interaction between the PI3K complex subunit, Atg14, and Atg13HORMA. This suggests that the Atg1 complex in any organism should be symmetric allowing for autophagy initiation to occur on both sides of the PAS, leading to a phagophore that can expand at both ends. In addition, the C- terminal early autophagy targeting/tethering (EAT) domain of S. cerevisiae Atg1 was shown to sense membrane curvature, dimerize, and tether lipid vesicles (Ragusa, Stanley and Hurley, 2012; Lin et al.,

2018); hence, Atg1 may also play a role in tethering Atg9 vesicles for phagophore formation. This aspect of Atg1 vesicle binding has not been explored to date in fission yeast.

The shift in evolution to remove genetic information encoding Atg29 and Atg31 proteins and the addition of Atg101 appears to coincide with a shift in vacuole/lysosome structure. While budding yeast, containing Atg29 and Atg31, have a single large vacuole located next to the PAS, mammals, containing

Atg101, have several smaller lysosomes. Fission yeast have been shown to also contain small fragmented vacuoles. Perhaps the ability to form larger vacuoles requires Atg29 and Atg31. Furthermore, sustaining the degradation requirements of the cell in a single structure rather than multiple smaller ones may be more difficult. The use of multiple smaller vacuoles and their connection to microtubules allows degradation to occur evenly in all areas of the cell while a central vacuole requires content required for degradation to travel longer distances; hence, organisms may have evolved to contain multiple vacuoles so that they can manage the degradation load of the cell during stress conditions.

Using current knowledge of the autophagy field and our findings, we can summarize that the architecture of the S. pombe Atg1 complex and the interactions it makes would allow for autophagy initiation to occur as follows: an elongated S. pombe Atg17 dimer would position Atg13HORMA at opposite ends of the Atg1 complex, enabling this domain to interact with PI3K complex components as well as Atg9.

Atg13 interacts with Atg17 at multiple interfaces (Figure 1.4C) allowing nucleation of the Atg1 complex to occur. This would cause a clustering of Atg9 containing vesicles at the PAS leading to autophagy initiation.

In this way Atg13HORMA could aid in recruiting the Atg1 complex to the PAS and subsequent recruitment of

98 the PI3K complex which has been proposed (Jao et al., 2013; S. W. Suzuki et al., 2015). Therefore, we propose that autophagy initiation acts via the “Ohsumi model” over the “Hurley model” whereby the complete Atg1 complex positions the HORMA domain of Atg13 to interact with downstream components required for phagophore biogenesis rather than the curvature of Atg17 being the factor that mediates vesicle recruitment (Figure 4.1)

Figure 4.1. Model of autophagy initiation. The symmetric dimer of Atg17 positions the HORMA domain at the ends of the Atg1 complex. The dotted line indicates symmetry. Atg29 and Atg31 are illustrated as translucent shapes as they are only present in the budding yeast system. The HORMA domain of Atg13 has been shown to interact with various autophagy related components including Atg9 vesicles, Atg101, and Atg14. Atg13 may switch binding partners based on the stage of phagophore biogenesis and autophagosome growth.

To validate this model of autophagy protein recruitment to the PAS and subsequent autophagy initiation, we should assess if fission yeast Atg13HORMA can indeed interact with Atg9 containing proteoliposomes. This can be done using a floatation assay whereby Atg9 is purified and incorporated into

20-30 nm lipid vesicles similar to those found in the cell (Ragusa, Stanley and Hurley, 2012). Lipid vesicles

99 incorporating Atg9 with the protein of interest can be separated using a sucrose gradient, the mixture is subject to centrifugation allowing for the vesicles to rise to the top of the gradient while unbound proteins and aggregates are forced to the bottom. Proteins that co-migrate with the Atg9-containing proteoliposomes can be inferred to interact with Atg9 containing vesicles. In addition to assessing the interaction between Atg13HORMA and Atg9, we can also assess if Atg9 interacts with other Atg1 complex components to evaluate if they can aid in Atg9 vesicle tethering. Furthermore, S. cerevisiae Atg1 has been shown to contain an EAT domain that functions to sense membrane curvature (Nguyen, Shteyn and Melia,

2017) and subsequently allows for liposome tethering (Goran Stjepanovic et al., 2014). Budding and fission yeast share ~35% identity between their Atg1 protein sequence, but it has yet to be shown if Atg1 in S. pombe interacts with vesicles. Floatation assays can be conducted with and without Atg9 to assess if Atg1 complex components are able to bind vesicles and if they have specificity for Atg9 containing proteoliposomes. Another interaction that must be confirmed to validate our model, is the interaction between fission yeast Atg13HORMA and Atg14. This can be assessed using pairwise interactions in vitro, as conducted in Chapters 2 and 3.

4.2 AP-MS to identify novel Atg101 interacting partners

To further understand the mechanism of an Atg101-containing ULK1/Atg1 complex, we set out to perform

LC-MS/MS on S. pombe cells expressing Atg101-GFP. We first ensured that our method was capable of efficiently isolating Atg101-GFP using LC-MS/MS on cells grown in YES media. We were able to detect peptides corresponding to Atg101, Atg1, and Atg13 illustrating that we were able to collect intact components of the fission yeast Atg1 complex. We next determined the difference in the Atg101 interactome under autophagy-inducing and non-autophagy-inducing conditions using nitrogen starvation with defined media EMM and EMM-N. Using defined media, we were unable to detect other autophagy- related proteins among the peptides that co-purified with Atg101-GFP, suggesting that the interaction between Atg101 and Atg1/Atg13 is not constitutive. It is interesting to note that although autophagy-

100 related genes in S. pombe have been shown to be upregulated during autophagy-inducing conditions

(personal communication, Li-Lin), cells grown in non-autophagy-inducing conditions, YES and EMM, recovered higher levels of peptides corresponding to Atg101 than cells grown in autophagy-inducing conditions (EMM-N). Since cells were first grown in YES complex media and then shifted to defined media with and without nitrogen, perhaps cells grow more optimally in richer media thus allowing Atg101 to be recovered in a higher quantity compared to nitrogen-starved media.

Our findings show that the complex media type, YES media, resulted in the greatest number of

Atg101 interactions. This may suggest that Atg101 has a greater potential to interact with proteins under these conditions or that proteins are better expressed. We can normalize our data to protein expression levels by measuring mRNA levels of our candidate genes in the specific growth conditions through qPCR.

Many proteins, 284, were identified to interact with Atg101 independent of the condition medium in which the cells were grown. Surprisingly, no Atg proteins other than our bait, Atg101, were identified in the peptides recovered from our affinity purification from cells grown in defined media. This could be due to a loss of interaction between Atg101 and Atg1/Atg13 during our experiments, or it could suggest that

Atg101 is not constitutively bound to the Atg1 complex and that it may contribute to other cellular functions. Some autophagy-related complexes were found in our enrichment analysis suggesting that

Atg101 does indeed affect autophagy. Together, our results suggest that Atg101 plays a role in autophagy as a component of the Atg1 complex, but also has other functions.

Although we were able to confirm 9 of the 16 interactions tested in vitro, these experiments were conducted in E. coli cells using a co-expression vector; hence, nutrient status does not play a role.

Furthermore, in some instances, the peptides detected from our fission yeast cells may not form a direct interaction with Atg101 and may be bridged by another yeast protein. These interactions may still be interesting as the bridging protein would also have an additional function forming a 3-component complex. These bridging interactions, although interesting, would be missed in our pairwise studies. For

101 a more thorough analysis, each candidate prey protein should also be tagged in vivo, for example with a

C-terminal FLAG tag in our S. pombe atg101-gfp strain. Cells should be grown in EMM and in EMM-N, the

FLAG tagged protein can be isolated and an interaction with Atg101-GFP can be detected via western blot.

In some cases we found prey genes to isolate with Atg101-GFP in autophagy-inducing conditions or non- inducting conditions exclusively. These findings should also be confirmed in vivo using the pull down technique described above using cells produced from the growth conditions in question.

Once interactions have been proven to occur in vitro and in vivo, we can further characterize the interaction to gain more detail on the strength and stability of the complex, this information could help us to understand if Atg101 has various distinct functions and if they compete with one another through protein binding. Techniques such as isothermal titration calorimetry (ITC) or microscale thermophoresis

(MST) can be used to determine the strength of an interaction. Structural techniques such as SAXS, EM or atomic force microscopy (AFM) can provide alternate information of the overall complex organization, oligomerization state and structure. We can also assess if the interaction stabilizes the partner protein using DSF (described in Chapter 2). These techniques can also be used to explore the dynamics and strength of the interaction of Atg101 with Atg1, Atg4, or Atg13.

The model of autophagy initiation for fission yeast described in the previous section shows that

Atg101 may disassociate from Atg13HORMA by competition with another protein so that the HORMA domain of Atg13 is free to interact with downstream components of the PI3K complex and Atg9 vesicles.

Atg1 may be one of these proteins although it was not pulled out from our LC-MS/MS studies using cells grown in defined media. We should test other proteins, such as Atg101, Atg9, and Atg14, to assess if these proteins can compete for Atg13HORMA binding. This can be done with in vitro affinity assays using purified tagged proteins or co-expression vectors with tags similar to the studies used in Chapter 2 and 3.

Furthermore, Atg13 was identified to have conserved arginine residues that may be used as a switch (Jao et al., 2013); this could potentially alter the structure of Atg13 changing its interactome; this switch may

102 act in response to growth conditions such as autophagy induction/progression. Hence, these conserved arginine residues may alter Atg13 allowing it to have dual binding properties. Further studies, involving mutagenesis of these arginine residues in vivo, are needed to further understand the function(s) of Atg13 and to confirm that Atg101 is required for other functions in the cell.

4.2.1 Implication of the interaction between Atg101 and Fkh1 in fission yeast

Using our AP-MS analysis we found that Atg101 forms a potential interaction with Fkh1. Since fkh1 has not been previously identified as an autophagy- related gene (Kohda et al., 2007; Sun et al., 2013) it is not likely part of the S. pombe core autophagy machinery. Furthermore, autophagy-related genes, when deleted are unable to sporulate. Although fkh1 is required for mating, it is not required for sporulation

(Weisman, Finkelstein and Choder, 2001). The Fkh1 homologue in S. cerevisiae, FKBP12 (FK506-binding protein of 12 kDa), forms a complex with rapamycin, restricting access to TOR’s kinase domain; thus, inhibiting TOR function (Loewith and Hall, 2011). However, rapamycin is not a physiologically relevant molecule as it is a natural product produced by bacterium Streptomyces hygroscopicus (Kim et al., 2014).

Furthermore, only S. pombe leucine auxotrophs have been demonstrated to show rapamycin sensitivity

(Weisman and Choder, 2001; Weisman et al., 2005). Hence, Fkh1 could be related to Atg101 through an autophagy-independent mechanism. Furthermore, the function of FKBP12 in the budding yeast occurs in the absence of Atg101; for these reasons, it may be worth exploring the interaction between fission yeast

Tor proteins, Fkh1, and Atg101.

We assessed the interaction interface between Atg101 and Fkh1 using mutational analysis. We could expand our studies by mutating the atg101 gene to include the WF94AA mutation and repeat the proteomic experiment. This would allow us to determine the difference in the interactome between

Atg101WT and Atg101WF94AA to further understand the importance of this unique WF finger. Furthermore, we could conduct mass spectrometry-based proteomics using Fkh1 as the bait protein to assess if Atg101 is within the discovered interactome. This experiment should be conducted under autophagy-inducing

103 and non-inducing conditions to determine if, in fact, the interaction between Atg101 and Fkh1 occurs under the stimulus of autophagy.

Our initial studies aimed to reconstitute the fission yeast Atg1 complex for structural analysis; however, generating full-length Atg13 and Atg1 proved difficult; therefore, we focused our studies on pairwise interactions between more “structured” domains of the Atg1 complex components. Now that we have learned more about which regions of the proteins are required for interactions, perhaps we can generate chimeric proteins to bias for complex formation and allow structural analysis of the full Atg1 complex in fission yeast. Structural analysis of this complex can be conducted via cryo-EM to obtain a structure with high-resolution view of the core complex devoid of the intrinsically disordered regions (Yu et al., 2018). We may also attempt to use cryo-EM to directly visualize how the core fission yeast Atg1 complex interact with Atg9 proteoliposomes. This information will help us understand how phagophore formation and expansion occurs. Structural studies can also be extended to include the Atg101-Fkh1 interaction to further understand details behind this novel complex. Furthermore, all interaction studies should be conducted in higher eukaryotes to determine if the discoveries made in yeast can be translated to mammalian systems. As autophagy has been linked to many devastating illnesses, further knowledge of these interactions can aid in developing effective therapeutics leading to advancement in the quality of life. Although work in this thesis contributed to improving the understanding of an Atg101-containing

ULK1/Atg1 complex, further experiments are necessary to delineate the mechanisms of action of autophagy initiation in the fission yeast Atg1 complex.

With the new findings from our LC-MS/MS studies, we update the model of autophagy initiation to include downstream functions of Atg101. As described above, the architecture of the S. pombe Atg1 complex and the interactions it makes, would regulate autophagy initiation. Atg13HORMA has many interaction partners such as Atg101, Atg9 containing proteoliposomes and Atg14. The Atg1 complex including the Atg13HORMA complex is recruited to the PAS with Atg9 and the PI3K complex (through the

104 association between Atg13HORMA and Atg14). Upon phagophore initiation, Atg101 may dissociate from

Atg13HORMA, possibly through a competing interaction with Atg1, Fkh1 or other Atg101 interacting proteins; these interactions may be specific to a particular growth condition. Exploring the validity of this model requires further study.

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Appendices

Table A1. Prey genes identified from proteomic mass spectrometry analysis of S. pombe cells expressing Atg101-GFP grown in YES media

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides aah1 Adenine deaminase (ADE) (EC 3.5.4.2) (Adenine 1 75.1 ------aminohydrolase) (AAH) aap1 Aspartyl aminopeptidase 1 (EC 3.4.11.21) ------1 99.58 ------aat2 Aspartate aminotransferase, cytoplasmic (EC 2.6.1.1) 3 99.58 2 99.58 2 99.58 (Transaminase A) ach1 Acetyl-CoA hydrolase (EC 3.1.2.1) (Acetyl-CoA ------1 69.26 deacylase) (Acetyl-CoA acylase) acp2 F-actin-capping protein subunit beta 2 99.58 2 99.58 2 99.58 act1 Actin 208 99.58 192 99.58 207 99.58 ade1 Bifunctional purine biosynthetic protein ADE1 3 99.58 3 99.58 3 99.58 [Includes: Phosphoribosylamine--glycine ligase (EC 6.3.4.13) (Glycinamide ribonucleotide synthetase) (GARS) (Phosphoribosylglycinamide synthetase); Phosphoribosylformylglycinamidine cyclo-ligase (EC 6.3.3.1) (AIR synthase) (AIRS) (Phosphoribosyl- aminoimidazole synthetase)] ade10 Bifunctional purine biosynthesis protein ade10 9 99.58 6 99.58 10 99.58 [Includes: Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) (5-aminoimidazole-4- carboxamide ribonucleotide formyltransferase) (AICAR transformylase); IMP cyclohydrolase (EC 3.5.4.10) (ATIC) (IMP synthase) (Inosinicase)] ade6 Phosphoribosylaminoimidazole carboxylase (EC 9 99.58 3 99.58 4 99.58 4.1.1.21) (AIR carboxylase) (AIRC) adh1 Alcohol dehydrogenase (EC 1.1.1.1) 32 99.58 38 99.58 40 99.58 adk1 Adenylate kinase (EC 2.7.4.3) (ATP-AMP 4 99.58 1 99.58 3 99.58 transphosphorylase) (ATP:AMP phosphotransferase) (Adenylate kinase cytosolic and mitochondrial) (Adenylate monophosphate kinase) adn1 Adhesion defective protein 1 1 96 ------

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides ado1 Adenosine kinase (EC 2.7.1.20) 2 99.58 2 99.58 1 99.28 aes1 Antisense-enhancing sequence 1 (EC 5.1.-.-) (AES 3 99.58 6 99.58 5 99.58 factor 1) ala1 Alanine--tRNA ligase (EC 6.1.1.7) (Alanyl-tRNA ------1 99.58 1 99.52 synthetase) (AlaRS) alm1 Abnormal long morphology protein 1 (Sp8) ------1 65.47 ------alp11 Cell polarity protein alp11 (Altered polarity protein 11) 1 72.26 ------anc1 ADP,ATP carrier protein (ADP/ATP translocase) 2 98.06 3 99.3 2 99.43 (Adenine nucleotide translocator) (ANT) ape1 Aminopeptidase 1 (EC 3.4.11.-) (Aminopeptidase I) ------1 99.58 ------apt1 Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) ------1 99.58 arf1 ADP-ribosylation factor 1 ------2 99.58 1 98.41 arg1 Probable acetylornithine aminotransferase, 3 99.58 5 99.58 4 99.58 mitochondrial (ACOAT) (EC 2.6.1.11) arg11 Protein arg11, mitochondrial [Cleaved into: N-acetyl- 2 99.58 5 99.58 2 99.58 gamma-glutamyl-phosphate reductase (EC 1.2.1.38) (N-acetyl-glutamate semialdehyde dehydrogenase) (NAGSA dehydrogenase); Acetylglutamate kinase (EC 2.7.2.8) (N-acetyl-L-glutamate 5-phosphotransferase) (NAG kinase) (AGK)] arg12 Argininosuccinate synthase (EC 6.3.4.5) (Citrulline------1 99.25 ------aspartate ligase) arg5 Carbamoyl-phosphate synthase arginine-specific small 7 99.58 6 99.58 2 99.58 chain (CPS-A) (EC 6.3.5.5) (Arginine-specific carbamoyl-phosphate synthetase, glutamine chain) aro1 Pentafunctional AROM polypeptide [Includes: 3------1 99.44 dehydroquinate synthase (DHQS) (EC 4.2.3.4); 3- phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19) (5-enolpyruvylshikimate-3-phosphate synthase) (EPSP synthase) (EPSPS); Shikimate kinase (SK) (EC 2.7.1.71); 3-dehydroquinate dehydratase (3- dehydroquinase) (EC 4.2.1.10); Shikimate dehydrogenase (EC 1.1.1.25)] aro4 Phospho-2-dehydro-3-deoxyheptonate aldolase, 11 99.58 8 99.58 12 99.58 tyrosine-inhibited (EC 2.5.1.54) (3-deoxy-D-arabino- heptulosonate 7-phosphate synthase) (DAHP

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides synthase) (Phospho-2-keto-3-deoxyheptonate aldolase) aru1 Arginase (EC 3.5.3.1) 2 91.93 2 96.75 1 99.58 asn1 Probable asparagine synthetase [glutamine- 6 99.58 4 99.58 4 99.58 hydrolyzing] (EC 6.3.5.4) (Glutamine-dependent asparagine synthetase) atg1 Serine/threonine-protein kinase atg1 (EC 2.7.11.1) ------3 96.26 (Autophagy-related protein 1) (Serine/threonine- protein kinase ppk36) atg13 Autophagy protein 13 (Meiotically up-regulated gene 8 99.58 14 99.58 10 99.58 78 protein) atg4 Probable cysteine protease atg4 (EC 3.4.22.-) 1 52.48 ------(Autophagy-related protein 4) atp1 ATP synthase subunit alpha, mitochondrial 8 99.58 7 99.58 7 99.58 atp16 ATP synthase subunit delta, mitochondrial (F-ATPase ------1 99.58 ------delta subunit) atp2 ATP synthase subunit beta, mitochondrial (EC 3.6.3.14) 14 99.58 11 99.58 17 99.58 bgs4 1,3-beta-glucan synthase component bgs4 (EC 1 99.58 2 99.47 3 97.65 2.4.1.34) (1,3-beta-D-glucan-UDP glucosyltransferase) bio2 Biotin synthase (EC 2.8.1.6) 2 99.58 2 99.58 1 99.58 bip1 78 kDa glucose-regulated protein homolog (GRP-78) 14 99.58 13 99.58 13 99.58 (Immunoglobulin heavy chain-binding protein homolog) (BiP) brr6 Nucleus export protein brr6 ------1 53.89 ------btf3 Nascent polypeptide-associated complex subunit beta 4 99.58 5 99.58 4 99.58 (NAC-beta) (Beta-NAC) but2 Uba3-binding protein but2 6 99.58 5 99.58 8 99.58 byr3 Cellular nucleic acid-binding protein homolog 3 99.58 3 99.58 2 99.58 cal1 Calnexin homolog 3 99.58 6 99.58 5 99.58 cam1 Calmodulin (CaM) 22 99.58 20 99.58 21 99.58 cam2 Myosin 1 light chain cam2 (Calmodulin-2) 5 99.58 8 99.58 7 99.58 cap1 Adenylyl cyclase-associated protein (CAP) 6 99.58 8 99.58 5 99.58 car2 Ornithine aminotransferase car2 (EC 2.6.1.13) 18 99.58 19 99.58 23 99.58 (Ornithine--oxo-acid aminotransferase) cbf12 Transcription factor cbf12 (C-promoter element------1 83.78 binding factor-like protein 12)

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides cbf5 H/ACA ribonucleoprotein complex subunit 4 (EC 4 99.58 5 99.58 8 99.58 5.4.99.-) (Centromere-binding factor 5 homolog) cct1 T-complex protein 1 subunit alpha (TCP-1-alpha) (CCT- 1 99.44 1 98.37 ------alpha) cct2 Probable T-complex protein 1 subunit beta (TCP-1- 14 99.58 13 99.58 8 99.58 beta) (CCT-beta) cct3 T-complex protein 1 subunit gamma (TCP-1-gamma) 6 99.58 9 99.58 5 99.58 (CCT-gamma) cct4 T-complex protein 1 subunit delta (TCP-1-delta) (CCT- 12 99.58 13 99.58 16 99.58 delta) cct5 T-complex protein 1 subunit epsilon (TCP-1-epsilon) 8 99.58 7 99.58 7 99.58 (CCT-epsilon) cct6 T-complex protein 1 subunit zeta (TCP-1-zeta) (CCT- 8 99.58 11 99.58 9 99.58 zeta) cct7 Probable T-complex protein 1 subunit eta (TCP-1-eta) 5 99.58 3 99.58 3 99.58 (CCT-eta) cct8 Probable T-complex protein 1 subunit theta (TCP-1- 5 99.58 6 99.58 4 99.58 theta) (CCT-theta) cdc13 G2/mitotic-specific cyclin cdc13 2 53.81 ------cdc22 Ribonucleoside-diphosphate reductase large chain (EC 7 99.58 7 99.58 8 99.58 1.17.4.1) (Ribonucleotide reductase) cdc3 Profilin 3 99.58 3 99.58 3 99.58 cdc48 Cell division cycle protein 48 14 99.58 14 99.58 13 99.58 cdc7 Cell division control protein 7 (EC 2.7.11.1) ------1 65.47 ------cdc8 Tropomyosin 9 99.58 7 99.58 7 99.58 cft1 Protein cft1 (Cleavage factor two protein 1) ------1 86.21 chc1 Probable clathrin heavy chain 9 99.58 10 99.58 11 99.58 chs1 Chitin synthase 1 (EC 2.4.1.16) (Chitin-UDP acetyl- 1 96 ------glucosaminyl transferase 1) clc1 Clathrin light chain (CLC) 5 99.58 2 98.05 2 93.92 cnd2 Condensin complex subunit 2 (Barren homolog) (CAPH ------1 66.09 ------homolog) (p105) cof1 Cofilin (Actin-depolymerizing factor 1) 8 99.58 8 99.58 5 99.58 cox6 Cytochrome c oxidase subunit 6, mitochondrial 1 99.24 ------(Cytochrome c oxidase polypeptide VI) csk1 Serine/threonine-protein kinase csk1 (EC 2.7.11.22) ------1 53.88 ------(CAK-activating kinase) (CAKAK)

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides cut6 Acetyl-CoA carboxylase (ACC) (EC 6.4.1.2) (Cell 30 99.58 26 99.58 26 99.58 untimely torn protein 6) [Includes: Biotin carboxylase (EC 6.3.4.14)] cut7 Kinesin-like protein cut7 (Cell untimely torn protein 7) 1 63.52 ------cwf24 Pre-mRNA-splicing factor cwf24 (Complexed with cdc5 2 64.91 ------1 64.51 protein 24) cyp4 Peptidyl-prolyl cis-trans isomerase B (PPIase B) (EC 1 99.58 1 99.58 ------5.2.1.8) (Cyclophilin 4) (Rotamase B) cyp9 Peptidyl-prolyl cis-trans isomerase 9 (PPIase cyp9) (EC ------1 74.22 ------5.2.1.8) (Cyclophilin 9) (Rotamase cyp9) dak1 Dihydroxyacetone kinase 1 (DHA kinase 1) (EC ------1 96.95 ------2.7.1.28) (EC 2.7.1.29) (Glycerone kinase 1) (Triokinase 1) (Triose kinase 1) dbp2 ATP-dependent RNA helicase dbp2 (EC 3.6.4.13) (p68------1 97.99 like protein) dbp5 ATP-dependent RNA helicase dbp5 (EC 3.6.4.13) 1 99.58 1 98.22 ------dcr1 Protein Dicer (Cell cycle control protein dcr1) (RNA ------1 53.81 ------interference pathway protein dcr1) [Includes: Endoribonuclease dcr1 (EC 3.1.26.-); ATP-dependent helicase dcr1 (EC 3.6.4.-)] ded1 ATP-dependent RNA helicase ded1 (EC 3.6.4.13) 43 99.58 35 99.58 41 99.58 (Multicopy suppressor of overexpressed cyr1 protein 2) dfr1 Dihydrofolate reductase (EC 1.5.1.3) 10 99.58 12 99.58 14 99.58 dps1 Aspartate--tRNA ligase, cytoplasmic (EC 6.1.1.12) 12 99.58 14 99.58 12 99.58 (Aspartyl-tRNA synthetase) (AspRS) dur3-3 Probable urea active transporter 3 ------1 63.58 ------eca39 Branched-chain-amino-acid aminotransferase, 6 99.58 6 99.58 9 99.58 mitochondrial (BCAT) (EC 2.6.1.42) ecm33 Cell wall protein ecm33 4 99.58 4 99.58 2 99.58 eft201; eft202 Elongation factor 2 (EF-2) 72 99.58 73 99.58 73 99.58 egd2 Nascent polypeptide-associated complex subunit 16 99.58 14 99.58 15 99.58 alpha (NAC-alpha) (Alpha-NAC) eif3f Eukaryotic translation initiation factor 3 subunit F 6 99.58 5 99.58 4 99.58 (eIF3f) eif3h Eukaryotic translation initiation factor 3 subunit H 7 99.58 6 99.58 7 99.58 (eIF3h)

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides elf1 mRNA export factor elf1 7 99.58 4 99.58 9 99.58 eno101 Enolase 1-1 (EC 4.2.1.11) (2-phospho-D-glycerate 49 99.58 49 99.58 53 99.58 hydro-lyase 1-1) (2-phosphoglycerate dehydratase 1- 1) ent1 Epsin-1 ------1 97.63 ------erg10 Acetyl-CoA acetyltransferase (EC 2.3.1.9) (Acetoacetyl- 17 99.58 18 99.58 14 99.58 CoA thiolase) (Ergosterol biosynthesis protein 10) erg11 Lanosterol 14-alpha demethylase erg11 (EC 2 99.58 3 99.58 3 99.58 1.14.13.70) (CYPLI) (Cytochrome P450 51) (Cytochrome P450-14DM) (Cytochrome P450-LIA1) (Sterol 14-alpha demethylase erg11) erg2 C-8 sterol isomerase erg2 (EC 5.-.-.-) (Delta-8--delta-7 2 98.6 ------sterol isomerase erg2) (Ergosterol biosynthesis protein 2) erg6 Sterol 24-C-methyltransferase erg6 (EC 2.1.1.41) ------1 99.58 3 99.58 (Delta(24)-sterol C-methyltransferase erg6) (Ergosterol biosynthesis protein 6) erm1 Putative endoplasmic reticulum metallopeptidase 1 1 53.3 ------(EC 3.4.-.-) (FXNA-like protease) fas1 Fatty acid synthase subunit beta (EC 2.3.1.86) 13 99.58 23 99.58 16 99.58 [Includes: 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59); Enoyl-[acyl-carrier-protein] reductase [NADH] (EC 1.3.1.9); [Acyl-carrier-protein] acetyltransferase (EC 2.3.1.38); [Acyl-carrier-protein] malonyltransferase (EC 2.3.1.39); S-acyl fatty acid synthase thioesterase (EC 3.1.2.14)] fas2 Fatty acid synthase subunit alpha (EC 2.3.1.86) 22 99.58 19 99.58 28 99.58 (p190/210) [Includes: Acyl carrier; 3-oxoacyl-[acyl- carrier-protein] reductase (EC 1.1.1.100) (Beta- ketoacyl reductase); 3-oxoacyl-[acyl-carrier-protein] synthase (EC 2.3.1.41) (Beta-ketoacyl synthase)] fba1 Fructose-bisphosphate aldolase (FBP aldolase) (FBPA) 17 99.58 24 99.58 19 99.58 (EC 4.1.2.13) (Fructose-1,6-bisphosphate aldolase) fib1 rRNA 2'-O-methyltransferase fibrillarin (EC 2.1.1.-) 8 99.58 4 99.58 9 99.58 (Histone-glutamine methyltransferase) fim1 Fimbrin 1 99.58 1 84.9 1 99.58

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides fkh1 Peptidyl-prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) ------2 99.58 (FK506-binding protein) (FKBP) fma2 Methionine aminopeptidase 2 (MAP 2) (MetAP 2) (EC 1 99.58 ------3.4.11.18) (Peptidase M) fmo1 Thiol-specific monooxygenase (EC 1.14.13.-) (Flavin- 3 99.58 4 99.58 ------dependent monooxygenase) fps1 Farnesyl pyrophosphate synthase (FPP synthase) (FPS) 2 86.74 2 87.36 ------(EC 2.5.1.10) ((2E,6E)-farnesyl diphosphate synthase) (Dimethylallyltranstransferase) (EC 2.5.1.1) (Farnesyl diphosphate synthase) (Geranyltranstransferase) frg1 Protein frg1 1 80.26 ------frs1 Phenylalanine--tRNA ligase beta subunit (EC 6.1.1.20) 1 98.06 2 60.31 3 96.39 (Phenylalanyl-tRNA synthetase beta subunit) (PheRS) frs2 Phenylalanine--tRNA ligase alpha subunit (EC 6.1.1.20) 2 86.74 ------1 87.39 (Phenylalanyl-tRNA synthetase alpha subunit) (PheRS) fyu1 Probable UTP--glucose-1-phosphate 11 99.58 10 99.58 12 99.58 uridylyltransferase (EC 2.7.7.9) (UDP-glucose pyrophosphorylase) (UDPGP) (UGPase) gar1 H/ACA ribonucleoprotein complex subunit 1 (snoRNP ------2 99.58 1 99.58 protein GAR1) gar2 Protein gar2 52 99.58 46 99.58 49 99.58 gas1 1,3-beta-glucanosyltransferase gas1 (EC 2.4.1.-) 2 99.58 4 99.58 2 99.58 gas2 1,3-beta-glucanosyltransferase gas2 (EC 2.4.1.-) 5 99.58 4 99.58 3 99.58 gcs1 Glutamate--cysteine ligase (EC 6.3.2.2) (Gamma-ECS) ------1 98.18 (GCS) (Gamma-glutamylcysteine synthetase) gdh1 NADP-specific glutamate dehydrogenase (NADP-GDH) 2 99.08 ------(EC 1.4.1.4) (NADP-dependent glutamate dehydrogenase) gdh2 Probable NAD-specific glutamate dehydrogenase ------1 99.58 (NAD-GDH) (EC 1.4.1.2) get3 ATPase get3 (EC 3.6.-.-) (Arsenical pump-driving ------3 99.58 3 99.58 ATPase) (Arsenite-stimulated ATPase) (Golgi to ER traffic protein 3) (Guided entry of tail-anchored proteins 3) ght5 High-affinity glucose transporter ght5 (Hexose ------1 99.58 1 99.58 transporter 5)

128

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides gln1 Glutamine synthetase (GS) (EC 6.3.1.2) (Glutamate-- 5 99.58 2 99.58 7 99.58 ammonia ligase) glt1 Putative glutamate synthase [NADPH] (EC 1.4.1.13) ------2 99.58 (NADPH-GOGAT) gly1 Probable low-specificity L-threonine aldolase (EC ------1 62.5 2 50.77 4.1.2.48) gpd1 Glycerol-3-phosphate dehydrogenase [NAD(+)] 1 (EC 6 99.58 5 99.58 6 99.58 1.1.1.8) (GPDH-C) (GPD-C) gpd2 Glycerol-3-phosphate dehydrogenase [NAD(+)] 2 (EC 2 99.58 1 99.58 2 99.58 1.1.1.8) gpd3 Glyceraldehyde-3-phosphate dehydrogenase 2 29 99.58 29 99.58 33 99.58 (GAPDH 2) (EC 1.2.1.12) gpm1 Phosphoglycerate mutase (PGAM) (EC 5.4.2.11) (BPG- 18 99.58 19 99.58 25 99.58 dependent PGAM) (MPGM) (Phosphoglyceromutase) gpx1 Glutathione peroxidase (EC 1.11.1.9) ------1 67.94 ------grs1 Putative glycine--tRNA ligase (EC 6.1.1.14) 15 99.58 12 99.58 8 99.58 (Diadenosine tetraphosphate synthetase) (AP-4-A synthetase) (Glycyl-tRNA synthetase) (GlyRS) gua1 Inosine-5'-monophosphate dehydrogenase (IMP 21 99.58 18 99.58 18 99.58 dehydrogenase) (IMPD) (IMPDH) (EC 1.1.1.205) gua1 GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2) 5 99.58 5 99.58 5 99.56 (GMP synthetase) (Glutamine amidotransferase) gus1 Probable glutamate--tRNA ligase, cytoplasmic (EC 12 99.58 11 99.58 10 99.58 6.1.1.17) (Glutamyl-tRNA synthetase) (GluRS) hap3 Transcriptional activator hap3 ------1 50.98 ------hcs1 Hydroxymethylglutaryl-CoA synthase (HMG-CoA ------1 53.15 1 66.31 synthase) (EC 2.3.3.10) (3-hydroxy-3-methylglutaryl coenzyme A synthase) hmt1 Heavy metal tolerance protein 2 98.3 5 99.49 12 99.52 hmt2 Sulfide:quinone oxidoreductase, mitochondrial (EC 1 99.58 ------1.8.5.-) (Cadmium resistance protein 1) (Heavy metal tolerance protein 2) hob1 Protein hob1 (Homolog of Bin1) 6 99.58 3 99.58 7 99.58 hob3 Protein hob3 (Homolog of Bin3) 3 96.15 3 97.42 1 62.13 hsp10 10 kDa heat shock protein, mitochondrial (HSP10) (10 1 84.99 2 99.54 2 75.96 kDa chaperonin)

129

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides hsp104 Heat shock protein 104 (Protein aggregation- 30 99.58 23 99.58 25 99.58 remodeling factor hsp104) hsp16 Heat shock protein 16 (16 kDa heat shock protein) 6 99.58 5 99.58 6 99.58 hsp60 Heat shock protein 60, mitochondrial (HSP60) 49 99.58 50 99.58 50 99.58 hsp78 Heat shock protein 78, mitochondrial 1 99.49 ------hst2 NAD-dependent protein deacetylase hst2 (EC 3.5.1.-) 2 66.09 ------1 50.27 (Homologous to sir2 protein 2) (Regulatory protein SIR2 homolog 2) hta1 Histone H2A-alpha (H2A.1) 1 84.8 2 83.27 1 81.97 htb1 Histone H2B-alpha (H2B.1) 2 65.47 5 99.58 6 99.58 hts1 Histidine--tRNA ligase, mitochondrial (EC 6.1.1.21) 4 99.58 3 98.44 3 99.58 (Histidyl-tRNA synthetase) (HisRS) hxk1 Hexokinase-1 (EC 2.7.1.1) 2 99.58 1 99.58 2 99.58 ilv1 Acetolactate synthase, mitochondrial (EC 2.2.1.6) 9 99.58 7 99.58 8 98.85 (AHAS) (ALS) (Acetohydroxy-acid synthase) ilv5 Probable ketol-acid reductoisomerase, mitochondrial 68 99.58 58 99.58 68 99.58 (EC 1.1.1.86) (Acetohydroxy-acid reductoisomerase) (Alpha-keto-beta-hydroxylacyl reductoisomerase) imp2 Septation protein imp2 1 99.28 ------int6 Eukaryotic translation initiation factor 3 subunit E 2 99.58 2 99.58 3 99.58 (eIF3e) irs1 Isoleucine--tRNA ligase, cytoplasmic (EC 6.1.1.5) 4 99.58 3 99.58 3 99.58 (Isoleucyl-tRNA synthetase) (IleRS) isn1 IMP-specific 5'-nucleotidase 1 (EC 3.1.3.-) 2 97.3 1 97.94 ------isp4 Sexual differentiation process protein isp4 2 99.58 4 99.58 3 99.58 kap114 Importin subunit beta-5 (114 kDa karyopherin) ------1 98.93 (Karyopherin subunit beta-5) (Karyopherin-114) kap123 Probable importin subunit beta-4 (Importin-123) ------1 99.58 ------(Karyopherin subunit beta-4) (Karyopherin-123) krs1 Lysine--tRNA ligase, cytoplasmic (EC 6.1.1.6) (Lysyl- 4 99.58 7 99.58 4 99.58 tRNA synthetase) (LysRS) ksg1 Serine/threonine-protein kinase ksg1 (EC 2.7.11.1) ------(PDK1-like kinase ksg1) las1 Pre-rRNA-processing protein las1 ------lat1 Dihydrolipoyllysine-residue acetyltransferase 34 99.58 33 99.58 30 99.58 component of pyruvate dehydrogenase complex, mitochondrial (EC 2.3.1.12) (Dihydrolipoamide

130

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides acetyltransferase component of pyruvate dehydrogenase complex) (Pyruvate dehydrogenase complex component E2) (PDC-E2) (PDCE2) let1 26S protease regulatory subunit 8 homolog (Protein 19 99.58 13 99.58 16 99.58 let1) leu1 3-isopropylmalate dehydrogenase (3-IPM-DH) (IMDH) 2 95.41 1 98.49 2 89.2 (EC 1.1.1.85) (Beta-IPM dehydrogenase) leu2 3-isopropylmalate dehydratase (EC 4.2.1.33) (Alpha- 37 99.58 32 99.58 39 99.58 IPM isomerase) (IPMI) (Isopropylmalate isomerase) leu3 2-isopropylmalate synthase (EC 2.3.3.13) (Alpha-IPM 1 95.26 3 99.58 3 99.58 synthase) (Alpha-isopropylmalate synthase) lid2 Lid2 complex component lid2 (Lid2C component lid2) 1 53.3 ------1 96.49 lrs1 Putative leucine--tRNA ligase, cytoplasmic (EC 6.1.1.4) 6 99.58 3 99.58 5 99.58 (Leucyl-tRNA synthetase) (LeuRS) lsm4 Probable U6 snRNA-associated Sm-like protein LSm4 ------1 88.85 lys1 L-2-aminoadipate reductase (EC 1.2.1.31) (EC 1.2.1.95) 6 99.58 7 99.58 3 99.58 (Alpha-aminoadipate reductase) (Alpha-AR) (L- aminoadipate-semialdehyde dehydrogenase) lys12 Homoisocitrate dehydrogenase (HICDH) (EC 1.1.1.87) 6 99.58 7 99.58 6 99.58 lys3 Saccharopine dehydrogenase [NAD(+), L-lysine- 1 99.58 3 98.11 3 99.58 forming] (SDH) (EC 1.5.1.7) (Lysine--2-oxoglutarate reductase) lys4 Homocitrate synthase, mitochondrial (EC 2.3.3.14) 30 99.58 24 99.58 28 99.58 lys9 Saccharopine dehydrogenase [NADP(+), L-glutamate- 14 99.58 15 99.58 14 99.58 forming] (EC 1.5.1.10) (Saccharopine reductase) mae2 NAD-dependent malic enzyme (NAD-ME) (EC 1.1.1.38) 7 99.58 9 99.58 6 99.58 mak3 Peroxide stress-activated histidine kinase mak3 (EC 1 81.77 ------2.7.13.3) (His-Asp phosphorelay kinase phk2) (Mcs4- associated kinase 3) mas5 Mitochondrial protein import protein mas5 31 99.58 30 99.58 23 99.58 mbf1 Multiprotein-bridging factor 1 ------3 99.58 met26 Probable 5-methyltetrahydropteroyltriglutamate-- 23 99.58 24 99.58 28 99.58 homocysteine methyltransferase (EC 2.1.1.14) (Cobalamin-independent methionine synthase) (Methionine synthase, vitamin-B12 independent isozyme) meu27 Meiotic expression up-regulated protein 27 ------1 61.22 ------

131

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides mge1 GrpE protein homolog, mitochondrial 2 99.58 4 99.58 3 99.58 mgr2 Protein mgr2 1 73.73 1 65.47 1 74.24 mlo3 mRNA export protein mlo3 (RNA-annealing protein ------1 78.23 mlo3) mmf1 Protein mmf1, mitochondrial (Isoleucine biosynthesis 7 99.58 4 99.58 8 99.58 and maintenance of intact mitochondria 1) (Maintenance of mitochondrial function 1) mnn9 Mannan polymerase complex subunit mnn9 3 99.58 3 99.58 3 99.58 moe1 Eukaryotic translation initiation factor 3 subunit D 4 99.58 3 99.58 5 99.58 (eIF3d) (Microtubule-destabilizing protein moe1) mpg1 Mannose-1-phosphate guanyltransferase (EC 2.7.7.13) 11 99.58 12 99.58 16 99.58 (GDP-mannose pyrophosphorylase) (GTP-mannose-1- phosphate guanylyltransferase) mrpl40 54S ribosomal protein L40, mitochondrial ------1 61.4 mrs1 Probable arginine--tRNA ligase, cytoplasmic (EC 4 99.58 1 58.78 3 99.58 6.1.1.19) (Arginyl-tRNA synthetase) (ArgRS) msd1 Aspartate--tRNA ligase, mitochondrial (EC 6.1.1.12) ------1 60.31 (Aspartyl-tRNA synthetase) (AspRS) mts2 26S protease regulatory subunit 4 homolog (Protein 19 99.58 21 99.58 29 99.58 mts2) mug157 Meiotically up-regulated gene 157 protein ------1 81.09 mug30 Probable E3 ubiquitin-protein ligase mug30 (EC 6.3.2.-) 1 54.54 ------(Meiotically up-regulated gene 30 protein) mug35 Meiotically up-regulated gene 35 protein ------2 95.97 ------mug4 Meiotically up-regulated gene 4 protein ------1 53.81 ------mug64 Meiotically up-regulated gene 64 protein 9 99.58 10 99.58 14 99.58 mug66 Meiotically up-regulated gene 66 protein (Autophagy- 38 99.58 42 99.58 38 99.58 related protein 101) mug82 Meiotically up-regulated gene 82 protein ------1 64.51 ------mug87 Meiotically up-regulated gene 87 protein 1 50.98 ------myo1 Myosin-1 (Class I unconventional myosin) (Type I 32 99.58 35 99.58 30 99.58 myosin) myo2 Myosin type-2 heavy chain 1 (Myosin type II heavy 6 99.58 1 99.58 3 99.58 chain 1) nat10 RNA cytidine acetyltransferase (EC 2.3.1.-) (18S rRNA 2 99.58 ------1 99.58 cytosine acetyltransferase) nda2 Tubulin alpha-1 chain 6 99.58 6 99.58 3 99.58

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Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides nda3 Tubulin beta chain (Beta-tubulin) 27 99.58 28 99.58 25 99.58 nip1 Eukaryotic translation initiation factor 3 subunit C 12 99.58 10 99.58 8 99.58 (eIF3c) (Eukaryotic translation initiation factor 3 93 kDa subunit homolog) (eIF3 p93) (Translation initiation factor eIF3, p93 subunit homolog) nog1 Probable nucleolar GTP-binding protein 1 ------1 97.5 ------nop56 Nucleolar protein 56 (Ribosome biosynthesis protein 4 99.58 7 99.58 6 99.58 sik1) nop58 Nucleolar protein 58 2 99.58 8 99.58 5 99.58 nro1 Negative regulator of ofd1 1 74.42 ------nrs1 Probable asparagine--tRNA ligase, cytoplasmic (EC 8 99.58 4 99.58 8 99.58 6.1.1.22) (Asparaginyl-tRNA synthetase) (AsnRS) obr1 P25 protein (Brefeldin A resistance protein) 3 90.51 4 99.58 4 99.58 oca8 Probable cytochrome b5 2 1 99.46 ------1 99.52 ogm4 Dolichyl-phosphate-mannose--protein ------1 73.73 ------mannosyltransferase 4 (EC 2.4.1.109) oxa101 Mitochondrial inner membrane protein oxa1-1 1 59.28 ------(Cytochrome oxidase biogenesis protein 1-1) (Sp1) p23fy Translationally-controlled tumor protein homolog 3 99.58 3 99.58 5 99.58 (TCTP) (p23fyp) paa1 Protein phosphatase PP2A regulatory subunit A 1 77.64 ------(Protein phosphatase 2A 65 kDa regulatory subunit) (PR65) pab1 Polyadenylate-binding protein, cytoplasmic and 33 99.58 41 99.58 47 99.58 nuclear (PABP) (Poly(A)-binding protein) (Polyadenylate tail-binding protein) pam1 Probable proteasome subunit beta type-6 (EC ------1 99.58 3.4.25.1) pam16 Mitochondrial import inner membrane translocase 1 99.29 2 99.58 2 99.58 subunit tim16 (Presequence translocated-associated motor subunit pam16) par2 Serine/threonine-protein phosphatase 2A 56 kDa 1 53.27 ------regulatory subunit delta 2 isoform (PP2A, B subunit, B' delta 2 isoform) pda1 Pyruvate dehydrogenase E1 component subunit alpha, 16 99.58 21 99.58 22 99.58 mitochondrial (PDHE1-A) (EC 1.2.4.1)

133

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides pdb1 Pyruvate dehydrogenase E1 component subunit beta, 37 99.58 41 99.58 39 99.58 mitochondrial (PDHE1-B) (EC 1.2.4.1) pex11 Peroxisomal biogenesis factor 11 3 99.58 3 99.58 4 99.58 pfk1 ATP-dependent 6-phosphofructokinase (ATP-PFK) 86 99.58 94 99.58 93 99.58 (Phosphofructokinase) (EC 2.7.1.11) (Phosphohexokinase) pgi1 Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) 1 96.12 1 99.04 ------(Phosphoglucose isomerase) (PGI) (Phosphohexose isomerase) (PHI) pgk1 Phosphoglycerate kinase (EC 2.7.2.3) 54 99.58 53 99.58 41 99.58 phf2 SWM histone demethylase complex subunit phf2 (PHD 1 99.43 ------1 61.22 finger domain-containing protein phf2) pho1 Acid phosphatase (EC 3.1.3.2) 3 99.58 ------1 99.58 pht1 Histone H2A.Z 1 75.1 1 78.07 1 91.18 pil1 Probable sphingolipid long chain base-responsive 53 99.58 52 99.58 41 99.58 protein pil1 (Protein kinase inhibitor pil1) plb1 Lysophospholipase 1 (EC 3.1.1.5) (Phospholipase B 1) 2 99.58 2 99.58 3 99.58 plr1 Pyridoxal reductase (PL reductase) (PL-red) (EC 3 83.77 ------1 93.01 1.1.1.65) pma1 Plasma membrane ATPase 1 (EC 3.6.3.6) (Proton pump 47 99.58 47 99.58 50 99.58 1) pmm1 Phosphomannomutase (PMM) (EC 5.4.2.8) 7 99.58 9 99.58 8 99.58 pmp20 Putative peroxiredoxin pmp20 (EC 1.11.1.15) 6 99.58 7 99.58 9 99.58 (Peroxisomal membrane protein pmp20) (Thioredoxin reductase) pob3 FACT complex subunit pob3 (Facilitates chromatin 1 90.87 ------transcription complex subunit pob3) ppa1 Inorganic pyrophosphatase (EC 3.6.1.1) 11 99.58 9 99.58 10 99.58 (Pyrophosphate phospho-hydrolase) (PPase) ppa2 Major serine/threonine-protein phosphatase PP2A-2 ------1 99.58 ------catalytic subunit (EC 3.1.3.16) ppi1 Peptidyl-prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) ------1 88.85 (Cyclophilin) (CPH) (Cyclosporin A-binding protein) (Rotamase) ppk30 Serine/threonine-protein kinase ppk30 (EC 2.7.11.1) ------1 57.98 ppt1 Serine/threonine-protein phosphatase T (PPT) (EC ------3 99.58 ------3.1.3.16)

134

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides pre10 Probable proteasome subunit alpha type-7 (EC ------1 99.44 ------3.4.25.1) prp1 Pre-mRNA-splicing factor prp1 ------1 55.76 1 77.89 prp19 Pre-mRNA-processing factor 19 (EC 6.3.2.-) ------1 65.25 (Complexed with cdc5 protein 8) prp39 Pre-mRNA-processing factor 39 ------1 67.58 prs1 Putative proline--tRNA ligase C19C7.06 (EC 6.1.1.15) 21 99.58 24 99.58 18 99.58 (Prolyl-tRNA synthetase) (ProRS) psi1 Protein psi1 (Protein psi) 5 99.58 2 99.58 3 99.58 pss1 Heat shock protein homolog pss1 15 99.58 9 99.58 17 99.58 ptc3 Protein phosphatase 2C homolog 3 (PP2C-3) (EC 3 99.58 5 99.58 5 99.58 3.1.3.16) ptr3 Ubiquitin-activating enzyme E1 1 (EC 6.2.1.45) 2 99.58 2 99.58 8 99.58 (Poly(A)+ RNA transport protein 3) pyk1 Pyruvate kinase (PK) (EC 2.7.1.40) 237 99.58 237 99.58 230 99.58 pyr1 Pyruvate carboxylase (EC 6.4.1.1) (Pyruvic carboxylase) 2 99.58 6 99.58 5 99.58 (PCB) qcr2 Cytochrome b-c1 complex subunit 2, mitochondrial ------2 99.58 (Complex III subunit 2) (Core protein II) (Ubiquinol- cytochrome-c reductase complex core protein 2) qcr6 Cytochrome b-c1 complex subunit 6 (Complex III 1 63.59 ------subunit 6) (Mitochondrial hinge protein) (Ubiquinol- cytochrome c reductase complex subunit 6) qcr9 Cytochrome b-c1 complex subunit 9 (Complex III 1 99.58 1 99.58 1 99.58 subunit 9) (Cytochrome c1 non-heme 7.3 kDa protein) (Ubiquinol-cytochrome c reductase complex 7.3 kDa protein) qrs1 Probable glutamine--tRNA ligase (EC 6.1.1.18) 4 99.58 1 79.25 ------(Glutaminyl-tRNA synthetase) (GlnRS) rad24 DNA damage checkpoint protein rad24 14 99.58 6 99.58 12 99.58 rad25 DNA damage checkpoint protein rad25 2 82.42 2 60.53 2 79.67 rae1 Poly(A)+ RNA export protein ------1 92.63 ------rar1 Probable methionine--tRNA ligase, cytoplasmic (EC 3 99.58 4 98.33 5 99.44 6.1.1.10) (Methionyl-tRNA synthetase) (MetRS) rfc2 Replication factor C subunit 2 (Replication factor C2) 1 67.22 ------(Activator 1 41 kDa subunit)

135

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides rhb1 GTP-binding protein rhb1 (GTP-binding protein Rheb ------2 99.58 homolog) rho1 GTP-binding protein rho1 5 99.58 3 99.58 6 99.58 rhp7 DNA repair protein rhp7 (RAD7 homolog) ------1 63.42 ------rib2 Diaminohydroxyphosphoribosylamino-pyrimidine ------1 51.31 ------deaminase (DRAP deaminase) (EC 3.5.4.26) (Riboflavin-specific deaminase) rkp1 Guanine nucleotide-binding protein subunit beta-like 17 99.58 19 99.58 17 99.58 protein (Receptor of activated protein kinase C) rli1 Translation initiation factor rli1 (ATP-binding cassette 1 97.86 1 56.21 2 64.98 sub-family E member rli1) (RNase L inhibitor) rnc1 RNA-binding protein rnc1 (RNA-binding protein that 1 99.44 ------1 99.44 suppresses calcineurin deletion 1) rpa34 DNA-directed RNA polymerase I subunit rpa34 (RNA ------2 99.58 2 99.58 polymerase I subunit A34) rpa43 DNA-directed RNA polymerase I subunit rpa43 (RNA ------1 98.82 ------polymerase I subunit A43) (DNA-dependent RNA polymerase 19 kDa polypeptide) rpl1001 60S ribosomal protein L10-A (QM protein homolog) 2 83.88 3 99.57 4 99.52 (SpQM) rpl1002 60S ribosomal protein L10-B 32 99.58 32 99.58 33 99.58 rpl101 60S ribosomal protein L1-B (L10a) 3 99.58 2 99.58 3 99.58 rpl102 60S ribosomal protein L1-A (L10a) 10 99.58 11 99.58 14 99.58 rpl1101 60S ribosomal protein L11-A 17 99.58 17 99.58 16 99.58 rpl1201 60S ribosomal protein L12-A 10 99.58 16 99.58 15 99.58 rpl13 60S ribosomal protein L13 19 99.58 20 99.58 25 99.58 rpl14 60S ribosomal protein L14 7 99.58 6 99.58 5 99.58 rpl15 60S ribosomal protein L15-A 1 99.58 ------2 99.48 rpl1502 60S ribosomal protein L15-B 14 99.58 23 99.58 18 99.58 rpl1601 60S ribosomal protein L16-B 1 73.13 1 77.72 2 75.41 rpl1602 60S ribosomal protein L16-A 17 99.58 15 99.58 10 99.58 rpl1701 60S ribosomal protein L17-A 32 99.58 34 99.58 29 99.58 rpl1702 60S ribosomal protein L17-B 5 98.51 5 98.95 5 94.65 rpl1801 60S ribosomal protein L18-A 14 99.58 18 99.58 13 99.58 rpl1802 60S ribosomal protein L18-B 13 99.58 12 99.58 12 99.58 rpl1901 60S ribosomal protein L19-A (YL15) 23 99.58 23 99.58 24 99.58 rpl1902 60S ribosomal protein L19-B 4 99.44 4 99.44 7 96.39

136

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides rpl2001 60S ribosomal protein L20-A (YL17) 22 99.58 23 99.58 17 99.58 rpl2101 60S ribosomal protein L21-A 8 99.58 7 99.58 6 99.58 rpl2102 60S ribosomal protein L21-B 8 99.58 13 99.58 11 99.58 rpl22 60S ribosomal protein L22 10 99.58 11 99.58 11 99.58 rpl2301 60S ribosomal protein L23-A 17 99.58 15 99.58 9 99.58 rpl2402 60S ribosomal protein L24-B 11 99.58 12 99.58 9 99.58 rpl2501 60S ribosomal protein L25-A 16 99.58 15 99.58 16 99.58 rpl26 60S ribosomal protein L26 14 99.58 18 99.58 16 99.58 rpl2701 60S ribosomal protein L27-A 14 99.58 17 99.58 12 99.58 rpl2702 60S ribosomal protein L27-B 6 99.58 6 99.58 3 99.58 rpl2801 60S ribosomal protein L28-B 11 99.58 14 99.58 12 99.58 rpl2802 60S ribosomal protein L28-A (L27A) (L29) 6 99.58 6 99.58 4 99.58 rpl3001 60S ribosomal protein L30-1 (L32) 7 99.58 9 99.58 7 99.58 rpl302 60S ribosomal protein L3-B 44 99.58 38 99.58 47 99.58 rpl31 60S ribosomal protein L31 8 99.58 10 99.58 7 99.58 rpl3201 60S ribosomal protein L32-B 5 99.58 6 99.58 5 99.58 rpl3202 60S ribosomal protein L32-A 3 99.58 3 99.58 4 99.58 rpl3402 60S ribosomal protein L34-B (60S ribosomal protein 7 99.58 6 99.44 11 99.58 L34-2) rpl35 60S ribosomal protein L35 17 99.58 12 99.58 12 99.58 rpl35a 60S ribosomal protein L33-B (L37B) 3 56.92 1 52.97 2 59.89 rpl3601 60S ribosomal protein L36-A 11 99.58 12 99.58 11 99.58 rpl3702 60S ribosomal protein L37-B (L37-2) (YL27) 4 99.58 5 99.58 4 99.58 rpl3801 60S ribosomal protein L38-1 1 97.22 3 99.43 1 99.43 rpl3802 60S ribosomal protein L38-2 ------3 94.96 1 94.2 rpl39 60S ribosomal protein L39 (YL36) ------1 77.06 rpl401 60S ribosomal protein L4-B 55 99.58 57 99.58 55 99.58 rpl402 60S ribosomal protein L4-A (L2) 13 99.58 13 99.58 14 99.58 rpl42 60S ribosomal protein L42 ------1 71.21 ------rpl4301 60S ribosomal protein L43-A (L37A) 8 99.48 4 98.21 9 96.82 rpl44 Probable 60S ribosomal protein L28e 23 99.58 20 99.58 21 99.58 rpl502 60S ribosomal protein L5-B 45 99.58 47 99.58 47 99.58 rpl6 60S ribosomal protein L6 14 99.58 18 99.58 15 99.58 rpl701 60S ribosomal protein L7-C 9 99.58 13 99.58 15 99.58 rpl702 60S ribosomal protein L7-B 6 99.58 6 99.58 7 99.58 rpl8 60S ribosomal protein L8 (L4) (L7A) 30 99.58 40 99.58 31 99.58

137

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides rpl801 60S ribosomal protein L2-A (K37) (K5) (KD4) 35 99.58 36 99.58 39 99.58 rpl901 60S ribosomal protein L9-A 4 99.58 5 99.58 6 99.58 rpl902 60S ribosomal protein L9-B 11 99.58 10 99.58 13 99.58 rpn1 26S proteasome regulatory subunit rpn1 (19S 3 89.5 2 99.44 3 99.58 regulatory cap region of 26S protease subunit 2) (Proteasome non-ATPase subunit mts4) rpn11 26S proteasome regulatory subunit rpn11 (Protein 7 99.58 10 99.58 8 99.58 pad1) rpn12 26S proteasome regulatory subunit rpn12 5 99.58 3 99.44 3 99.58 rpn2 26S proteasome regulatory subunit rpn2 2 65.36 1 81.09 ------rpn3 Probable 26S proteasome regulatory subunit rpn3 6 99.58 4 99.58 3 99.58 rpn501; rpn502 26S proteasome regulatory subunit rpn5 1 98.95 3 99.58 3 99.58 rpn6 Probable 26S proteasome regulatory subunit rpn6 4 99.58 7 99.58 4 99.58 rpn7 Probable 26S proteasome regulatory subunit rpn7 ------1 98.83 rpn8 26S proteasome regulatory subunit rpn8 9 99.58 7 99.58 10 99.58 rpn9 Probable 26S proteasome regulatory subunit rpn9 4 99.58 4 99.58 3 99.58 rpp0 60S acidic ribosomal protein P0 20 99.58 22 99.58 19 99.58 rpp101 60S acidic ribosomal protein P1-alpha 1 (A1) 6 99.58 5 99.58 4 99.58 rpp103 60S acidic ribosomal protein P1-alpha 5 4 99.58 3 99.58 3 99.58 rpp201 60S acidic ribosomal protein P2-alpha (A2) (L12EI) 4 99.58 4 99.58 4 99.58 (L40C) rpp203 60S acidic ribosomal protein P2-C 4 99.58 2 63.73 2 96.63 rps001 40S ribosomal protein S0-A 7 99.58 5 99.58 6 99.58 rps002 40S ribosomal protein S0-B 11 99.58 9 99.58 12 99.58 rps1001 40S ribosomal protein S10-A 2 99.58 3 99.58 3 99.58 rps1002 40S ribosomal protein S10-B 3 99.58 11 99.58 11 99.58 rps101 40S ribosomal protein S1-A (S3aE-A) 19 99.58 18 99.58 16 99.58 rps102 40S ribosomal protein S1-B (S3aE-B) 14 99.58 13 99.58 14 99.58 rps1101 40S ribosomal protein S11-A 23 99.58 19 99.58 15 99.58 rps1201 40S ribosomal protein S12-A 12 99.58 17 99.58 13 99.58 rps1202 40S ribosomal protein S12-B 5 99.58 7 99.58 8 99.58 rps13 40S ribosomal protein S13 21 99.58 17 99.58 20 99.58 rps1402 40S ribosomal protein S14-B 11 99.58 18 99.58 16 99.58 rps1501 40S ribosomal protein S15-A 9 99.58 8 99.58 9 99.58 rps1502 40S ribosomal protein S15-B 2 99.58 2 99.44 2 99.44 rps1601 40S ribosomal protein S16-A 12 99.58 11 99.58 17 99.58

138

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides rps1701 40S ribosomal protein S17-A 10 99.58 9 99.58 10 99.58 rps1801 40S ribosomal protein S18-A 10 99.58 10 99.58 15 99.58 rps1901 40S ribosomal protein S19-A (S16-A) 13 99.58 13 99.58 16 99.58 rps1902 40S ribosomal protein S19-B (S16-B) 4 99.58 ------rps2 40S ribosomal protein S2 9 99.58 11 99.58 13 99.58 rps20 40S ribosomal protein S20 10 97.46 10 97.07 7 97.03 rps21 40S ribosomal protein S21 (S28) 13 99.58 12 99.58 14 99.58 rps2201 40S ribosomal protein S22-A 9 99.58 14 99.58 14 99.58 rps2302 40S ribosomal protein S23-B 21 99.58 26 99.58 16 99.58 rps2401 40S ribosomal protein S24-A 3 99.58 4 99.58 6 99.58 rps2402 40S ribosomal protein S24-B 8 99.58 7 99.58 7 99.58 rps2502 40S ribosomal protein S25-A (S31-A) 8 99.28 5 98.61 3 99.44 rps2601 40S ribosomal protein S26-A ------2 98.32 2 99.58 rps2602 40S ribosomal protein S26-B 3 99.58 4 99.58 3 99.58 rps27 40S ribosomal protein S27 4 57.65 3 81.26 3 86.48 rps2801 40S ribosomal protein S28-A (S33) 12 99.58 10 99.58 12 99.58 rps29 40S ribosomal protein S29 2 87.69 3 83.71 2 54.98 rps3 40S ribosomal protein S3 26 99.58 27 99.58 29 99.58 rps3001 40S ribosomal protein S30-A 3 95.95 4 95.09 4 96.1 rps402 40S ribosomal protein S4-B 7 99.58 8 99.58 4 99.58 rps403 40S ribosomal protein S4-C 12 99.58 13 99.58 14 99.58 rps5 40S ribosomal protein S5-A 31 99.58 23 99.58 25 99.58 rps502 40S ribosomal protein S5-B ------1 80.65 2 85.16 rps602 40S ribosomal protein S6-B 34 99.58 49 99.58 41 99.58 rps7 40S ribosomal protein S7 38 99.58 38 99.58 35 99.58 rps802 40S ribosomal protein S8-B 22 99.58 21 99.58 18 99.58 rps901 40S ribosomal protein S9-A 3 99.58 5 99.58 6 99.58 rps902 40S ribosomal protein S9-B 13 99.58 16 99.58 14 99.58 rpt1 26S protease regulatory subunit 7 homolog 32 99.58 31 99.58 34 99.58 rpt3 26S protease regulatory subunit 6B homolog 11 99.58 11 99.58 13 99.58 rpt4 Probable 26S protease subunit rpt4 5 99.58 9 99.58 9 99.58 rqh1 ATP-dependent DNA helicase hus2/rqh1 (EC 3.6.4.12) ------1 52.48 ------rtp1 RNA polymerase II assembly factor rtp1 ------1 60.31 rvb1 RuvB-like helicase 1 (EC 3.6.4.12) 3 99.58 4 99.58 5 99.58 rvb2 RuvB-like helicase 2 (EC 3.6.4.12) 12 99.58 14 99.58 12 99.58 sal3 Importin subunit beta-3 (Importin beta sal3) 1 99.58 1 99.58 2 99.58

139

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides sam1 S-adenosylmethionine synthase (AdoMet synthase) 10 99.58 11 99.58 8 99.58 (EC 2.5.1.6) (Methionine adenosyltransferase) (MAT) sbp1 Ran-specific GTPase-activating protein 1 (Ran-binding 2 99.52 3 99 4 98.32 protein 1) (RANBP1) (Spi1-binding protein) sce3 Probable RNA-binding protein sce3 ------2 94.73 2 87.39 sdo1 Ribosome maturation protein sdo1 1 63.38 ------sec17 Probable vesicular-fusion protein sec17 homolog ------1 69.41 sec18 Vesicular-fusion protein sec18 ------2 99.45 ------sec27 Probable coatomer subunit beta' (Beta'-coat protein) 2 99.48 2 99.25 1 99.58 (Beta'-COP) sen34 Probable tRNA-splicing endonuclease subunit sen34 ------1 92.22 ------(EC 4.6.1.16) (tRNA-intron endonuclease sen34) sfc3 Transcription factor tau subunit sfc3 (TFIIIC subunit 1 58 1 55.62 1 57.99 sfc3) (Transcription factor C subunit 3) sgt2 Small glutamine-rich tetratricopeptide repeat- 16 99.58 12 99.58 11 99.58 containing protein 2 shm2 Serine hydroxymethyltransferase, mitochondrial 1 99.58 2 99.58 2 99.58 (SHMT) (EC 2.1.2.1) (Glycine hydroxymethyltransferase) (Serine methylase) sir1 Sulfite reductase [NADPH] subunit beta (EC 1.8.1.2) 8 99.58 10 99.58 7 99.54 skp1 Suppressor of kinetochore protein 1 (P19/Skp1 ------1 99.58 homolog) sks2 Heat shock protein sks2 (Heat shock cognate protein 108 99.58 101 99.58 103 99.58 hsc1) sla1 Actin cytoskeleton-regulatory complex protein sla1 2 99.58 2 99.58 1 99.58 sla1 La protein homolog (La autoantigen homolog) (La 2 99.54 ------ribonucleoprotein) slm1 Cytoskeletal signaling protein slm1 5 99.58 5 99.58 6 99.58 slt1 Protein slt1 4 99.58 4 99.58 3 99.58 sly1 Protein sly1 ------1 77.13 ------snd1 Staphylococcal nuclease domain-containing protein 1 1 61.12 ------3 99.58 (4SNc-Tudor domain protein) snu13 13 kDa ribonucleoprotein-associated protein 5 99.58 2 99.58 4 99.52 snz1 Probable pyridoxal 5'-phosphate synthase subunit 1 78.94 2 81.6 2 86.17 PDX1 (PLP synthase subunit PDX1) (EC 4.3.3.6) spac1039.02 Uncharacterized protein C1039.02 1 53.81 ------spac105.02c Ankyrin repeat-containing protein C105.02c 2 87.48 ------1 99.58

140

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides spac11d3.02c UPF0039 protein C11D3.02c 2 98.21 1 51.69 1 74.6 spac11e3.14 Uncharacterized protein C11E3.14 ------1 52.41 spac12g12.07c Uncharacterized protein C12G12.07c 2 99.58 5 99.58 3 99.54 spac13c5.05c Phosphoacetylglucosamine mutase 1 (PAGM) (EC 1 99.58 ------5.4.2.3) (Acetylglucosamine phosphomutase) (N- acetylglucosamine-phosphate mutase) spac1565.05 Uncharacterized protein C1565.05 1 99.58 ------spac1635.01 Probable mitochondrial outer membrane protein 6 99.58 7 99.58 3 99.58 porin spac17g6.01 Putative metal ion transporter C17A12.14 2 94.54 2 63.28 3 95.88 spac17h9.14c Protein disulfide-isomerase C17H9.14c (EC 5.3.4.1) 2 99.58 2 99.56 2 99.57 spac18b11.02c Uncharacterized protein C18B11.02c 1 55.52 1 82.23 ------spac1f12.07 Putative phosphoserine aminotransferase (PSAT) (EC 6 99.58 4 99.58 9 99.58 2.6.1.52) (Phosphohydroxythreonine aminotransferase) spac1f5.02 Putative protein disulfide-isomerase C1F5.02 (EC 3 99.58 5 99.58 6 99.58 5.3.4.1) spac1f8.04c Uncharacterized protein C1F8.04c 1 50.98 ------spac1f8.07c Probable pyruvate decarboxylase C1F8.07c (EC 4.1.1.1) 51 99.58 38 99.58 48 99.58 spac212.06c RecQ DNA helicase-like protein C212.06c ------1 53.81 ------spac222.01 Probable phosphatase C1687.21 (EC 3.1.3.-) 2 99.58 ------spac222.08c Uncharacterized glutaminase C222.08c (EC 3.5.1.2) 1 89.78 ------2 78.57 spac22a12.16 Probable ATP-citrate synthase subunit 2 (EC 2.3.3.8) 3 99.58 4 99.58 3 99.58 (ATP-citrate (pro-S-)-lyase 2) (Citrate cleavage enzyme subunit 2) spac22h10.11c Uncharacterized protein C22H10.11c 2 92.05 2 73.73 2 65.47 spac24b11.07c Uncharacterized protein C24B11.07c 1 74.91 ------spac24b11.12c Putative phospholipid-transporting ATPase ------1 89.48 C24B11.12c (EC 3.6.3.1) spac24c9.06c Aconitate hydratase, mitochondrial (Aconitase) (EC 10 99.58 10 99.58 8 99.58 4.2.1.3) (Citrate hydro-lyase) spac24c9.12c Probable serine hydroxymethyltransferase, cytosolic 38 99.58 48 99.58 46 99.58 (SHMT) (EC 2.1.2.1) (Glycine hydroxymethyltransferase) (Serine methylase) spac24h6.10c Putative phospho-2-dehydro-3-deoxyheptonate 8 99.58 5 99.58 7 99.58 aldolase (EC 2.5.1.54) (3-deoxy-D-arabino- heptulosonate 7-phosphate synthase) (DAHP

141

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides synthase) (Phospho-2-keto-3-deoxyheptonate aldolase) spac24h6.13 Uncharacterized membrane protein C24H6.13 1 67.74 ------spac25b8.12c Uncharacterized hydrolase C25B8.12c (EC 3.-.-.-) 3 99.58 3 99.58 3 99.58 spac25b8.17 Probable intramembrane protease C25B8.17 (EC ------1 73.56 ------3.4.23.-) spac25g10.08 Eukaryotic translation initiation factor 3 subunit B 18 99.58 20 99.58 19 99.58 (eIF3b) (Eukaryotic translation initiation factor 3 90 kDa subunit homolog) (eIF3 p90) (Translation initiation factor eIF3 p90 subunit homolog) (spPrt1) spac26f1.07 Uncharacterized oxidoreductase C26F1.07 (EC 1.-.-.-) ------1 68.28 1 72.02 spac27e2.03c Obg-like ATPase 1 4 99.58 1 98.17 2 99.58 spac27f1.06c Probable peptidyl-prolyl cis-trans isomerase C27F1.06c 2 99.58 ------1 99.58 (PPIase) (EC 5.2.1.8) (Rotamase) spac29a4.15 Serine--tRNA ligase, cytoplasmic (EC 6.1.1.11) (Seryl- 3 99.58 3 99.58 3 99.58 tRNA synthetase) (SerRS) (Seryl-tRNA(Ser/Sec) synthetase) spac30.10c Probable cysteine--tRNA ligase (EC 6.1.1.16) (Cysteinyl- 1 99.58 ------1 99.58 tRNA synthetase) (CysRS) spac30c2.04 tRNA-aminoacylation cofactor arc1 5 99.58 7 99.58 6 99.58 spac31g5.05c Ribulose-phosphate 3-epimerase (EC 5.1.3.1) 1 62.41 ------(Pentose-5-phosphate 3-epimerase) (PPE) (RPE) spac3a12.13c Probable eukaryotic translation initiation factor 3 ------1 99.58 subunit J (eIF3j) (Eukaryotic translation initiation factor 3 30 kDa subunit) (eIF-3 30 kDa) spac3g6.03c Maf-like protein C3G6.03c 1 83.08 ------spac3h1.02c Uncharacterized protein C3H1.02c 1 81.09 ------spac458.02c Uncharacterized protein C458.02c 8 99.58 8 99.58 9 99.58 spac4a8.14 Ribose-phosphate pyrophosphokinase 1 (EC 2.7.6.1) 5 99.58 4 99.58 6 99.58 (Phosphoribosyl pyrophosphate synthase 1) spac4g9.12 Probable gluconokinase (EC 2.7.1.12) (Gluconate 2 99.47 2 99.58 2 99.58 kinase) spac4h3.03c Uncharacterized protein C4H3.03c 1 61.87 ------spac4h3.04c MEMO1 family protein C4H3.04c ------1 82.23 ------spac521.04c Putative cation exchanger C521.04c 2 97.94 2 95.84 1 95.52 spac56e4.03 Aromatic amino acid aminotransferase C56E4.03 (EC 1 99.58 ------1 99.44 2.6.1.57)

142

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides spac56f8.03 Eukaryotic translation initiation factor 5B (eIF-5B) (EC 3 99.52 2 99.58 2 98.32 3.6.5.3) (Translation initiation factor IF-2) spac57a7.12 Heat shock protein 70 homolog C57A7.12 40 99.58 40 99.58 41 99.58 spac57a7.13 Uncharacterized RNA-binding protein C57A7.13 ------1 53.81 spac589.06c Uncharacterized protein C589.06c 3 99.58 2 99.58 3 99.58 spac5h10.03 Probable phosphatase SPAC5H10.03 (EC 3.1.3.-) 3 99.58 4 99.58 5 99.58 spac607.08c Uncharacterized membrane protein C6F6.13c 3 99.58 3 99.58 4 99.58 spac644.13c Protein YIP4 (YPT-interacting protein 4) ------2 99.58 ------spac694.02 Uncharacterized helicase C694.02 (EC 3.6.4.-) ------1 64.65 spac6c3.08 Ankyrin repeat-containing protein C6C3.08 1 66.83 ------spac6g10.04c Probable proteasome subunit alpha type-6 (EC 2 99.58 1 98.8 3 99.58 3.4.25.1) spac750.01 Putative aryl-alcohol dehydrogenase C750.01 (EC 4 95.95 2 95.49 3 95.89 1.1.1.-) spac9.07c Uncharacterized GTP-binding protein C9.07c 1 70.77 3 99.58 3 99.58 spac955.02c Uncharacterized protein C139.01c 2 99.58 2 99.58 1 98.85 spac9e9.09c Putative aldehyde dehydrogenase-like protein 15 99.58 20 99.58 22 99.58 C9E9.09c (EC 1.2.1.-) spacunk4.10 Putative 2-hydroxyacid dehydrogenase UNK4.10 (EC 3 99.58 1 99.58 1 99.58 1.-.-.-) spap27g11.14c Uncharacterized protein SPAP27G11.14c ------1 84.78 spapb1a10.13 Uncharacterized protein PB1A10.13 ------1 68.52 spapb1e7.01c Uncharacterized protein PB1E7.01c ------1 73.73 1 58.03 spapb24d3.08c Zinc-type alcohol dehydrogenase-like protein 4 99.58 ------2 99.58 PB24D3.08c (EC 1.-.-.-) spapj696.02 SH3 domain-containing protein PJ696.02 8 99.58 5 99.58 11 99.58 spbc119.03 Probable catechol O-methyltransferase 1 (EC 2.1.1.6) 4 99.58 ------spbc13g1.02 Probable mannose-1-phosphate guanyltransferase (EC 10 99.58 8 99.58 9 99.58 2.7.7.13) (GDP-mannose pyrophosphorylase) (GTP- mannose-1-phosphate guanylyltransferase) spbc15c4.03 Uncharacterized Rab geranylgeranyltransferase ------1 92.22 ------C15C4.03 spbc1677.03c Threonine dehydratase, mitochondrial (EC 4.3.1.19) ------1 94.54 ------(Threonine deaminase) spbc1683.04 Putative beta-glucosidase (EC 3.2.1.21) (Beta-D------1 53.3 ------glucoside glucohydrolase) (Cellobiase) (Gentiobiase) spbc16a3.08c Uncharacterized protein C16A3.08c 8 99.58 12 99.58 12 99.58

143

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides spbc16g5.05c Vesicle-associated membrane protein-associated 1 83.1 2 86.42 1 51.5 protein C16G5.05c (VAMP-associated protein C16G5.05c) (VAP homolog 1) spbc16h5.08c Uncharacterized ABC transporter ATP-binding protein 19 99.58 23 99.58 21 99.58 C16H5.08c spbc1703.03c Uncharacterized ARM-like repeat-containing protein ------1 85.17 C1703.03c spbc1711.08 Uncharacterized protein C1711.08 2 99.26 5 99.58 2 98.18 spbc19f5.04 Probable aspartokinase (EC 2.7.2.4) (Aspartate kinase) ------1 56.97 spbc19f8.03c ENTH domain-containing protein C19F8.03c 1 96.63 2 97.87 ------spbc19g7.10c DNA topoisomerase 2-associated protein pat1 ------1 87.52 1 96.88 (Decapping activator and translational repressor pat1) (Topoisomerase II-associated protein pat1) spbc21c3.15c Putative aldehyde dehydrogenase-like protein C21C3 ------1 52.48 (EC 1.2.1.-) spbc21d10.11c Probable cysteine desulfurase, mitochondrial (EC 7 99.58 5 99.58 4 99.58 2.8.1.7) spbc24c6.04 Probable delta-1-pyrroline-5-carboxylate 16 99.58 14 99.58 21 99.58 dehydrogenase (P5C dehydrogenase) (EC 1.2.1.88) (L- glutamate gamma-semialdehyde dehydrogenase) spbc25h2.16c Probable ADP-ribosylation factor-binding protein ------1 87.08 C25H2.16c spbc26h8.11c Uncharacterized protein C26H8.11c 3 99.58 1 98.85 2 99.14 spbc2d10.11c Putative nucleosome assembly protein C2D10.11C 3 99.58 3 99.58 4 99.58 spbc2g5.05 Probable transketolase (TK) (EC 2.2.1.1) 9 99.58 12 99.58 10 99.58 spbc30d10.05c Uncharacterized oxidoreductase C30D10.05c (EC 1.-.-.- 2 92.98 2 97.2 2 94.36 ) spbc32f12.10 Phosphoglucomutase (PGM) (EC 5.4.2.2) (Glucose 8 99.58 6 99.58 5 99.58 phosphomutase) spbc342.04 Uncharacterized protein C342.04 4 99.58 2 99.58 3 99.58 spbc3e7.07c Protein PBDC1 homolog ------2 96.95 1 95.4 spbc4.03c Uncharacterized protein C4.03c 1 50.95 ------spbc543.02c DnaJ homolog subfamily C member 7 homolog ------1 99.58 ------spbc582.08 Putative alanine aminotransferase (EC 2.6.1.2) ------1 98.37 2 65.12 (Glutamate pyruvate transaminase) (GPT) (Glutamic-- alanine transaminase) (Glutamic--pyruvic transaminase)

144

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides spbc660.16 6-phosphogluconate dehydrogenase, decarboxylating 14 99.58 13 99.58 16 99.58 (EC 1.1.1.44) spbc713.03 Putative D-lactate dehydrogenase C713.03, ------1 95.61 mitochondrial (EC 1.1.2.4) spbc713.09 Uncharacterized protein C713.09 1 97.74 2 97.14 ------spbc725.08 Zinc finger protein C725.08 2 99.58 2 99.58 2 99.58 spbc800.10c Uncharacterized calcium-binding protein C800.10c 1 67.44 ------1 63.38 spbc83.16c Mitochondrial outer membrane protein C83.16c ------1 98.03 ------spbc839.16 C-1-tetrahydrofolate synthase, cytoplasmic (C1-THF ------1 64.9 ------synthase) [Includes: Methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5); Methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9); Formyltetrahydrofolate synthetase (EC 6.3.4.3)] spbc8d2.18c Adenosylhomocysteinase (AdoHcyase) (EC 3.3.1.1) (S- 8 99.58 6 99.58 9 99.58 adenosyl-L-homocysteine hydrolase) spbc902.04 Uncharacterized RNA-binding protein C902.04 2 99.58 1 99.58 2 99.58 spbcpt2r1.02 Uncharacterized protein CPT2R1.02 ------1 97.37 ------spbp16f5.06 Uncharacterized RNA-binding protein P16F5.06 1 59.6 ------spbp23a10.11c Uncharacterized serine-rich protein P23A10.11c ------2 99.58 1 96.73 spbp4g3.01 Putative inorganic phosphate transporter C8E4.01c ------2 99.58 1 90.99 spbp4h10.15 Homocitrate dehydratase, mitochondrial (EC 4.2.1.-) 5 99.58 4 99.58 4 99.58 (Aconitase 2) spbpj4664.04 Putative coatomer subunit alpha (Alpha-coat protein) ------2 99.58 (Alpha-COP) spcc1223.09 Uricase (EC 1.7.3.3) (Urate oxidase) ------1 99.44 spcc1259.09c Probable pyruvate dehydrogenase protein X 2 99.58 7 99.58 1 99.58 component, mitochondrial (Dihydrolipoamide dehydrogenase-binding protein of pyruvate dehydrogenase complex) spcc1322.09 Maintenance of telomere capping protein 1 2 99.44 2 99.44 1 78.62 spcc1393.09c RWD domain-containing protein C1393.09c 1 99.58 ------spcc1494.08c Uncharacterized protein C1494.08c ------1 52.48 spcc1620.06c Ribose-phosphate pyrophosphokinase 2 (EC 2.7.6.1) 5 99.58 5 99.58 6 99.58 (Phosphoribosyl pyrophosphate synthase 2) spcc1620.08 Probable succinyl-CoA ligase [GDP-forming] subunit 2 99.07 2 97.76 2 97.52 beta, mitochondrial (EC 6.2.1.4) (Succinyl-CoA synthetase beta chain) (SCS-beta)

145

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides spcc1672.09 Probable lipase C1672.09 (EC 3.1.1.-) ------1 89.94 spcc16a11.16c Probable proteasomal ubiquitin receptor ADRM1 ------1 54.8 homolog spcc1795.05c Uridylate kinase (UK) (EC 2.7.4.14) (ATP:UMP 3 99.58 3 99.58 3 99.58 phosphotransferase) (Deoxycytidylate kinase) (CK) (dCMP kinase) (Uridine monophosphate kinase) (UMP kinase) (UMPK) spcc1827.03c Putative peroxisomal-coenzyme A synthetase (EC 6.-.- 23 99.58 22 99.58 26 99.58 .-) spcc18b5.05c Putative 1 90.87 2 68.78 1 86.93 hydroxymethylpyrimidine/phosphomethylpyrimidine kinase C18B5.05c (EC 2.7.1.49) (EC 2.7.4.7) (Hydroxymethylpyrimidine kinase) (HMP kinase) (Hydroxymethylpyrimidine phosphate kinase) (HMP-P kinase) (HMP-phosphate kinase) (HMPP kinase) spcc297.01 Chorismate synthase (EC 4.2.3.5) (5- 1 99.58 2 99.58 2 99.58 enolpyruvylshikimate-3-phosphate phospholyase) spcc320.06 Uncharacterized protein C320.06 1 86.93 ------spcc330.03c Uncharacterized heme-binding protein C330.03c 2 99.58 3 99.58 3 99.58 spcc364.06 Putative nucleosome assembly protein C364.06 9 99.58 8 99.58 7 99.58 spcc417.14c Probable acetyl-coenzyme A synthetase (EC 6.2.1.1) 3 99.58 3 99.58 3 99.58 (Acetate--CoA ligase) (Acyl-activating enzyme) spcc4g3.01 Putative D-3-phosphoglycerate dehydrogenase (3- 15 99.58 13 99.58 13 99.58 PGDH) (EC 1.1.1.95) spcc4g3.03 Uncharacterized protein C4G3.03 ------1 61.51 ------spcc550.11 Probable importin c550.11 ------1 99.58 ------spcc584.01c Probable sulfite reductase [NADPH] flavoprotein 2 99.58 5 99.58 2 99.58 component (EC 1.8.1.2) spcc63.14 Uncharacterized protein C63.14 18 99.58 16 99.58 16 99.58 spcc70.03c Probable proline dehydrogenase, mitochondrial (EC 1 98.08 1 99.58 ------1.5.5.2) (Probable proline oxidase) spcc794.11c ENTH domain-containing protein C794.11c 2 99.58 1 99.43 ------spcc825.01 Uncharacterized ABC transporter ATP-binding protein 1 98.85 1 99.58 4 99.58 C825.01 spcc965.12 Uncharacterized dipeptidase C965.12 (EC 3.4.13.19) 1 61.87 ------spcpb16a4.05c Uncharacterized urease accessory protein ureG-like 3 99.58 ------3 99.58 spi1 GTP-binding nuclear protein spi1 8 99.58 9 99.58 7 99.58

146

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides spmit.02 Uncharacterized cox1 intron-1 45.6 kDa protein 1 54.23 ------(Probable maturase) spp27 Upstream activation factor subunit spp27 (Upstream ------1 99.44 ------activation factor 27 KDa subunit) (p27) (Upstream activation factor 30 KDa subunit) (p30) (Upstream activation factor subunit uaf30) srp1 Pre-mRNA-splicing factor srp1 14 99.58 13 99.58 11 99.58 srp2 Pre-mRNA-splicing factor srp2 5 99.46 6 99.58 9 99.58 srp54 Signal recognition particle 54 kDa protein homolog 1 99.52 1 99.48 ------(SRP54) ssa1 Probable heat shock protein ssa1 53 99.58 50 99.58 52 99.58 ssa2 Probable heat shock protein ssa2 46 99.58 39 99.58 43 99.58 ssp1 Heat shock 70 kDa protein, mitochondrial 32 99.58 27 99.58 33 99.58 ssp2 SNF1-like protein kinase ssp2 (EC 2.7.11.1) ------1 99.58 ------sti1 Heat shock protein sti1 homolog 46 99.58 46 99.58 47 99.58 sty1 Mitogen-activated protein kinase sty1 (MAP kinase ------2 99.58 sty1) (EC 2.7.11.24) (MAP kinase spc1) sua1 Sulfate adenylyltransferase (EC 2.7.7.4) (ATP- 5 99.44 3 99.58 2 99.58 sulfurylase) (Sulfate adenylate transferase) (SAT) sui1 Protein translation factor sui1 4 99.58 4 99.58 5 99.58 sum1 Eukaryotic translation initiation factor 3 subunit I 11 99.58 18 99.58 15 99.58 (eIF3i) (Eukaryotic translation initiation factor 3 39 kDa subunit homolog) (eIF-3 39 kDa subunit homolog) (eIF3 p39) (Suppressor of uncontrolled mitosis 1) sum2 Protein sum2 6 99.58 7 99.58 6 99.58 sup35 Eukaryotic peptide chain release factor GTP-binding 8 99.58 8 99.58 8 99.58 subunit (ERF-3) (ERF3) (ERF2) (Polypeptide release factor 3) (Translation release factor 3) sup45 Eukaryotic peptide chain release factor subunit 1 ------1 99.58 ------(Eukaryotic release factor 1) (eRF1) swi2 Mating-type switching protein swi2 ------1 60.31 ------swo1 Heat shock protein 90 homolog 131 99.58 111 99.58 137 99.58 tal1 Transaldolase (EC 2.2.1.2) 9 99.58 6 99.58 7 99.44 tam10 Uncharacterized protein tam10 (Transcripts altered in 4 98.53 3 79.59 3 99.46 meiosis protein 10) tbp1 26S protease regulatory subunit 6A 12 99.58 12 99.58 12 99.58

147

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides tcg1 Single-stranded TG1-3 DNA-binding protein 1 99.58 2 92.43 2 99.58 (Meiotically up-regulated gene 187 protein) tdh1 Glyceraldehyde-3-phosphate dehydrogenase 1 456 99.58 458 99.58 434 99.58 (GAPDH 1) (EC 1.2.1.12) tef101 Elongation factor 1-alpha-A (EF-1-alpha-A) 308 99.58 311 99.58 273 99.58 tef102 Elongation factor 1-alpha-B (EF-1-alpha-B) 2 99.58 2 99.58 2 99.58 tef3 Elongation factor 3 (EF-3) 93 99.58 89 99.58 98 99.58 tef3 Elongation factor 1-gamma (EF-1-gamma) (eEF-1B 7 99.58 5 99.58 9 99.58 gamma) tef5 Elongation factor 1-beta (EF-1-beta) 21 99.58 20 99.58 23 99.58 thi5 Thiamine repressible genes regulatory protein thi5 ------1 53.81 ------(Transcription factor ntf1 5) thrc Threonine synthase (TS) (EC 4.2.3.1) 4 99.58 2 99.52 3 99.44 ths1 Threonine--tRNA ligase, cytoplasmic (EC 6.1.1.3) 19 99.58 16 99.58 13 99.58 (Threonyl-tRNA synthetase) (ThrRS) tif1 ATP-dependent RNA helicase eIF4A (EC 3.6.4.13) 22 99.58 16 99.58 15 99.58 (Eukaryotic initiation factor 4A) (eIF-4A) (Translation initiation factor 1) tif211 Eukaryotic translation initiation factor 2 subunit alpha 3 99.58 9 99.58 7 99.58 (eIF-2-alpha) tif212 Probable eukaryotic translation initiation factor 2 ------1 99.58 1 99.58 subunit beta (eIF-2-beta) tif213 Eukaryotic translation initiation factor 2 subunit 3 99.58 6 99.58 4 99.58 gamma (eIF-2-gamma) tif223 Probable translation initiation factor eIF-2B subunit ------2 99.58 2 99.58 gamma (eIF-2B GDP-GTP exchange factor subunit gamma) tif224 Probable translation initiation factor eIF-2B subunit 1 87.2 ------delta (eIF-2B GDP-GTP exchange factor subunit delta) tif225 Probable translation initiation factor eIF-2B subunit 7 99.58 6 99.58 7 99.58 epsilon (eIF-2B GDP-GTP exchange factor subunit epsilon) tif32 Eukaryotic translation initiation factor 3 subunit A 5 99.52 6 98.85 7 99.58 (eIF3a) (Eukaryotic translation initiation factor 3 110 kDa subunit) (eIF3 p110) (Translation initiation factor eIF3, p110 subunit)

148

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides tif35 Eukaryotic translation initiation factor 3 subunit G 15 99.58 14 99.58 12 99.58 (eIF3g) (Eukaryotic translation initiation factor 3 RNA- binding subunit) (eIF-3 RNA-binding subunit) (Translation initiation factor eIF3 p33 subunit homolog) (eIF3 p33 homolog) tif412 ATP-dependent RNA helicase fal1 (EC 3.6.4.13) 5 99.58 11 99.58 10 99.58 tif471 Eukaryotic translation initiation factor 4 gamma (eIF-4- 4 99.58 4 99.58 6 99.58 gamma) (eIF-4G) tif5 Probable eukaryotic translation initiation factor 5 (eIF- 4 99.58 5 99.58 9 99.58 5) tif51b Eukaryotic translation initiation factor 5A-2 (eIF-5A-2) 7 99.58 6 99.58 5 99.58 tif6 Eukaryotic translation initiation factor 6 (eIF-6) 3 99.58 3 99.58 4 99.58 tol1 3'(2'),5'-bisphosphate nucleotidase (EC 3.1.3.7) 2 99.58 3 99.58 ------(3'(2'),5-bisphosphonucleoside 3'(2')- phosphohydrolase) (DPNPase) (Halotolerance protein tol1) (Target of lithium protein 1) tpi1 Triosephosphate isomerase (TIM) (EC 5.3.1.1) (Triose- 3 99.44 4 98.18 3 99.17 phosphate isomerase) tpx1 Peroxiredoxin tpx1 (EC 1.11.1.15) (Peroxiredoxin tsa1) 16 99.58 18 99.58 15 99.58 (Thioredoxin peroxidase) trp1 Multifunctional tryptophan biosynthesis protein 3 99.57 3 80.64 ------[Includes: Anthranilate synthase component 2 (AS) (EC 4.1.3.27) (Anthranilate synthase, glutamine amidotransferase component); Indole-3-glycerol phosphate synthase (IGPS) (EC 4.1.1.48); N-(5'- phosphoribosyl)anthranilate isomerase (PRAI) (EC 5.3.1.24)] trp2 Tryptophan synthase (EC 4.2.1.20) 9 99.58 9 99.58 9 99.58 trp3 Probable anthranilate synthase component 1 (EC 9 99.58 5 99.58 4 99.58 4.1.3.27) (Anthranilate synthase component I) trp4 Anthranilate phosphoribosyltransferase (EC 2.4.2.18) 1 50.71 ------trr1 Thioredoxin reductase (EC 1.8.1.9) (Caffeine resistance 5 99.58 3 99.58 3 99.58 protein 4) tsr1 Ribosome biogenesis protein tsr1 1 77.13 ------tuf1 Elongation factor Tu, mitochondrial 3 99.58 6 99.58 2 99.54 tvp15 Golgi apparatus membrane protein tvp15 1 99.55 2 99.58 2 99.58

149

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides tys1 Tyrosine--tRNA ligase, cytoplasmic (EC 6.1.1.1) 2 82.35 1 68.75 3 99.58 (Tyrosyl-tRNA synthetase) (TyrRS) uap56 ATP-dependent RNA helicase uap56 (EC 3.6.4.13) 6 99.44 4 99.58 9 99.58 ubi3 Ubiquitin-40S ribosomal protein S27a [Cleaved into: 3 92.33 2 70 2 91.87 Ubiquitin; 40S ribosomal protein S27a] ubp6 Ubiquitin carboxyl-terminal hydrolase 6 (EC 3.4.19.12) 2 99.58 4 99.58 3 99.58 (Deubiquitinating enzyme 6) (Ubiquitin thiollesterase 6) (Ubiquitin-specific-processing protease 6) uep1 Ubiquitin-60S ribosomal protein L40 [Cleaved into: 2 99.58 ------Ubiquitin; 60S ribosomal protein L40 (CEP52)] ura1 Protein ura1 [Includes: Glutamine-dependent 23 99.58 31 99.58 24 99.58 carbamoyl-phosphate synthase (EC 6.3.5.5); Aspartate carbamoyltransferase (EC 2.1.3.2)] ura7 CTP synthase (EC 6.3.4.2) (CTP synthetase) (UTP-- 5 99.58 5 99.58 4 99.58 ammonia ligase) utp13 Probable U3 small nucleolar RNA-associated protein ------1 71.57 ------13 (U3 snoRNA-associated protein 13) vas2 Valine--tRNA ligase (EC 6.1.1.9) (Valyl-tRNA ------2 77.24 1 97.59 synthetase) (ValRS) vgl1 Vigilin 1 (KH domain-containing protein vgl1) 17 99.58 19 99.58 23 99.58 vip1 Protein vip1 19 99.58 16 99.58 15 99.58 vma1 V-type proton ATPase catalytic subunit A (V-ATPase 2 99.52 4 99.58 2 94.75 subunit A) (EC 3.6.3.14) (V-ATPase 67 kDa subunit) (Vacuolar proton pump subunit alpha) vma13 V-type proton ATPase subunit H (V-ATPase subunit H) 4 99.58 2 98.54 3 99.58 (V-ATPase 54 kDa subunit) (Vacuolar proton pump subunit H) vma2 V-type proton ATPase subunit B (V-ATPase subunit B) 5 99.58 7 99.58 5 99.58 (V-ATPase 57 kDa subunit) (Vacuolar proton pump subunit B) vma8 V-type proton ATPase subunit D (V-ATPase subunit D) ------1 91.19 (Vacuolar proton pump subunit D) vps1 Vacuolar protein sorting-associated protein 1 2 99.26 3 99.58 3 99.58 vps16 Probable vacuolar protein sorting-associated protein ------1 83.4 ------16 homolog vtc2 Vacuolar transporter chaperone 2 3 99.58 3 99.58 3 99.58 vtc4 Vacuolar transporter chaperone 4 ------1 87.69 1 85.7

150

Replicate 1 Replicate 2 Replicate 3 Total Total Total Prey Gene Gene Description Probability Probability Probability Number of Number of Number of Score Score Score Peptides Peptides Peptides vti1 Vesicle transport v-SNARE protein vti1 ------1 99.58 wbp1 Dolichyl-diphosphooligosaccharide--protein 2 99.58 4 99.58 3 99.58 glycosyltransferase subunit wbp1 (Oligosaccharyl transferase 48 kDa subunit) (EC 2.4.99.18) (Oligosaccharyl transferase subunit beta) win1 MAP kinase kinase kinase win1 (EC 2.7.11.25) ------1 61.34 ------wis2 40 kDa peptidyl-prolyl cis-trans isomerase (PPIase) (EC ------1 81.41 1 99.58 5.2.1.8) (Cyclophilin-40) (CYP-40) (Rotamase) wpl1 Wings apart-like protein homolog 1 2 96.26 1 96.1 2 97.33 wrs1 Tryptophan--tRNA ligase, cytoplasmic (EC 6.1.1.2) 2 99.58 ------(Tryptophanyl-tRNA synthetase) (TrpRS) ypt1 GTP-binding protein ypt1 1 99.58 ------3 99.58 ypt2 GTP-binding protein ypt2 (SEC4 homolog) 4 85.01 4 86.93 2 81.77 ypt3 GTP-binding protein ypt3 (RAB) 1 93.32 3 97.2 1 61.12 ypt5 GTP-binding protein ypt5 3 99.58 4 99.58 2 99.58 zuo1 Zuotin (DnaJ-related protein zuo1) (J protein zuo1) 11 99.58 9 99.58 9 99.58 (Ribosome-associated complex subunit zuo1)

Table A2. Prey genes identified from proteomic mass spectrometry analysis of S. pombe cells expressing Atg101-GFP in EMM and EMM-N

151

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides acp2 3 99.58 1 99.58 2 99.58 3 98.12 3 99.58 3 99.58 act1 264 99.58 238 99.58 250 99.58 160 99.58 165 99.58 208 99.58 ade1 3 99.58 4 99.58 2 99.58 1 71.4 ------2 94.84 ade1 4 99.58 5 99.58 4 99.58 6 99.58 7 99.58 4 99.58 ade3 1 99.58 ------2 99.58 1 99.44 ------2 99.58 ade6 2 92.6 3 96.27 3 97.61 2 94.76 3 97.87 3 96.23 ade9 ------1 65.65 1 78.03 ------adh1 23 99.58 24 99.58 23 99.58 21 99.58 19 99.58 20 99.58 adk1 1 76.68 ------ado1 ------1 98.93 1 99.58 2 99.1 4 99.58 2 99.58 aes1 1 83.13 1 74.6 6 99.44 2 88.7 4 99.58 4 95.67 aif1 ------1 94.1 ------air1 ------1 50.98 1 54.19 1 54.91 ala1 5 99.58 2 99.29 5 99.58 2 94.24 2 99.11 2 96.04 alg5 1 54.54 ------alp7 1 95.41 ------alr1 ------1 53.62 ------anp1 ------1 52.22 ------ape1 4 99.58 ------1 89.46 ------apt1 ------1 99.44 ------arb1 1 68.74 1 81.12 ------arf1 3 98.32 2 99.17 3 95.83 2 99.58 3 99.12 3 98.85 arg11 10 99.58 4 99.58 9 99.58 3 94.52 3 98.49 2 99.58 arg5 11 99.58 17 99.58 13 99.58 8 99.58 4 99.58 3 99.58 aro1 3 89.56 ------1 95.1 2 93.13 1 99.58 ------aro4 2 99.58 5 99.58 5 99.58 ------asn1 ------1 55.57 2 85.68 atp1 5 99.08 3 95.74 4 92.19 4 97.59 6 92.93 5 99.58 atp2 3 99.58 8 99.58 6 99.58 2 94.53 3 99.58 3 93.16

152

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides bag101 2 86.74 2 98.61 ------2 90.65 2 86.46 ------bgs4 3 60.18 1 99.58 2 96.53 1 99.24 ------bip1 1 57.66 ------2 74.53 1 96.07 bms1 1 97.46 ------btf3 ------3 99.58 2 99.58 2 99.58 4 99.58 1 94.11 bun107 ------1 70.08 ------1 99.17 but2 9 99.58 10 99.58 5 99.58 8 99.58 9 99.58 4 99.58 byr3 6 99.58 7 99.58 8 99.58 6 99.58 6 99.58 8 99.58 caf1 2 99.58 ------cam1 ------1 99.58 2 99.58 2 99.44 4 99.58 cap1 2 99.58 5 99.58 5 99.58 5 99.58 4 99.58 5 99.58 car2 4 99.58 4 99.58 4 99.58 3 99.44 2 97.22 2 94.12 cbf5 71 99.58 78 99.58 65 99.58 125 99.58 109 99.58 146 99.58 cct1 1 94.06 ------1 65.19 ------cct2 8 99.58 13 99.58 12 99.58 7 99.58 7 99.58 8 99.58 cct3 10 99.58 11 99.58 7 99.58 5 99.58 4 99.58 5 99.58 cct4 4 99.58 3 99.58 2 99.58 ------cct5 3 99.58 4 99.58 3 99.58 4 99.43 3 99.58 2 99.58 cct6 4 99.58 2 73.95 3 99.58 1 99.58 2 82.21 3 99.52 cct7 3 99.58 3 99.58 3 99.58 2 89.87 3 99.44 3 96.04 cct8 2 99.58 1 99.58 2 99.58 2 99.58 2 99.58 2 99.58 cdb4 1 61.69 ------cdc13 ------1 56.21 ------cdc15 ------1 79.96 ------2 99.58 2 99.58 2 98.18 cdc22 2 99.44 3 99.58 4 99.44 ------1 99.24 ------cdc28 ------1 57.16 ------1 64.06 ------cdc3 2 75.37 3 97.86 1 50.77 ------1 61.19 ------cdc37 ------2 59.6 cdc42 2 98.91 1 63.38 2 94.8 1 88.51 1 97.45 2 99.58

153

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides cdc48 2 99.58 2 99.58 ------2 95.24 3 98.05 3 99.25 chc1 5 99.58 4 99.58 ------3 99.58 4 99.58 4 99.58 cip2 1 52.93 ------cit1 1 99.58 3 99.58 2 99.58 ------1 91.92 clc1 ------2 99.12 4 99.58 ------3 99.58 cmb1 ------1 92.95 2 85.41 1 95.56 2 83.41 ------cmc2 ------1 68.78 ------cof1 2 99.58 2 99.58 ------2 99.58 2 99.58 2 99.58 cog8 ------2 72.09 ------cox6 1 96.73 4 81.73 1 99.52 1 77.35 ------cpy1 3 97.22 3 83.38 2 99.14 2 94.42 1 86.98 1 99.44 crn1 2 99.58 2 99.55 2 99.58 ------cta1 ------1 60.84 1 94.76 1 93.21 cut15 ------2 99.58 cut6 5 99.58 4 99.58 4 99.58 7 99.58 8 99.58 7 99.58 cwf10 ------1 53.88 ------cyp4 ------1 95.6 ------1 54.64 dak1 1 98.62 1 99.58 2 98.56 ------1 99.58 ------dbp2 7 99.58 5 99.58 9 99.58 4 99.58 7 99.45 5 99.49 dbp3 13 99.58 8 99.58 11 99.44 9 97.86 2 96.26 5 97.13 dbp5 ------1 99.29 ------ded1 20 99.58 23 99.58 19 99.58 12 99.58 14 99.58 16 99.58 dfr1 1 99.24 ------3 90.59 ------did2 ------1 80.62 2 86.15 dld1 ------2 80.35 ------dot2 ------1 50.98 ------dps1 22 99.58 29 99.58 25 99.58 25 99.58 23 99.58 24 99.58 dug1 2 99.58 2 99.58 2 99.58 2 79.48 2 99.58 ------eca39 1 99.58 ------2 99.58 2 99.58 2 99.58 2 99.58

154

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides ecm33 2 99.58 1 99.58 3 99.58 ------3 99.58 eft201; eft202 15 99.58 19 99.58 15 99.58 16 99.58 15 99.58 16 99.58 egd2 4 99.58 4 99.58 4 99.58 3 99.04 2 98.21 3 99.58 eif3f 6 99.58 6 99.58 5 99.58 1 99.58 1 76.81 2 97.46 eif3h 1 99.58 2 99.58 2 99.58 ------2 99.58 elf1 10 99.58 10 99.58 5 99.58 7 98.66 4 99.58 3 98.78 end4 9 99.58 8 99.58 8 99.58 5 99.58 6 99.58 8 99.58 eno101 56 99.58 53 99.58 49 99.58 27 99.58 35 99.58 34 99.58 eno102 1 99.45 2 99.58 2 99.58 2 99.58 3 99.58 3 99.58 ent1 ------1 98.59 ------erg10 4 99.58 8 99.58 6 99.58 5 99.58 2 98.54 3 99.58 erg6 2 99.58 2 99.58 ------fas1 1 99.58 3 99.58 2 99.58 2 99.08 ------fas2 12 99.58 10 99.58 13 99.58 4 99.58 3 95.19 4 99.58 fba1 7 99.58 6 99.58 5 99.58 2 84.98 1 99.58 1 99.58 fbp1 ------1 97.59 ------fet5 ------1 99.58 1 99.58 fib1 42 99.58 33 99.58 46 99.58 32 99.58 28 99.58 20 99.58 fim1 ------2 99.44 ------fkh1 ------2 73.95 2 90.51 1 79.69 for3 ------1 50.98 ------fyu1 29 99.58 38 99.58 26 99.58 25 99.58 24 99.58 28 99.58 gal1 ------1 99.58 2 99.44 ------1 99.58 gar1 22 99.58 21 99.58 16 99.58 19 99.58 20 99.58 35 99.58 gar2 116 99.58 122 99.58 117 99.58 140 99.58 133 99.58 133 99.58 gas1 2 99.58 2 99.58 3 99.58 2 99.58 2 99.58 1 95.97 gas5 14 99.58 18 99.58 9 99.58 7 99.58 11 99.2 11 99.58 gcn2 ------1 86.51 ------1 81.09 ------

155

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides gdh1 3 99.58 2 99.58 5 99.58 3 99.58 3 99.58 5 99.58 gef1 ------1 54.93 ------gef2 ------1 69.15 ------ght5 2 99.19 1 98.2 ------1 66.52 2 99.58 ------gld1 7 99.58 6 99.58 3 99.58 3 99.45 5 99.58 7 99.58 gln1 ------1 81.96 1 51.91 1 99.44 glt1 ------2 99.58 4 99.58 3 99.58 2 99.58 gpd1 2 99.58 2 99.58 3 99.48 2 99.58 2 99.58 2 99.58 gpd2 2 99.44 2 96.04 4 97.73 1 88.41 2 90.51 ------gpd3 37 99.58 40 99.58 34 99.58 28 99.58 32 99.58 27 99.58 gpm1 23 99.58 23 99.58 23 99.58 13 99.58 17 99.58 18 99.58 grn1 ------1 50.6 ------grs1 19 99.58 21 99.58 21 99.58 11 99.58 7 99.58 11 99.58 grx4 ------1 59.28 ------1 63.03 gua1 2 99.58 2 99.58 ------1 92.01 2 99.25 gua1 10 99.58 10 99.58 10 99.58 5 95.62 5 99.58 3 98.64 gus1 8 99.58 11 99.58 8 99.58 2 99.58 13 99.58 9 99.58 his2 1 99.4 ------2 99.58 ------1 64.16 ------his5 ------1 50.78 ------1 74.6 hob1 2 99.58 3 99.29 ------3 99.58 1 69.69 3 99.44 hob3 1 97.94 2 99.58 ------3 99.58 1 60.71 1 81.09 hsp10 3 96.39 2 99.58 3 99.44 2 99.44 3 99.58 1 97.03 hsp104 4 99.58 5 99.58 2 98.83 7 99.58 2 99.58 7 99.58 hsp16 2 98.53 2 95.89 2 97.14 2 92.13 2 98.14 2 98.85 hsp3101 ------1 98.12 ------2 97.25 hsp60 70 99.58 71 99.58 66 99.58 26 99.58 32 99.58 25 99.58 hsp78 ------2 99.58 ------1 97.82 1 96.75 ------hst2 ------1 76.07 ------htb1 2 99.04 ------1 78.43 ------

156

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides hts1 1 67.16 ------1 86.56 ------hxk1 ------1 55.77 ------hxk2 ------1 98.63 idh2 2 99.58 2 99.58 4 99.58 3 99.58 4 99.58 2 99.58 idp1 5 99.58 5 99.58 3 99.45 ------1 65.59 ------ilv1 5 99.58 8 99.58 9 99.58 4 99.58 1 97.95 1 60.53 ilv5 17 99.58 17 99.58 19 99.58 14 99.58 12 99.58 16 99.58 imp2 10 99.58 9 99.58 8 99.58 ------2 89.87 2 71.38 int6 ------1 98.85 ------irs1 ------3 99.58 2 99.04 ------1 99.58 1 97.58 isn1 6 99.58 5 99.58 7 99.58 ------2 98.85 ------iws1 ------1 85.89 ------kap114 ------2 99.58 2 99.58 ------ker1 3 99.58 2 99.58 3 98.3 1 53.28 ------3 80.23 kes1 ------1 99.58 1 99.58 1 99.58 2 99.58 kgd1 ------1 99.58 ------kri1 2 88.72 1 96.92 2 92.05 2 95.73 3 91.61 1 95.2 krs1 5 98.06 7 99.58 5 99.58 4 99.58 2 99.58 4 99.58 lat1 ------1 94.64 ------lcf1 2 99.58 ------lcf2 ------1 58.67 3 99.58 ------2 95.89 let1 5 99.58 3 99.58 4 99.58 ------2 99.58 ------leu1 3 99.58 4 99.58 ------1 99.44 leu2 13 99.58 15 99.58 12 99.58 12 99.58 17 99.58 13 99.58 lia1 ------3 99.58 3 99.58 ------lid2 ------1 53.3 ------lrs1 3 79.02 4 99.55 2 99.58 ------2 52.03 lsd90 14 99.58 17 99.58 15 99.58 21 99.58 29 99.58 29 99.58 lvs1 ------1 65.47 ------1 90.87 ------

157

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides lys1 ------1 81.09 ------2 98.53 lys12 5 99.58 3 99.58 3 99.58 3 99.58 2 99.58 3 99.58 lys4 29 99.58 27 99.58 30 99.58 21 99.58 18 99.58 21 99.58 lys9 12 99.58 12 99.58 12 99.58 11 99.58 11 99.58 9 99.58 mae2 7 99.58 3 90.93 3 99.58 4 99.58 3 99.44 1 80.09 mas5 1 92.59 3 99.58 3 99.43 ------2 97.25 mbf1 6 99.58 4 99.58 6 99.58 4 99.58 6 99.58 5 99.58 mbo1 ------1 65.47 mcs2 ------1 50.98 ------MDH1 ------2 99.58 2 99.58 4 99.58 5 99.58 2 99.58 met26 13 99.58 12 99.58 17 99.58 6 99.58 6 99.58 6 99.58 met6 1 52.48 ------meu7 ------1 51.95 mge1 2 99.58 3 97.86 4 99.58 3 96.11 1 97.06 2 96.75 mgr2 ------1 70.77 ------mic60 ------1 56.66 ------mis15 ------1 53.27 ------mlo3 2 99.55 3 99.08 1 70.77 2 99.44 2 93.94 2 94.24 mmf1 9 99.58 7 99.58 9 99.45 7 99.58 6 99.58 5 99.48 mnn9 ------2 99.58 2 99.58 mod5 ------1 60.33 moe1 1 98.82 2 98.53 2 96.15 ------1 99.19 ------mpg1 6 99.58 5 99.58 6 99.58 2 99.58 3 99.58 3 99.58 mrs1 1 99.11 ------1 73.13 ------mrt4 ------1 99.58 ------mst2 ------1 58.03 mts2 5 99.58 7 99.58 7 99.58 1 96.44 3 96.41 4 96.75 mug35 1 71.62 ------2 94.7 ------1 53.92 mug64 9 99.58 10 99.58 5 99.58 4 95.8 4 99.17 3 98.65

158

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides mug66 6 99.58 4 99.58 3 99.58 1 99.58 3 99.58 3 99.58 mug70 ------1 77.89 ------mvd1 ------1 98.12 2 99.58 1 99.58 myo2 ------1 81.08 ------nab2 ------1 51.96 ------nat10 2 99.58 2 99.58 2 99.58 2 94.48 1 91.58 ------nda2 7 99.58 5 99.58 9 99.58 5 99.58 9 99.58 11 99.58 nda3 18 99.58 12 99.58 13 99.58 6 99.58 8 99.58 7 99.58 ndk1 ------1 65.97 ------nhp2 15 99.58 13 99.58 14 99.58 9 99.58 8 99.58 8 99.58 nhp6 ------2 99.21 2 99.09 nip1 3 99.58 3 99.58 ------3 99.58 3 99.58 3 99.58 nog1 3 99.58 3 99.58 2 99.58 3 99.58 2 99.58 2 99.58 nog2 2 99.58 3 99.58 1 99.44 1 99.58 1 66.54 ------nop10 3 99.58 1 65.75 ------1 63.38 6 99.43 4 98.06 nop56 27 99.58 26 99.58 23 99.58 23 99.58 21 99.58 26 99.58 nop58 36 99.58 36 99.58 39 99.58 27 99.58 34 99.58 24 99.58 nop9 5 99.58 4 99.58 ------4 99.58 3 99.58 not1 ------2 99.44 ------nrs1 3 99.43 4 99.52 4 99.57 2 99.44 2 94.48 3 99.58 nsa2 ------1 89.71 ------1 51.92 ntp1 1 94.28 2 99.58 2 99.58 3 99.29 2 98.49 2 99.58 nup211 ------2 99.58 ------3 99.58 3 99.58 3 99.58 nup60 ------1 65.8 ------obp1 ------2 99.08 3 85.81 2 99.58 2 99.58 obr1 8 99.58 8 99.58 8 99.58 2 95.66 6 99.58 5 99.58 p23fy ------2 99.44 1 87.11 ------paa1 ------1 83.38 pab1 ------2 99.58 2 95.25 ------1 52.03 ------

159

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides pac1 2 97.05 1 98.62 1 86.98 ------1 99.58 ------pam16 ------2 97.71 ------pan1 1 99.58 2 99.58 2 99.58 3 94.65 3 98.82 3 99.58 pdb1 3 99.52 3 99.58 4 99.58 5 97.74 3 97.63 3 69.78 pdf1 ------1 72.71 ------pex11 2 99.58 2 71.37 2 98.83 ------pex14 ------2 98.17 ------2 98.24 pfk1 44 99.58 49 99.58 44 99.58 37 99.58 28 99.58 37 99.58 pgi1 8 99.58 6 99.58 6 99.58 3 99.52 4 99.36 7 99.58 pgk1 29 99.58 24 99.58 28 99.58 10 99.58 10 99.58 10 99.58 pil1 4 99.58 5 99.3 7 99.58 4 99.12 5 98.17 5 99.12 plb1 2 99.58 3 99.58 2 99.58 1 96.6 2 99.58 2 99.58 plr1 1 75.4 4 93.13 3 82.78 ------1 65.38 4 75.41 pma1 69 99.58 67 99.58 59 99.58 40 99.58 50 99.58 49 99.58 pmd1 ------1 67.56 ------pmp20 3 99.58 3 99.58 5 99.58 5 99.58 5 99.58 3 99.58 ppa1 1 79.17 2 99.58 3 99.44 3 99.58 3 99.58 2 99.58 ppi1 9 99.58 6 99.58 6 99.58 3 99.58 7 98.06 6 97.77 ppk19 ------1 54.06 1 63.99 ------1 65.47 ppk25 ------1 53.89 ------ppm2 ------1 67.82 ------ppn1 ------2 99.08 2 95.89 ppt1 9 99.58 13 99.58 12 99.58 3 99.58 8 99.58 3 99.58 pre10 ------1 96.27 ------2 98.88 ------pre6 1 73.73 ------prp19 1 84.81 2 89.36 2 94.58 ------1 94.11 1 53.79 prp43 ------1 91.42 1 75.85 ------1 71.41 prs1 10 99.58 11 99.58 10 99.58 7 99.58 6 99.58 9 99.58 pss1 4 99.58 3 99.58 ------2 99.58 1 99.44 ------

160

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides ptc3 2 97.94 3 99.58 2 96.23 2 99.58 1 98.95 4 96.74 ptr3 ------1 94.84 ------pxr1 ------1 99.58 ------2 99.52 ------pyk1 133 99.58 130 99.58 135 99.58 94 99.58 91 99.58 95 99.58 pyr1 1 99.58 4 99.58 2 99.44 1 95.79 ------qcr9 ------1 99.54 ------qrs1 3 99.58 ------rad24 17 99.58 16 99.58 9 99.58 10 99.58 16 99.58 14 99.58 rad25 8 99.58 11 99.58 4 99.58 13 99.57 7 99.58 6 99.47 rar1 1 79.4 3 99.58 3 99.58 ------1 93.86 2 99.58 ret2 ------1 90.93 ------rex4 ------1 61.34 ------rga7 ------2 99.44 ------rgf2 ------1 76.59 ------rho1 5 99.58 6 99.58 11 99.58 9 99.58 5 99.58 4 99.58 rhp16 ------1 80.75 rki1 ------1 56.93 1 95.22 ------1 65.56 ------rkp1 7 99.58 6 99.58 6 99.58 3 99.58 3 99.58 2 99.58 rnc1 ------2 99.19 3 99.58 2 99.29 2 99.58 rnp24 ------1 82.21 3 92.33 ------2 98.11 ------rpa1 19 99.58 19 99.58 19 99.58 5 99.58 7 99.58 9 99.58 rpa12 7 99.58 5 99.58 6 99.58 6 99.58 4 99.58 7 99.58 rpa2 5 99.58 8 99.58 9 99.58 5 99.58 1 94.84 7 99.58 rpa34 14 99.58 13 99.58 14 99.58 17 99.58 15 99.58 13 99.58 rpa49 ------1 99.58 ------rpc19 2 99.58 2 98.34 2 99.58 2 99.58 2 99.44 2 99.58 rpc40 3 99.58 8 99.58 3 99.58 ------5 99.58 5 99.44 rpl1002 17 99.58 14 99.58 14 99.58 8 99.58 7 99.58 7 99.58 rpl101 2 99.48 2 96.1 1 94.14 2 99.47 2 99.3 2 99.58

161

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides rpl102 1 57.95 ------rpl1101 3 99.58 6 99.58 7 99.58 5 99.58 3 99.58 3 99.58 rpl1201 3 99.58 4 99.58 3 99.58 3 99.58 3 99.58 3 99.58 rpl13 4 98.37 4 99.57 3 99.58 6 99.52 4 99.44 6 99.58 rpl14 2 99.12 3 75.19 ------2 86.48 ------rpl1502 5 99.58 3 96.16 3 97.58 1 54.98 1 65.42 1 82.78 rpl1602 3 99.58 3 99.58 4 99.58 4 99.58 3 99.58 2 99.58 rpl1701 4 99.58 2 99.58 2 99.58 2 99.58 3 99.44 2 99.58 rpl1801 3 99.58 5 99.52 4 99.58 2 99.47 5 99.58 2 99.58 rpl1802 3 99.25 4 99.44 2 99.58 2 99.58 3 99.58 2 99.58 rpl1901 7 99.58 4 98.32 3 97.99 5 99.58 5 99.58 5 99.58 rpl1902 1 95.52 ------1 95.84 1 95.52 1 95.84 ------rpl2001 11 99.58 6 99.58 5 99.58 7 99.58 5 98.06 8 99.58 rpl2101 4 99.58 4 97.7 4 99.58 7 99.58 3 99.58 5 94.64 rpl2102 1 96.16 1 53.47 ------3 94.12 1 53.81 1 94.75 rpl22 6 99.58 6 99.58 5 99.58 5 99.58 6 99.58 5 99.58 rpl2301 4 99.58 5 99.58 4 99.58 3 99.58 4 99.58 2 99.58 rpl2402 ------2 99.52 ------3 99.44 rpl2501 13 99.58 16 99.58 17 99.58 11 99.58 13 99.58 7 99.58 rpl26 4 99.58 4 99.58 6 99.44 6 99.58 8 99.58 4 99.58 rpl2701 4 99.44 2 99.58 2 99.58 1 95.57 2 97.95 ------rpl2702 2 86.74 3 99.58 4 99.58 2 99.44 5 99.44 ------rpl2801 5 99.58 5 99.58 4 99.58 6 99.58 5 99.58 2 99.58 rpl2802 1 99.58 3 99.58 3 99.58 5 99.58 3 99.58 3 99.58 rpl3001 7 99.58 5 99.58 6 99.58 2 99.58 6 99.58 4 99.58 rpl302 20 99.58 15 99.58 20 99.58 14 99.58 17 99.58 12 99.58 rpl31 2 92.91 2 85.2 2 92.98 2 96.6 2 96.75 2 90.98 rpl3402 4 97.33 6 98.82 7 99 3 89.5 5 99.08 3 99.58 rpl35 1 85.06 1 60.66 3 99.58 ------3 98.59 ------

162

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides rpl35a 1 56.8 2 58.89 1 57.21 1 62.36 1 63.03 2 67.01 rpl3601 ------2 99.58 1 75.36 ------rpl3702 5 99.58 4 99.58 5 99.58 3 99.58 5 99.58 4 99.58 rpl401 15 99.58 19 99.58 20 99.58 22 99.58 19 99.58 21 99.58 rpl402 ------2 99.58 1 99.44 2 99.58 rpl44 12 99.58 5 99.58 9 99.58 4 99.58 4 99.58 10 99.58 rpl502 3 89.5 1 99.31 ------1 66.04 2 94.55 4 88.12 rpl6 7 94.75 5 97.99 11 94.48 4 90.84 2 73.95 3 92.33 rpl701 3 99.58 2 99.58 2 99.58 3 97.67 3 99.58 3 99.58 rpl702 ------2 97.34 3 94.69 3 90.99 ------1 84.46 rpl8 11 99.58 14 99.31 9 99.58 6 99.44 6 99.44 7 99.44 rpl801 18 99.58 14 99.58 16 99.58 14 99.58 13 99.58 15 99.58 rpl902 ------1 80.75 ------rpn1 4 99.56 4 99.58 6 99.58 ------2 99.08 2 99.32 rpn11 7 99.58 9 99.58 8 99.58 2 99.17 ------3 85.09 rpn12 2 77.06 2 95.24 1 83.68 1 54.88 ------1 97.13 rpn2 ------2 98.44 2 94.28 2 78.3 2 89.88 ------rpn3 2 99.58 1 99.58 3 99.58 3 99.58 3 99.58 ------rpn501; rpn502 1 60.76 2 99.58 2 81 3 99.58 3 99.58 3 99.58 rpn6 2 96.1 2 92.16 1 90.16 ------rpn8 3 99.58 9 99.58 7 99.58 7 99.58 6 99.58 7 99.58 rpn9 3 99.12 5 99.58 5 99.58 ------3 99.58 3 99.58 rpp0 1 56.7 1 81.1 1 54.93 ------1 63.92 1 61.33 rpp101 4 99.58 3 99.58 4 99.58 ------3 91.5 rpp103 3 99.58 4 99.58 3 97.94 ------1 68.78 rps001 ------1 99.58 ------rps002 2 99.54 3 99.58 3 99.44 1 77.13 3 97.87 1 56.09 rps1001 ------3 94.75 1 67.54 2 97.27

163

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides rps1002 2 99.58 1 82.42 2 91.49 1 84.11 ------rps101 9 99.1 7 99.58 3 99.44 1 98.97 5 98.94 3 99.21 rps102 10 99.58 7 99.58 6 99.58 4 99.58 8 99.58 8 99.44 rps1101 3 86.21 5 99.58 6 99.58 6 89.75 6 94.73 4 99.58 rps1201 6 99.58 3 99.58 6 99.58 9 99.58 7 99.58 6 99.58 rps1202 1 99.58 ------3 99.58 4 99.58 2 99.58 5 99.58 rps13 8 99.58 6 99.58 8 99.58 4 98.62 6 99.58 3 99.58 rps1402 17 99.58 15 99.58 18 99.58 16 99.58 13 99.58 16 99.58 rps1502 1 67.76 1 79.05 ------1 63.95 rps1601 5 99.58 5 99.58 5 99.58 3 99.58 3 99.58 1 99.08 rps1701 4 97.89 3 99.12 5 97.14 4 98.78 3 92.66 2 98.88 rps1801 ------1 99.58 1 99.08 ------rps1901 7 99.58 5 99.58 7 99.58 7 99.58 7 99.58 6 99.58 rps1902 3 99.58 3 99.58 3 99.58 2 88.41 3 99.58 2 98.83 rps2 7 99.58 8 99.58 5 99.58 3 99.58 5 99.58 4 99.58 rps20 3 95.76 4 66 2 97.69 2 93.63 3 92.51 3 87.17 rps21 6 99.58 9 99.58 6 99.58 7 99.58 7 99.58 5 99.58 rps2201 2 93.66 ------1 99.4 1 93.92 3 96.39 rps2302 2 60.33 ------1 52.03 ------1 74.63 2 77.97 rps2402 2 70.77 3 99.29 1 79.77 1 95.57 2 99.17 ------rps2502 1 99.44 3 99.58 5 99.08 2 99.44 3 99.58 2 98.49 rps2602 ------1 98.59 1 53.88 ------2 98.37 ------rps27 2 89.25 2 97.59 3 97.45 ------rps2801 7 99.58 5 99.58 5 99.58 3 99.58 3 99.58 3 99.58 rps29 2 81.23 2 80.11 2 85.93 2 73.11 2 88.59 1 66.83 rps3 6 99.58 13 99.58 9 99.58 6 99.58 4 99.58 5 99.58 rps3001 2 92.99 ------2 88.43 ------rps403 1 99.11 1 99.58 ------rps5 9 99.58 8 99.58 8 99.58 2 85.09 3 99.58 1 85.17

164

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides rps602 9 99.58 11 99.58 11 99.58 11 99.58 16 99.58 17 99.58 rps7 6 99.3 5 99.58 3 93.35 1 84.47 3 97.69 1 92.74 rps802 5 99.58 8 99.58 7 99.58 8 99.58 11 99.58 11 99.58 rps901 ------1 87.07 1 97.76 ------rps902 1 99.58 ------rpt1 17 99.58 10 99.58 8 99.58 10 99.58 9 99.58 4 99.58 rpt3 7 99.58 9 99.58 7 99.58 3 99.58 4 99.58 3 99.58 rpt4 3 98.55 4 99.09 3 93.74 ------3 97.87 5 97.3 rrp5 ------1 54.98 1 55.57 ------rtn1 2 85.28 2 94.75 3 94.52 ------1 96.93 2 97.43 rvb1 12 99.58 11 99.58 9 99.58 10 99.58 14 99.58 12 99.58 rvb2 44 99.58 40 99.58 37 99.58 12 99.58 14 99.58 13 99.58 sal3 ------1 91.29 ------1 95.77 ------sam1 12 99.58 12 99.58 10 99.58 8 99.58 6 99.58 6 99.58 sce3 ------4 99.46 ------sdh1 ------1 94.65 1 82.99 4 99.44 sec14 ------1 69.99 1 92.53 ------sec18 ------1 99.58 1 99.58 ------1 99.44 sec231 ------2 99.58 3 99.58 3 99.58 3 95.95 3 99.58 sec72 ------1 65.5 ------sec8 ------1 52.48 1 52.48 ------sfc4 ------1 53.27 ------sgt2 13 99.58 12 99.58 9 99.58 10 99.58 12 99.58 10 99.58 shm2 ------1 97.9 2 98.36 ------shq1 9 99.58 5 99.58 6 99.58 5 99.58 6 99.58 5 99.58 sir1 3 92.6 2 94.07 ------2 92.63 2 95.3 2 98.11 skp1 ------1 98.62 2 99.58 ------sks2 69 99.58 63 99.58 59 99.58 28 99.58 31 99.58 25 99.58 sla1 2 98.93 ------2 99.58 2 99.58

165

166

167

168

169

170

171

172

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides spcc63. 06 2 94.7 2 95.25 2 89.75 2 82.81 1 63.56 1 86.13 spcc63. 14 33 99.58 28 99.58 42 99.58 37 99.58 36 99.58 48 99.58 spcc663 .09c 2 99.58 5 99.58 3 99.58 4 99.58 6 99.58 3 99.58 spcc663 .13c ------1 99.24 ------1 90.69 spcc663 .18 1 59.6 ------spcc70. 05c ------1 51.06 ------spcc794 .01c ------1 51.04 ------spcc830 .11c 4 99.58 4 99.58 5 99.58 ------spcpb16 a4.05c 1 99.58 ------1 96.44 ------spi1 2 98.14 1 83.64 ------3 80.8 1 76.79 2 78.85 spp27 ------1 75.02 ------spt7 ------1 50.98 ------srp1 3 99.58 3 98.81 3 99.43 ------1 72.82 ------srp14 2 99.58 1 99.58 2 99.58 1 99.58 3 99.58 3 99.58 srp2 1 99.58 3 99.58 3 99.58 4 99.58 3 99.58 3 99.58 ssa1 27 99.58 23 99.58 16 99.58 22 99.58 19 99.58 19 99.58 ssa2 25 99.58 16 99.58 19 99.58 17 99.58 15 99.58 17 99.58 ssp1 4 99.58 4 99.58 5 99.55 4 99.36 5 99.58 5 99.58 sti1 9 99.58 8 99.58 11 99.58 3 99.58 7 99.58 6 99.58 stt4 ------1 79.57 sty1 1 98.17 ------2 99.58 ------2 99.58 ------sua1 9 99.58 6 99.58 8 99.58 5 99.58 5 99.31 6 99.22

173

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides sum1 3 99.09 3 99.58 3 97.99 ------3 99.44 2 98.59 sup35 2 99.58 3 99.58 2 99.58 ------svf2 ------1 65.97 ------swo1 51 99.58 52 99.58 49 99.58 41 99.58 45 99.58 33 99.58 tal1 6 99.58 8 99.58 4 99.58 2 99.08 2 99.44 2 99.58 tam10 5 99.58 8 99.44 9 98.03 8 99.58 7 97.14 6 97.69 tbp1 6 99.58 6 99.58 8 99.58 5 99.58 7 99.58 5 99.44 tcg1 3 99.58 3 99.58 3 99.58 ------2 99.58 3 99.58 tdh1 417 99.58 443 99.58 456 99.58 487 99.58 468 99.58 514 99.58 tea1 ------1 85.57 ------tef101 139 99.58 151 99.58 149 99.58 141 99.58 126 99.58 130 99.58 tef3 49 99.58 51 99.58 46 99.58 46 99.58 39 99.58 43 99.58 tef3 5 99.44 5 97.5 5 99.58 2 79.45 2 85.16 1 90.98 tef5 2 99.45 8 99.58 9 99.58 6 99.44 3 99.58 4 99.58 thrc 1 61.44 ------1 74.64 2 78.03 1 91.29 ths1 5 99.58 3 99.58 3 99.56 6 99.44 4 99.44 4 99.58 tif1 5 99.58 6 99.58 4 99.24 4 98.95 3 97.55 4 98.82 tif11 3 99.58 2 99.58 2 99.58 3 99.58 4 99.58 2 97.92 tif211 6 99.58 7 99.58 6 99.58 2 98.18 5 99.58 2 97.8 tif223 2 99.46 ------2 98.37 3 97.83 2 99.58 3 99.04 tif225 1 99.58 2 99.58 1 99.58 2 99.58 3 99.58 3 99.58 tif32 4 98.66 3 98.85 4 99.58 4 98.77 1 99.1 2 98.37 tif35 7 99.58 5 99.58 4 99.58 5 99.58 5 99.58 5 99.58 tif471 2 99.58 2 99.58 3 99.54 2 94.95 3 99.44 5 99.52 tif5 2 99.58 ------3 99.58 ------tif51b ------4 99.58 2 99.58 5 99.58 1 71.19 3 99.58 tif6 4 99.44 2 99.58 3 99.18 3 99.58 2 98.56 ------tim13 1 98.11 ------1 99.28 3 99.58 2 99.58 2 99.58 tip1 ------2 95.8 ------

174

EMM EMM-N Replicate 1 Replicate 2 Replicate 3 Replicate 1 Replicate 2 Replicate 3 Total Total Total Total Total Total Number Probability Number Probability Number Probability Number Probability Number Probability Number Probability Prey of Score of Score of Score of Score of Score of Score Gene Peptides Peptides Peptides Peptides Peptides Peptides tom70 ------1 92.22 ------1 99.58 ------tpi1 4 99.58 5 99.58 5 99.58 3 99.58 3 99.58 4 99.12 tpr1 ------1 82.37 ------tpx1 4 99.58 6 99.58 5 99.58 4 99.58 6 99.58 7 99.58 trp1 3 98.43 6 99.04 5 99.58 1 78.75 ------1 56.97 trp2 ------2 99.58 2 99.58 3 99.58 2 99.58 2 99.58 trp3 ------2 99.58 ------trr1 2 99.58 3 99.58 3 99.58 4 99.58 4 99.52 3 99.58 trx1 ------2 99.58 1 94.91 ------1 72.48 trx2 1 99.58 ------tsc1 1 94.35 1 85.7 2 99.58 2 99.1 1 97.52 2 99.58 tuf1 5 99.57 4 99.58 2 99.58 ------1 91.2 tup11 ------2 99.58 tup12 ------1 80.17 ------tys1 5 99.58 2 99.58 3 99.58 3 99.58 1 99.58 3 99.58 uap56 ------1 85.18 ------1 84.07 1 99.58 ubc4 ------1 62.22 1 68.79 1 60.53 ------1 66.1 ubp16 ------1 99.58 ------ubr11 ------1 62.52 ------uep1 2 99.58 2 99.58 2 99.58 2 99.58 3 99.58 3 99.58 ura1 67 99.58 61 99.58 60 99.58 23 99.58 24 99.58 26 99.58 ura7 6 99.37 4 99.46 8 99.58 3 99.44 3 99.44 2 99.58 urg1 ------30 99.58 26 99.58 28 99.58 urg2 ------7 99 5 94.7 5 99.58 urg3 ------1 99.58 utp10 2 98.14 4 99.44 2 98.91 2 96.27 2 70.74 1 58.44 utp23 ------1 98.81 ------2 99.44 2 65.42 ------utp4 2 99.58 2 99.58 2 99.58 1 99.44 ------2 99.58 utp5 6 99.58 9 99.58 9 99.58 2 99.58 1 99.58 1 73.01

175

176

Table A3. Prey genes identified from cells grown in EMM and EMM-N with associated descriptions

Prey Gene Description aah1 Adenine deaminase (ADE) (EC 3.5.4.2) (Adenine aminohydrolase) (AAH) aah1 Alpha-amylase 1 (EC 3.2.1.1) (1,4-alpha-D-glucan glucanohydrolase) aat2 Aspartate aminotransferase, cytoplasmic (EC 2.6.1.1) (Transaminase A) ach1 Acetyl-CoA hydrolase (EC 3.1.2.1) (Acetyl-CoA deacylase) (Acetyl-CoA acylase) acp2 F-actin-capping protein subunit beta act1 Actin ade1 Bifunctional purine biosynthetic protein ADE1 [Includes: Phosphoribosylamine-- glycine ligase (EC 6.3.4.13) (Glycinamide ribonucleotide synthetase) (GARS) (Phosphoribosylglycinamide synthetase); Phosphoribosylformylglycinamidine cyclo-ligase (EC 6.3.3.1) (AIR synthase) (AIRS) (Phosphoribosyl-aminoimidazole synthetase)] ade10 Bifunctional purine biosynthesis protein ade10 [Includes: Phosphoribosylaminoimidazolecarboxamide formyltransferase (EC 2.1.2.3) (5- aminoimidazole-4-carboxamide ribonucleotide formyltransferase) (AICAR transformylase); IMP cyclohydrolase (EC 3.5.4.10) (ATIC) (IMP synthase) (Inosinicase)] ade3 Probable phosphoribosylformylglycinamidine synthase (FGAM synthase) (FGAMS) (EC 6.3.5.3) (Formylglycinamide ribonucleotide amidotransferase) (FGAR amidotransferase) (FGAR-AT) (Formylglycinamide ribotide amidotransferase) ade6 Phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) (AIR carboxylase) (AIRC) ade9 C-1-tetrahydrofolate synthase, mitochondrial (C1-THF synthase) [Includes: Methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5); Methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9); Formyltetrahydrofolate synthetase (EC 6.3.4.3)] adh1 Alcohol dehydrogenase (EC 1.1.1.1) adk1 Adenylate kinase (EC 2.7.4.3) (ATP-AMP transphosphorylase) (ATP:AMP phosphotransferase) (Adenylate kinase cytosolic and mitochondrial) (Adenylate monophosphate kinase) ado1 Adenosine kinase (EC 2.7.1.20) aes1 Antisense-enhancing sequence 1 (EC 5.1.-.-) (AES factor 1) aif1 Apoptosis-inducing factor 1 (EC 1.-.-.-) air1 Protein air1 ala1 Alanine--tRNA ligase (EC 6.1.1.7) (Alanyl-tRNA synthetase) (AlaRS) alg5 Dolichyl-phosphate beta-glucosyltransferase (DolP-glucosyltransferase) (EC 2.4.1.117) (Asparagine-linked glycosylation protein 5) alp7 Microtubule protein alp7 (Altered polarity protein 7) (Transforming acidic coiled-coil protein mia1) (TACC protein mia1) alr1 Alanine racemase, catabolic (EC 5.1.1.1) anp1 Mannan polymerase II complex anp1 subunit (M-Pol II subunit anp1) ape1 Aminopeptidase 1 (EC 3.4.11.-) (Aminopeptidase I) apt1 Adenine phosphoribosyltransferase (APRT) (EC 2.4.2.7) arb1 Argonaute-binding protein 1 arf1 ADP-ribosylation factor 1 arg11 Protein arg11, mitochondrial [Cleaved into: N-acetyl-gamma-glutamyl- phosphate reductase (EC 1.2.1.38) (N-acetyl-glutamate semialdehyde dehydrogenase) (NAGSA dehydrogenase); Acetylglutamate kinase (EC 2.7.2.8) (N-acetyl-L-glutamate 5-phosphotransferase) (NAG kinase) (AGK)] arg5 Carbamoyl-phosphate synthase arginine-specific small chain (CPS-A) (EC 6.3.5.5) (Arginine-specific carbamoyl-phosphate synthetase, glutamine chain)

177

Prey Gene Description aro1 Pentafunctional AROM polypeptide [Includes: 3-dehydroquinate synthase (DHQS) (EC 4.2.3.4); 3-phosphoshikimate 1-carboxyvinyltransferase (EC 2.5.1.19) (5-enolpyruvylshikimate-3-phosphate synthase) (EPSP synthase) (EPSPS); Shikimate kinase (SK) (EC 2.7.1.71); 3-dehydroquinate dehydratase (3- dehydroquinase) (EC 4.2.1.10); Shikimate dehydrogenase (EC 1.1.1.25)] aro4 Phospho-2-dehydro-3-deoxyheptonate aldolase, tyrosine-inhibited (EC 2.5.1.54) (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) (DAHP synthase) (Phospho-2-keto-3-deoxyheptonate aldolase) asn1 Probable asparagine synthetase [glutamine-hydrolyzing] (EC 6.3.5.4) (Glutamine-dependent asparagine synthetase) atp1 ATP synthase subunit alpha, mitochondrial atp2 ATP synthase subunit beta, mitochondrial (EC 3.6.3.14) bag101 BAG family molecular chaperone regulator 1A (BAG-1A) bgs4 1,3-beta-glucan synthase component bgs4 (EC 2.4.1.34) (1,3-beta-D-glucan-UDP glucosyltransferase) bip1 78 kDa glucose-regulated protein homolog (GRP-78) (Immunoglobulin heavy chain-binding protein homolog) (BiP) bms1 Ribosome biogenesis protein bms1 btf3 Nascent polypeptide-associated complex subunit beta (NAC-beta) (Beta-NAC) bun107 UBP9-binding protein bun107 (Binding ubp9 protein of 107 kDa) but2 Uba3-binding protein but2 byr3 Cellular nucleic acid-binding protein homolog caf1 Poly(A) ribonuclease pop2 (EC 3.1.13.4) (CCR4-associated factor 1) cam1 Calmodulin (CaM) cap1 Adenylyl cyclase-associated protein (CAP) car2 Ornithine aminotransferase car2 (EC 2.6.1.13) (Ornithine--oxo-acid aminotransferase) cbf5 H/ACA ribonucleoprotein complex subunit 4 (EC 5.4.99.-) (Centromere-binding factor 5 homolog) cct1 T-complex protein 1 subunit alpha (TCP-1-alpha) (CCT-alpha) cct2 Probable T-complex protein 1 subunit beta (TCP-1-beta) (CCT-beta) cct3 T-complex protein 1 subunit gamma (TCP-1-gamma) (CCT-gamma) cct4 T-complex protein 1 subunit delta (TCP-1-delta) (CCT-delta) cct5 T-complex protein 1 subunit epsilon (TCP-1-epsilon) (CCT-epsilon) cct6 T-complex protein 1 subunit zeta (TCP-1-zeta) (CCT-zeta) cct7 Probable T-complex protein 1 subunit eta (TCP-1-eta) (CCT-eta) cct8 Probable T-complex protein 1 subunit theta (TCP-1-theta) (CCT-theta) cdb4 Curved DNA-binding protein (42 kDa protein) cdc13 G2/mitotic-specific cyclin cdc13 cdc15 Cell division control protein 15 cdc22 Ribonucleoside-diphosphate reductase large chain (EC 1.17.4.1) (Ribonucleotide reductase) cdc28 Pre-mRNA-splicing factor ATP-dependent RNA helicase-like protein cdc28 (EC 3.6.4.13) (Pre-mRNA-processing protein 8) cdc3 Profilin cdc37 Hsp90 co-chaperone Cdc37 (Cell division control protein 37) (Hsp90 chaperone protein kinase-targeting subunit) cdc42 Cell division control protein 42 homolog (CDC42Sp) cdc48 Cell division cycle protein 48 chc1 Probable clathrin heavy chain cip2 RNA-binding post-transcriptional regulator cip2 (Csx1-interacting protein 2) cit1 Probable citrate synthase, mitochondrial (EC 2.3.3.16) clc1 Clathrin light chain (CLC) cmb1 Mismatch-binding protein cmb1 cmc2 COX assembly mitochondrial protein 2 (Cx9C motif-containing protein 2)

178

Prey Gene Description cof1 Cofilin (Actin-depolymerizing factor 1) cog8 Conserved oligomeric Golgi complex subunit 8 (COG complex subunit 8) (Component of oligomeric Golgi complex 8) cox6 Cytochrome c oxidase subunit 6, mitochondrial (Cytochrome c oxidase polypeptide VI) cpy1 Carboxypeptidase Y (CPY) (EC 3.4.16.5) crn1 Coronin-like protein crn1 cta1 Catalase (EC 1.11.1.6) cut15 Importin subunit alpha-1 (Cell untimely torn protein 15) (Karyopherin subunit alpha-1) (Serine-rich RNA polymerase I suppressor protein) cut6 Acetyl-CoA carboxylase (ACC) (EC 6.4.1.2) (Cell untimely torn protein 6) [Includes: Biotin carboxylase (EC 6.3.4.14)] cwf10 Pre-mRNA-splicing factor cwf10 (114 kDa U5 small nuclear ribonucleoprotein component homolog) (Complexed with cdc5 protein 10) cyp4 Peptidyl-prolyl cis-trans isomerase B (PPIase B) (EC 5.2.1.8) (Cyclophilin 4) (Rotamase B) dak1 Dihydroxyacetone kinase 1 (DHA kinase 1) (EC 2.7.1.28) (EC 2.7.1.29) (Glycerone kinase 1) (Triokinase 1) (Triose kinase 1) dbp2 ATP-dependent RNA helicase dbp2 (EC 3.6.4.13) (p68-like protein) dbp3 ATP-dependent RNA helicase dbp3 (EC 3.6.4.13) dbp5 ATP-dependent RNA helicase dbp5 (EC 3.6.4.13) ded1 ATP-dependent RNA helicase ded1 (EC 3.6.4.13) (Multicopy suppressor of overexpressed cyr1 protein 2) dfr1 Dihydrofolate reductase (EC 1.5.1.3) did2 Vacuolar protein-sorting-associated protein 46 (Charged multivesicular body protein 1) (Doa4-independent degradation protein 2) dld1 Dihydrolipoyl dehydrogenase, mitochondrial (EC 1.8.1.4) (Dihydrolipoamide dehydrogenase) (DLDH) dot2 Vacuolar-sorting protein dot2 (Defective organization of telomere protein 2) (ELL-associated protein of 30 kDa homolog dot2) dps1 Aspartate--tRNA ligase, cytoplasmic (EC 6.1.1.12) (Aspartyl-tRNA synthetase) (AspRS) dug1 Cys-Gly metallodipeptidase dug1 (EC 3.4.13.-) (GSH degradosomal complex subunit DUG1) eca39 Branched-chain-amino-acid aminotransferase, mitochondrial (BCAT) (EC 2.6.1.42) ecm33 Cell wall protein ecm33 eft201; eft202 Elongation factor 2 (EF-2) egd2 Nascent polypeptide-associated complex subunit alpha (NAC-alpha) (Alpha- NAC) eif3f Eukaryotic translation initiation factor 3 subunit F (eIF3f) eif3h Eukaryotic translation initiation factor 3 subunit H (eIF3h) elf1 mRNA export factor elf1 end4 Endocytosis protein end4 (SLA2 protein homolog) eno101 Enolase 1-1 (EC 4.2.1.11) (2-phospho-D-glycerate hydro-lyase 1-1) (2- phosphoglycerate dehydratase 1-1) eno102 Enolase 1-2 (EC 4.2.1.11) (2-phospho-D-glycerate hydro-lyase 1-2) (2- phosphoglycerate dehydratase 1-2) ent1 Epsin-1 erg10 Acetyl-CoA acetyltransferase (EC 2.3.1.9) (Acetoacetyl-CoA thiolase) (Ergosterol biosynthesis protein 10) erg6 Sterol 24-C-methyltransferase erg6 (EC 2.1.1.41) (Delta(24)-sterol C- methyltransferase erg6) (Ergosterol biosynthesis protein 6) fas1 Fatty acid synthase subunit beta (EC 2.3.1.86) [Includes: 3-hydroxyacyl-[acyl- carrier-protein] dehydratase (EC 4.2.1.59); Enoyl-[acyl-carrier-protein] reductase [NADH] (EC 1.3.1.9); [Acyl-carrier-protein] acetyltransferase (EC 2.3.1.38); [Acyl-

179

Prey Gene Description carrier-protein] malonyltransferase (EC 2.3.1.39); S-acyl fatty acid synthase thioesterase (EC 3.1.2.14)] fas2 Fatty acid synthase subunit alpha (EC 2.3.1.86) (p190/210) [Includes: Acyl carrier; 3-oxoacyl-[acyl-carrier-protein] reductase (EC 1.1.1.100) (Beta-ketoacyl reductase); 3-oxoacyl-[acyl-carrier-protein] synthase (EC 2.3.1.41) (Beta- ketoacyl synthase)] fba1 Fructose-bisphosphate aldolase (FBP aldolase) (FBPA) (EC 4.1.2.13) (Fructose- 1,6-bisphosphate aldolase) fbp1 Fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11) (D-fructose-1,6- bisphosphate 1-phosphohydrolase) fet5 GPN-loop GTPase 3 (EC 3.6.5.-) (Factor of eukaryotic transcription 5) fib1 rRNA 2'-O-methyltransferase fibrillarin (EC 2.1.1.-) (Histone-glutamine methyltransferase) fim1 Fimbrin fkh1 Peptidyl-prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) (FK506-binding protein) (FKBP) for3 Formin-3 fyu1 Probable UTP--glucose-1-phosphate uridylyltransferase (EC 2.7.7.9) (UDP- glucose pyrophosphorylase) (UDPGP) (UGPase) gal1 Galactokinase (EC 2.7.1.6) (Galactose kinase) gar1 H/ACA ribonucleoprotein complex subunit 1 (snoRNP protein GAR1) gar2 Protein gar2 gas1 1,3-beta-glucanosyltransferase gas1 (EC 2.4.1.-) gas5 1,3-beta-glucanosyltransferase gas5 gcn2 eIF-2-alpha kinase GCN2 (Serine/threonine-protein kinase gcn2) (EC 2.7.11.1) (Serine/threonine-protein kinase ppk28) gdh1 NADP-specific glutamate dehydrogenase (NADP-GDH) (EC 1.4.1.4) (NADP- dependent glutamate dehydrogenase) gef1 Rho guanine nucleotide exchange factor gef1 gef2 Rho guanine nucleotide exchange factor gef2 ght5 High-affinity glucose transporter ght5 (Hexose transporter 5) gld1 Glycerol dehydrogenase 1 (GDH) (GLDH) (EC 1.1.1.6) gln1 Glutamine synthetase (GS) (EC 6.3.1.2) (Glutamate--ammonia ligase) glt1 Putative glutamate synthase [NADPH] (EC 1.4.1.13) (NADPH-GOGAT) gpd1 Glycerol-3-phosphate dehydrogenase [NAD(+)] 1 (EC 1.1.1.8) (GPDH-C) (GPD-C) gpd2 Glycerol-3-phosphate dehydrogenase [NAD(+)] 2 (EC 1.1.1.8) gpd3 Glyceraldehyde-3-phosphate dehydrogenase 2 (GAPDH 2) (EC 1.2.1.12) gpm1 Phosphoglycerate mutase (PGAM) (EC 5.4.2.11) (BPG-dependent PGAM) (MPGM) (Phosphoglyceromutase) grn1 GTPase grn1 (GTPase in ribosomal export from the nucleolus protein 1) (Nuclear GTP-binding protein grn1) grs1 Putative glycine--tRNA ligase (EC 6.1.1.14) (Diadenosine tetraphosphate synthetase) (AP-4-A synthetase) (Glycyl-tRNA synthetase) (GlyRS) grx4 Monothiol glutaredoxin-4 gua1 GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2) (GMP synthetase) (Glutamine amidotransferase) gua1 Inosine-5'-monophosphate dehydrogenase (IMP dehydrogenase) (IMPD) (IMPDH) (EC 1.1.1.205) gus1 Probable glutamate--tRNA ligase, cytoplasmic (EC 6.1.1.17) (Glutamyl-tRNA synthetase) (GluRS) his2 Histidinol dehydrogenase (HDH) (EC 1.1.1.23) his5 Imidazoleglycerol-phosphate dehydratase (IGPD) (EC 4.2.1.19) hob1 Protein hob1 (Homolog of Bin1) hob3 Protein hob3 (Homolog of Bin3) hsp10 10 kDa heat shock protein, mitochondrial (HSP10) (10 kDa chaperonin) hsp104 Heat shock protein 104 (Protein aggregation-remodeling factor hsp104)

180

Prey Gene Description hsp16 Heat shock protein 16 (16 kDa heat shock protein) hsp3101 Glutathione-independent glyoxalase hsp3101 (EC 4.2.1.130) (Glyoxalase 3 homolog 1) (Heat shock protein 31 homolog 1) hsp60 Heat shock protein 60, mitochondrial (HSP60) hsp78 Heat shock protein 78, mitochondrial hst2 NAD-dependent protein deacetylase hst2 (EC 3.5.1.-) (Homologous to sir2 protein 2) (Regulatory protein SIR2 homolog 2) htb1 Histone H2B-alpha (H2B.1) hts1 Histidine--tRNA ligase, mitochondrial (EC 6.1.1.21) (Histidyl-tRNA synthetase) (HisRS) hxk1 Hexokinase-1 (EC 2.7.1.1) hxk2 Hexokinase-2 (EC 2.7.1.1) idh2 Isocitrate dehydrogenase [NAD] subunit 2, mitochondrial (EC 1.1.1.41) (Isocitric dehydrogenase) (NAD(+)-specific ICDH) idp1 Probable isocitrate dehydrogenase [NADP], mitochondrial (IDH) (EC 1.1.1.42) (IDP) (NADP(+)-specific ICDH) (Oxalosuccinate decarboxylase) ilv1 Acetolactate synthase, mitochondrial (EC 2.2.1.6) (AHAS) (ALS) (Acetohydroxy- acid synthase) ilv5 Probable ketol-acid reductoisomerase, mitochondrial (EC 1.1.1.86) (Acetohydroxy-acid reductoisomerase) (Alpha-keto-beta-hydroxylacyl reductoisomerase) imp2 Septation protein imp2 int6 Eukaryotic translation initiation factor 3 subunit E (eIF3e) irs1 Isoleucine--tRNA ligase, cytoplasmic (EC 6.1.1.5) (Isoleucyl-tRNA synthetase) (IleRS) isn1 IMP-specific 5'-nucleotidase 1 (EC 3.1.3.-) iws1 Transcription factor iws1 kap114 Importin subunit beta-5 (114 kDa karyopherin) (Karyopherin subunit beta-5) (Karyopherin-114) ker1 DNA-directed RNA polymerase I subunit rpa14 (RNA polymerase I subunit A14) (DNA-directed RNA polymerase I 17 kDa polypeptide) (Nucleolar protein ker1) kes1 Protein kes1 kgd1 2-oxoglutarate dehydrogenase, mitochondrial (EC 1.2.4.2) (2-oxoglutarate dehydrogenase complex component E1) (OGDC-E1) (Alpha-ketoglutarate dehydrogenase) kri1 Protein kri1 krs1 Lysine--tRNA ligase, cytoplasmic (EC 6.1.1.6) (Lysyl-tRNA synthetase) (LysRS) lat1 Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (EC 2.3.1.12) (Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex) (Pyruvate dehydrogenase complex component E2) (PDC-E2) (PDCE2) lcf1 Long-chain-fatty-acid--CoA ligase 1 (EC 6.2.1.3) (Fatty acid activator 1) (Long- chain acyl-CoA synthetase 1) lcf2 Long-chain-fatty-acid--CoA ligase 2 (EC 6.2.1.3) (Fatty acid activator 2) (Long- chain acyl-CoA synthetase 2) let1 26S protease regulatory subunit 8 homolog (Protein let1) leu1 3-isopropylmalate dehydrogenase (3-IPM-DH) (IMDH) (EC 1.1.1.85) (Beta-IPM dehydrogenase) leu2 3-isopropylmalate dehydratase (EC 4.2.1.33) (Alpha-IPM isomerase) (IPMI) (Isopropylmalate isomerase) lia1 Deoxyhypusine hydroxylase (DOHH) (EC 1.14.99.29) (Deoxyhypusine dioxygenase) (Deoxyhypusine monooxygenase) lid2 Lid2 complex component lid2 (Lid2C component lid2) lrs1 Putative leucine--tRNA ligase, cytoplasmic (EC 6.1.1.4) (Leucyl-tRNA synthetase) (LeuRS) lsd90 Protein lsd90 (90kDa large and small daughter protein)

181

Prey Gene Description lvs1 Beige protein homolog 1 lys1 L-2-aminoadipate reductase (EC 1.2.1.31) (EC 1.2.1.95) (Alpha-aminoadipate reductase) (Alpha-AR) (L-aminoadipate-semialdehyde dehydrogenase) lys12 Homoisocitrate dehydrogenase (HICDH) (EC 1.1.1.87) lys4 Homocitrate synthase, mitochondrial (EC 2.3.3.14) lys9 Saccharopine dehydrogenase [NADP(+), L-glutamate-forming] (EC 1.5.1.10) (Saccharopine reductase) mae2 NAD-dependent malic enzyme (NAD-ME) (EC 1.1.1.38) mas5 Mitochondrial protein import protein mas5 mbf1 Multiprotein-bridging factor 1 mbo1 Microtubule organizer protein 1 (Morphology defective protein 20) mcs2 Cyclin mcs2 (Mitotic catastrophe suppressor 2) MDH1 Malate dehydrogenase, mitochondrial (EC 1.1.1.37) met26 Probable 5-methyltetrahydropteroyltriglutamate--homocysteine methyltransferase (EC 2.1.1.14) (Cobalamin-independent methionine synthase) (Methionine synthase, vitamin-B12 independent isozyme) met6 Homoserine O-acetyltransferase (EC 2.3.1.31) (Homoserine O-trans-acetylase) meu7 Alpha-amylase 4 (EC 3.2.1.1) (1,4-alpha-D-glucan glucanohydrolase) (Meiotic expression up-regulated protein 7) mge1 GrpE protein homolog, mitochondrial mgr2 Protein mgr2 mic60 MICOS complex subunit mic60 (Mitofilin) mis15 Kinetochore protein mis15 (Sim4 complex subunit mis15) mlo3 mRNA export protein mlo3 (RNA-annealing protein mlo3) mmf1 Protein mmf1, mitochondrial (Isoleucine biosynthesis and maintenance of intact mitochondria 1) (Maintenance of mitochondrial function 1) mnn9 Mannan polymerase complex subunit mnn9 mod5 Cell polarity protein mod5 (Tea1-anchoring protein mod5) moe1 Eukaryotic translation initiation factor 3 subunit D (eIF3d) (Microtubule- destabilizing protein moe1) mpg1 Mannose-1-phosphate guanyltransferase (EC 2.7.7.13) (GDP-mannose pyrophosphorylase) (GTP-mannose-1-phosphate guanylyltransferase) mrs1 Probable arginine--tRNA ligase, cytoplasmic (EC 6.1.1.19) (Arginyl-tRNA synthetase) (ArgRS) mrt4 Ribosome assembly factor mrt4 (mRNA turnover protein 4) mst2 Histone acetyltransferase mst2 (EC 2.3.1.48) mts2 26S protease regulatory subunit 4 homolog (Protein mts2) mug35 Meiotically up-regulated gene 35 protein mug64 Meiotically up-regulated gene 64 protein mug66 Meiotically up-regulated gene 66 protein (Autophagy-related protein 101) mug70 Meiotically up-regulated gene 70 protein mvd1 Diphosphomevalonate decarboxylase (EC 4.1.1.33) (Mevalonate pyrophosphate decarboxylase) (Mevalonate-5-diphosphate decarboxylase) (MDDase) myo2 Myosin type-2 heavy chain 1 (Myosin type II heavy chain 1) nab2 Nuclear polyadenylated RNA-binding protein nab2 nat10 RNA cytidine acetyltransferase (EC 2.3.1.-) (18S rRNA cytosine acetyltransferase) nda2 Tubulin alpha-1 chain nda3 Tubulin beta chain (Beta-tubulin) ndk1 Nucleoside diphosphate kinase (NDK) (NDP kinase) (EC 2.7.4.6) nhp2 H/ACA ribonucleoprotein complex subunit 2 (H/ACA snoRNP protein NHP2) (High mobility group-like nuclear protein 2) (P17-nhp2) nhp6 Non-histone chromosomal protein 6 nip1 Eukaryotic translation initiation factor 3 subunit C (eIF3c) (Eukaryotic translation initiation factor 3 93 kDa subunit homolog) (eIF3 p93) (Translation initiation factor eIF3, p93 subunit homolog)

182

Prey Gene Description nog1 Probable nucleolar GTP-binding protein 1 nog2 Nucleolar GTP-binding protein 2 nop10 H/ACA ribonucleoprotein complex subunit 3 (Nucleolar protein 10) (Nucleolar protein family A member 3) (snoRNP protein nop10) nop56 Nucleolar protein 56 (Ribosome biosynthesis protein sik1) nop58 Nucleolar protein 58 nop9 Nucleolar protein 9 (Pumilio domain-containing protein nop9) not1 General negative regulator of transcription subunit 1 nrs1 Probable asparagine--tRNA ligase, cytoplasmic (EC 6.1.1.22) (Asparaginyl-tRNA synthetase) (AsnRS) nsa2 Ribosome biogenesis protein nsa2 ntp1 Neutral trehalase (EC 3.2.1.28) (Alpha,alpha-trehalase) (Alpha,alpha-trehalose glucohydrolase) nup211 Nucleoporin nup211 (Nuclear pore protein nup211) nup60 Nucleoporin nup60 (Nuclear pore protein nup60) obp1 Oxysterol-binding protein-like protein 1 obr1 P25 protein (Brefeldin A resistance protein) p23fy Translationally-controlled tumor protein homolog (TCTP) (p23fyp) paa1 Protein phosphatase PP2A regulatory subunit A (Protein phosphatase 2A 65 kDa regulatory subunit) (PR65) pab1 Polyadenylate-binding protein, cytoplasmic and nuclear (PABP) (Poly(A)-binding protein) (Polyadenylate tail-binding protein) pac1 Double-strand-specific pac1 ribonuclease (EC 3.1.26.3) (Protein hcs) pam16 Mitochondrial import inner membrane translocase subunit tim16 (Presequence translocated-associated motor subunit pam16) pan1 Actin cytoskeleton-regulatory complex protein pan1 pdb1 Pyruvate dehydrogenase E1 component subunit beta, mitochondrial (PDHE1-B) (EC 1.2.4.1) pdf1 Palmitoyl-protein thioesterase-dolichyl pyrophosphate phosphatase fusion 1 [Cleaved into: Palmitoyl-protein thioesterase (PPT) (EC 3.1.2.22) (Palmitoyl- protein hydrolase); Dolichyldiphosphatase (EC 3.6.1.43) (Dolichyl pyrophosphate phosphatase)] pex11 Peroxisomal biogenesis factor 11 pex14 Peroxisomal membrane protein pex14 (Peroxin-14) pfk1 ATP-dependent 6-phosphofructokinase (ATP-PFK) (Phosphofructokinase) (EC 2.7.1.11) (Phosphohexokinase) pgi1 Glucose-6-phosphate isomerase (GPI) (EC 5.3.1.9) (Phosphoglucose isomerase) (PGI) (Phosphohexose isomerase) (PHI) pgk1 Phosphoglycerate kinase (EC 2.7.2.3) pil1 Probable sphingolipid long chain base-responsive protein pil1 (Protein kinase inhibitor pil1) plb1 Lysophospholipase 1 (EC 3.1.1.5) (Phospholipase B 1) plr1 Pyridoxal reductase (PL reductase) (PL-red) (EC 1.1.1.65) pma1 Plasma membrane ATPase 1 (EC 3.6.3.6) (Proton pump 1) pmd1 Leptomycin B resistance protein pmd1 pmp20 Putative peroxiredoxin pmp20 (EC 1.11.1.15) (Peroxisomal membrane protein pmp20) (Thioredoxin reductase) ppa1 Inorganic pyrophosphatase (EC 3.6.1.1) (Pyrophosphate phospho-hydrolase) (PPase) ppi1 Peptidyl-prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) (Cyclophilin) (CPH) (Cyclosporin A-binding protein) (Rotamase) ppk19 Serine/threonine-protein kinase ppk19 (EC 2.7.11.1) ppk25 Serine/threonine-protein kinase ppk25 (EC 2.7.11.1) ppm2 tRNA wybutosine-synthesizing protein 4 (tRNA-yW synthesizing protein 4) (EC 2.1.1.290) (EC 2.3.1.231) (Leucine carboxyl methyltransferase 2) (tRNA(Phe) (7- (3-amino-3-(methoxycarbonyl)propyl)wyosine(37)-N)-

183

Prey Gene Description methoxycarbonyltransferase) (tRNA(Phe) (7-(3-amino-3- carboxypropyl)wyosine(37)-O)-methyltransferase) ppn1 Endopolyphosphatase (EC 3.6.1.10) ppt1 Serine/threonine-protein phosphatase T (PPT) (EC 3.1.3.16) pre10 Probable proteasome subunit alpha type-7 (EC 3.4.25.1) pre6 Probable proteasome subunit alpha type-4 (EC 3.4.25.1) prp19 Pre-mRNA-processing factor 19 (EC 6.3.2.-) (Complexed with cdc5 protein 8) prp43 Probable pre-mRNA-splicing factor ATP-dependent RNA helicase prp43 (EC 3.6.4.13) prs1 Putative proline--tRNA ligase C19C7.06 (EC 6.1.1.15) (Prolyl-tRNA synthetase) (ProRS) pss1 Heat shock protein homolog pss1 ptc3 Protein phosphatase 2C homolog 3 (PP2C-3) (EC 3.1.3.16) ptr3 Ubiquitin-activating enzyme E1 1 (EC 6.2.1.45) (Poly(A)+ RNA transport protein 3) pxr1 Protein pxr1 (PinX1-related protein 1) pyk1 Pyruvate kinase (PK) (EC 2.7.1.40) pyr1 Pyruvate carboxylase (EC 6.4.1.1) (Pyruvic carboxylase) (PCB) qcr9 Cytochrome b-c1 complex subunit 9 (Complex III subunit 9) (Cytochrome c1 non-heme 7.3 kDa protein) (Ubiquinol-cytochrome c reductase complex 7.3 kDa protein) qrs1 Probable glutamine--tRNA ligase (EC 6.1.1.18) (Glutaminyl-tRNA synthetase) (GlnRS) rad24 DNA damage checkpoint protein rad24 rad25 DNA damage checkpoint protein rad25 rar1 Probable methionine--tRNA ligase, cytoplasmic (EC 6.1.1.10) (Methionyl-tRNA synthetase) (MetRS) ret2 Coatomer subunit delta (Delta-coat protein) (Delta-COP) rex4 RNA exonuclease 4 (EC 3.1.-.-) rga7 Probable Rho-GTPase-activating protein 7 rgf2 Rho1 guanine nucleotide exchange factor 2 rho1 GTP-binding protein rho1 rhp16 ATP-dependent helicase rhp16 (EC 3.6.4.-) (DNA repair protein rhp16) (RAD16 homolog) rki1 Ribose-5-phosphate isomerase (EC 5.3.1.6) (D-ribose-5-phosphate ketol- isomerase) (Phosphoriboisomerase) rkp1 Guanine nucleotide-binding protein subunit beta-like protein (Receptor of activated protein kinase C) rnc1 RNA-binding protein rnc1 (RNA-binding protein that suppresses calcineurin deletion 1) rnp24 RNA-binding protein rnp24 rpa1 DNA-directed RNA polymerase I subunit rpa1 (EC 2.7.7.6) (DNA-directed RNA polymerase I 190 kDa polypeptide) (DNA-directed RNA polymerase I largest subunit) rpa12 DNA-directed RNA polymerase I subunit RPA12 (DNA-directed RNA polymerase I 13.1 kDa polypeptide) rpa2 Probable DNA-directed RNA polymerase I subunit RPA2 (EC 2.7.7.6) (DNA- directed RNA polymerase I polypeptide 2) (RNA polymerase I subunit 2) rpa34 DNA-directed RNA polymerase I subunit rpa34 (RNA polymerase I subunit A34) rpa49 DNA-directed RNA polymerase I subunit rpa49 (RNA polymerase I subunit A49) (DNA-directed RNA polymerase I 49 kDa polypeptide) rpc19 DNA-directed RNA polymerases I and III subunit RPAC2 (RNA polymerases I and III subunit AC2) (AC19) (DNA-directed RNA polymerases I and III 14 kDa polypeptide)

184

Prey Gene Description rpc40 DNA-directed RNA polymerases I and III subunit RPAC1 (RNA polymerases I and III subunit AC1) (DNA-directed RNA polymerases I and III 40 kDa polypeptide) (AC40) (RPC39) rpl1002 60S ribosomal protein L10-B rpl101 60S ribosomal protein L1-B (L10a) rpl102 60S ribosomal protein L1-A (L10a) rpl1101 60S ribosomal protein L11-A rpl1201 60S ribosomal protein L12-A rpl13 60S ribosomal protein L13 rpl14 60S ribosomal protein L14 rpl1502 60S ribosomal protein L15-B rpl1602 60S ribosomal protein L16-A rpl1701 60S ribosomal protein L17-A rpl1801 60S ribosomal protein L18-A rpl1802 60S ribosomal protein L18-B rpl1901 60S ribosomal protein L19-A (YL15) rpl1902 60S ribosomal protein L19-B rpl2001 60S ribosomal protein L20-A (YL17) rpl2101 60S ribosomal protein L21-A rpl2102 60S ribosomal protein L21-B rpl22 60S ribosomal protein L22 rpl2301 60S ribosomal protein L23-A rpl2402 60S ribosomal protein L24-B rpl2501 60S ribosomal protein L25-A rpl26 60S ribosomal protein L26 rpl2701 60S ribosomal protein L27-A rpl2702 60S ribosomal protein L27-B rpl2801 60S ribosomal protein L28-B rpl2802 60S ribosomal protein L28-A (L27A) (L29) rpl3001 60S ribosomal protein L30-1 (L32) rpl302 60S ribosomal protein L3-B rpl31 60S ribosomal protein L31 rpl3402 60S ribosomal protein L34-B (60S ribosomal protein L34-2) rpl35 60S ribosomal protein L35 rpl35a 60S ribosomal protein L33-B (L37B) rpl3601 60S ribosomal protein L36-A rpl3702 60S ribosomal protein L37-B (L37-2) (YL27) rpl401 60S ribosomal protein L4-B rpl402 60S ribosomal protein L4-A (L2) rpl44 Probable 60S ribosomal protein L28e rpl502 60S ribosomal protein L5-B rpl6 60S ribosomal protein L6 rpl701 60S ribosomal protein L7-C rpl702 60S ribosomal protein L7-B rpl8 60S ribosomal protein L8 (L4) (L7A) rpl801 60S ribosomal protein L2-A (K37) (K5) (KD4) rpl902 60S ribosomal protein L9-B rpn1 26S proteasome regulatory subunit rpn1 (19S regulatory cap region of 26S protease subunit 2) (Proteasome non-ATPase subunit mts4) rpn11 26S proteasome regulatory subunit rpn11 (Protein pad1) rpn12 26S proteasome regulatory subunit rpn12 rpn2 26S proteasome regulatory subunit rpn2 rpn3 Probable 26S proteasome regulatory subunit rpn3 rpn501; rpn502 26S proteasome regulatory subunit rpn5 rpn6 Probable 26S proteasome regulatory subunit rpn6

185

Prey Gene Description rpn8 26S proteasome regulatory subunit rpn8 rpn9 Probable 26S proteasome regulatory subunit rpn9 rpp0 60S acidic ribosomal protein P0 rpp101 60S acidic ribosomal protein P1-alpha 1 (A1) rpp103 60S acidic ribosomal protein P1-alpha 5 rps001 40S ribosomal protein S0-A rps002 40S ribosomal protein S0-B rps1001 40S ribosomal protein S10-A rps1002 40S ribosomal protein S10-B rps101 40S ribosomal protein S1-A (S3aE-A) rps102 40S ribosomal protein S1-B (S3aE-B) rps1101 40S ribosomal protein S11-A rps1201 40S ribosomal protein S12-A rps1202 40S ribosomal protein S12-B rps13 40S ribosomal protein S13 rps1402 40S ribosomal protein S14-B rps1502 40S ribosomal protein S15-B rps1601 40S ribosomal protein S16-A rps1701 40S ribosomal protein S17-A rps1801 40S ribosomal protein S18-A rps1901 40S ribosomal protein S19-A (S16-A) rps1902 40S ribosomal protein S19-B (S16-B) rps2 40S ribosomal protein S2 rps20 40S ribosomal protein S20 rps21 40S ribosomal protein S21 (S28) rps2201 40S ribosomal protein S22-A rps2302 40S ribosomal protein S23-B rps2402 40S ribosomal protein S24-B rps2502 40S ribosomal protein S25-A (S31-A) rps2602 40S ribosomal protein S26-B rps27 40S ribosomal protein S27 rps2801 40S ribosomal protein S28-A (S33) rps29 40S ribosomal protein S29 rps3 40S ribosomal protein S3 rps3001 40S ribosomal protein S30-A rps403 40S ribosomal protein S4-C rps5 40S ribosomal protein S5-A rps602 40S ribosomal protein S6-B rps7 40S ribosomal protein S7 rps802 40S ribosomal protein S8-B rps901 40S ribosomal protein S9-A rps902 40S ribosomal protein S9-B rpt1 26S protease regulatory subunit 7 homolog rpt3 26S protease regulatory subunit 6B homolog rpt4 Probable 26S protease subunit rpt4 rrp5 rRNA biogenesis protein rrp5 (Ribosomal RNA-processing protein 5) (U3 small nucleolar RNA-associated protein rrp5) (U3 snoRNA-associated protein rrp5) rtn1 Reticulon-like protein 1 (Cell lysis protein cwl1) rvb1 RuvB-like helicase 1 (EC 3.6.4.12) rvb2 RuvB-like helicase 2 (EC 3.6.4.12) sal3 Importin subunit beta-3 (Importin beta sal3) sam1 S-adenosylmethionine synthase (AdoMet synthase) (EC 2.5.1.6) (Methionine adenosyltransferase) (MAT) sce3 Probable RNA-binding protein sce3

186

Prey Gene Description sdh1 Probable succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial (EC 1.3.5.1) (Flavoprotein subunit of complex II) (FP) sec14 Sec14 cytosolic factor (Phosphatidylinositol/phosphatidyl-choline transfer protein) (PI/PC TP) (Sporulation-specific protein 20) sec18 Vesicular-fusion protein sec18 sec231 Protein transport protein sec23-1 sec72 Protein transport protein sec72 sec8 Exocyst complex component sec8 sfc4 Transcription factor tau subunit sfc4 (TFIIIC subunit sfc4) (Transcription factor C subunit 4) sgt2 Small glutamine-rich tetratricopeptide repeat-containing protein 2 shm2 Serine hydroxymethyltransferase, mitochondrial (SHMT) (EC 2.1.2.1) (Glycine hydroxymethyltransferase) (Serine methylase) shq1 Protein shq1 sir1 Sulfite reductase [NADPH] subunit beta (EC 1.8.1.2) skp1 Suppressor of kinetochore protein 1 (P19/Skp1 homolog) sks2 Heat shock protein sks2 (Heat shock cognate protein hsc1) sla1 Actin cytoskeleton-regulatory complex protein sla1 sla1 La protein homolog (La autoantigen homolog) (La ribonucleoprotein) slm1 Cytoskeletal signaling protein slm1 slp1 WD repeat-containing protein slp1 slt1 Protein slt1 sly1 Protein sly1 snf22 SWI/SNF chromatin-remodeling complex subunit snf22 (EC 3.6.4.-) (ATP- dependent helicase snf22) (SWI/SNF complex subunit snf22) snf7 Vacuolar-sorting protein snf7 (Vacuolar protein-sorting-associated protein 32) snz1 Probable pyridoxal 5'-phosphate synthase subunit PDX1 (PLP synthase subunit PDX1) (EC 4.3.3.6) sod1 Superoxide dismutase [Cu-Zn] (EC 1.15.1.1) sod2 Superoxide dismutase [Mn], mitochondrial (EC 1.15.1.1) sof1 Protein sof1 (U3 small nucleolar RNA-associated protein sof1) (U3 snoRNA- associated protein sof1) spac10f6.17c Protein phosphatase 2C homolog C10F6.17c (EC 3.1.3.16) (Pyruvate dehydrogenase (Lipoamide) phosphatase C10F6.17c) spac11d3.02c UPF0039 protein C11D3.02c spac11d3.11c Uncharacterized transcriptional regulatory protein C11D3.11c spac13c5.05c Phosphoacetylglucosamine mutase 1 (PAGM) (EC 5.4.2.3) (Acetylglucosamine phosphomutase) (N-acetylglucosamine-phosphate mutase) spac144.05 Uncharacterized ATP-dependent helicase C144.05 (EC 3.6.4.-) spac1565.05 Uncharacterized protein C1565.05 spac15e1.04 Probable thymidylate synthase (TS) (TSase) (EC 2.1.1.45) spac16e8.13 RING finger protein ETP1 homolog (BRAP2 homolog) spac17h9.07 Uncharacterized protein C17H9.07 spac17h9.14c Protein disulfide-isomerase C17H9.14c (EC 5.3.4.1) spac186.04c Uncharacterized protein C186.04c spac18g6.01c Uncharacterized protein C18G6.01c spac19g12.09 NAD/NADP-dependent indole-3-acetaldehyde reductase (EC 1.1.1.190) (EC 1.1.1.191) (AKR3C2) spac1f12.05 Uncharacterized protein C1F12.05 spac1f12.07 Putative phosphoserine aminotransferase (PSAT) (EC 2.6.1.52) (Phosphohydroxythreonine aminotransferase) spac1f5.02 Putative protein disulfide-isomerase C1F5.02 (EC 5.3.4.1) spac1f8.07c Probable pyruvate decarboxylase C1F8.07c (EC 4.1.1.1) spac222.08c Uncharacterized glutaminase C222.08c (EC 3.5.1.2)

187

Prey Gene Description spac22a12.16 Probable ATP-citrate synthase subunit 2 (EC 2.3.3.8) (ATP-citrate (pro-S-)-lyase 2) (Citrate cleavage enzyme subunit 2) spac23a1.17 SH3 domain-containing protein C23A1.17 spac24c9.06c Aconitate hydratase, mitochondrial (Aconitase) (EC 4.2.1.3) (Citrate hydro-lyase) spac24c9.12c Probable serine hydroxymethyltransferase, cytosolic (SHMT) (EC 2.1.2.1) (Glycine hydroxymethyltransferase) (Serine methylase) spac24h6.10c Putative phospho-2-dehydro-3-deoxyheptonate aldolase (EC 2.5.1.54) (3-deoxy- D-arabino-heptulosonate 7-phosphate synthase) (DAHP synthase) (Phospho-2- keto-3-deoxyheptonate aldolase) spac25h1.01c Uncharacterized protein C23H3.15c spac26f1.07 Uncharacterized oxidoreductase C26F1.07 (EC 1.-.-.-) spac27f1.06c Probable peptidyl-prolyl cis-trans isomerase C27F1.06c (PPIase) (EC 5.2.1.8) (Rotamase) spac29a4.15 Serine--tRNA ligase, cytoplasmic (EC 6.1.1.11) (Seryl-tRNA synthetase) (SerRS) (Seryl-tRNA(Ser/Sec) synthetase) spac2c4.18 Uncharacterized RNA-binding protein C25G10.01 spac30c2.04 tRNA-aminoacylation cofactor arc1 spac3a11.10c Uncharacterized dipeptidase C3A11.10c (EC 3.4.13.19) spac3g9.11c Putative pyruvate decarboxylase C3G9.11c (EC 4.1.1.1) spac3h8.01 Putative pyruvate decarboxylase C13A11.06 (EC 4.1.1.1) spac3h8.05c Uncharacterized protein C3H8.05c spac3h8.07c Probable prefoldin subunit 3 spac458.02c Uncharacterized protein C458.02c spac4d7.02c Phosphatidylglycerol phospholipase C (EC 3.1.4.-) spac4h3.07c Putative thiosulfate sulfurtransferase, mitochondrial (EC 2.8.1.1) (Rhodanese- like protein) spac4h3.08 Uncharacterized oxidoreductase C4H3.08 (EC 1.-.-.-) spac521.03 NADP-dependent 3-hydroxy acid dehydrogenase (L-allo-threonine dehydrogenase) (EC 1.1.1.381) spac56e4.03 Aromatic amino acid aminotransferase C56E4.03 (EC 2.6.1.57) spac57a7.01 Uncharacterized protein C167.05 spac57a7.12 Heat shock protein 70 homolog C57A7.12 spac5h10.03 Probable phosphatase SPAC5H10.03 (EC 3.1.3.-) spac694.02 Uncharacterized helicase C694.02 (EC 3.6.4.-) spac750.01 Putative aryl-alcohol dehydrogenase C750.01 (EC 1.1.1.-) spac750.07c Uncharacterized GPI-anchored protein SPAC750.07c spac8c9.04 Meiotically up-regulated protein C8C9.04 spac9.07c Uncharacterized GTP-binding protein C9.07c spac926.08c Ribosome production factor 2 homolog (Brix domain-containing protein 1 homolog) (Ribosome biogenesis protein RPF2 homolog) spac9e9.09c Putative aldehyde dehydrogenase-like protein C9E9.09c (EC 1.2.1.-) spac9g1.05 Uncharacterized WD repeat-containing protein C9G1.05 spapb17e12.14c Probable 6-phosphofructo-2-kinase PB17E12.14c (EC 2.7.1.105) spapb24d3.08c Zinc-type alcohol dehydrogenase-like protein PB24D3.08c (EC 1.-.-.-) spb1 AdoMet-dependent rRNA methyltransferase spb1 (EC 2.1.1.-) (2'-O-ribose RNA methyltransferase) (S-adenosyl-L-methionine-dependent methyltransferase) spbc1198.01 Zinc-type alcohol dehydrogenase-like protein C1198.01 (EC 1.-.-.-) spbc1198.05 Guanylate kinase (EC 2.7.4.8) (GMP kinase) spbc13g1.02 Probable mannose-1-phosphate guanyltransferase (EC 2.7.7.13) (GDP-mannose pyrophosphorylase) (GTP-mannose-1-phosphate guanylyltransferase) spbc14c8.04 Probable acetolactate synthase small subunit (Acetohydroxy-acid synthase small subunit) (AHAS) (ALS) spbc16a3.08c Uncharacterized protein C16A3.08c spbc16h5.08c Uncharacterized ABC transporter ATP-binding protein C16H5.08c

188

Prey Gene Description spbc1703.07 Probable ATP-citrate synthase subunit 1 (EC 2.3.3.8) (ATP-citrate (pro-S-)-lyase 1) (Citrate cleavage enzyme subunit 1) spbc1711.05 LisH domain-containing protein C1711.05 spbc1711.08 Uncharacterized protein C1711.08 spbc17d11.08 Uncharacterized WD repeat-containing protein C17D11.08 spbc17d11.09 Uncharacterized protein C17D1.01 spbc19f8.03c ENTH domain-containing protein C19F8.03c spbc215.06c UPF0743 protein C215.06c spbc215.10 Uncharacterized hydrolase C215.10 (EC 3.-.-.-) spbc215.11c Uncharacterized oxidoreductase C215.11c (EC 1.-.-.-) spbc21b10.08c Uncharacterized protein C21B10.08c spbc21d10.11c Probable cysteine desulfurase, mitochondrial (EC 2.8.1.7) spbc24c6.04 Probable delta-1-pyrroline-5-carboxylate dehydrogenase (P5C dehydrogenase) (EC 1.2.1.88) (L-glutamate gamma-semialdehyde dehydrogenase) spbc25b2.10 Universal stress protein A family protein C25B2.10 spbc25h2.16c Probable ADP-ribosylation factor-binding protein C25H2.16c spbc29a3.06 Probable U3 small nucleolar RNA-associated protein 18 (U3 snoRNA-associated protein 18) spbc29a3.16 Ribosome biogenesis regulatory protein homolog spbc2d10.11c Putative nucleosome assembly protein C2D10.11C spbc2f12.05c Oxysterol-binding protein homolog C2F12.05c spbc2g5.02c Probable casein kinase II subunit beta-2 (CK II beta-2) spbc2g5.05 Probable transketolase (TK) (EC 2.2.1.1) spbc32f12.10 Phosphoglucomutase (PGM) (EC 5.4.2.2) (Glucose phosphomutase) spbc354.10 CUE domain-containing protein C354.10 spbc365.09c KIN17-like protein spbc428.11 O-acetylhomoserine (thiol)-lyase (EC 2.5.1.49) (Homocysteine synthase) (O- acetylhomoserine sulfhydrylase) (OAH sulfhydrylase) spbc4c3.03 Probable homoserine kinase (HK) (HSK) (EC 2.7.1.39) spbc577.10 Probable proteasome subunit beta type-7 (EC 3.4.25.1) spbc582.08 Putative alanine aminotransferase (EC 2.6.1.2) (Glutamate pyruvate transaminase) (GPT) (Glutamic--alanine transaminase) (Glutamic--pyruvic transaminase) spbc660.16 6-phosphogluconate dehydrogenase, decarboxylating (EC 1.1.1.44) spbc713.13c Uncharacterized protein C216.01c spbc725.01 Aspartate aminotransferase, mitochondrial (EC 2.6.1.1) (Transaminase A) spbc776.03 Probable homoserine dehydrogenase (HDH) (EC 1.1.1.3) spbc8d2.16c Putative methyltransferase SPBC8D2.16c (EC 2.1.1.-) spbc8d2.18c Adenosylhomocysteinase (AdoHcyase) (EC 3.3.1.1) (S-adenosyl-L-homocysteine hydrolase) spbp16f5.06 Uncharacterized RNA-binding protein P16F5.06 spbp4h10.15 Homocitrate dehydratase, mitochondrial (EC 4.2.1.-) (Aconitase 2) spbp8b7.05c Carbonic anhydrase (EC 4.2.1.1) (Carbonate dehydratase) spbp8b7.26 Uncharacterized protein P8B7.26 spcc1020.07 Putative uncharacterized hydrolase C1020.07 (EC 3.-.-.-) spcc126.12 Protein NIF3 homolog spcc162.06c Uncharacterized protein C162.06c spcc1620.06c Ribose-phosphate pyrophosphokinase 2 (EC 2.7.6.1) (Phosphoribosyl pyrophosphate synthase 2) spcc1795.05c Uridylate kinase (UK) (EC 2.7.4.14) (ATP:UMP phosphotransferase) (Deoxycytidylate kinase) (CK) (dCMP kinase) (Uridine monophosphate kinase) (UMP kinase) (UMPK) spcc1827.03c Putative peroxisomal-coenzyme A synthetase (EC 6.-.-.-) spcc1827.06c Probable aspartate-semialdehyde dehydrogenase (ASA dehydrogenase) (ASADH) (EC 1.2.1.11) (Aspartate-beta-semialdehyde dehydrogenase)

189

Prey Gene Description spcc18b5.05c Putative hydroxymethylpyrimidine/phosphomethylpyrimidine kinase C18B5.05c (EC 2.7.1.49) (EC 2.7.4.7) (Hydroxymethylpyrimidine kinase) (HMP kinase) (Hydroxymethylpyrimidine phosphate kinase) (HMP-P kinase) (HMP-phosphate kinase) (HMPP kinase) spcc364.06 Putative nucleosome assembly protein C364.06 spcc417.13 Uncharacterized protein C191.01 spcc417.14c Probable acetyl-coenzyme A synthetase (EC 6.2.1.1) (Acetate--CoA ligase) (Acyl- activating enzyme) spcc4g3.01 Putative D-3-phosphoglycerate dehydrogenase (3-PGDH) (EC 1.1.1.95) spcc4g3.17 HD domain-containing protein C4G3.17 spcc550.11 Probable importin c550.11 spcc584.01c Probable sulfite reductase [NADPH] flavoprotein component (EC 1.8.1.2) spcc63.06 Uncharacterized WD repeat-containing protein C63.06 spcc63.14 Uncharacterized protein C63.14 spcc663.09c Uncharacterized oxidoreductase C663.09c (EC 1.-.-.-) spcc663.13c Uncharacterized N-acetyltransferase C663.13c (EC 2.3.1.-) spcc663.18 SCOCO-like protein 1 spcc70.05c Probable serine/threonine-protein kinase C70.05c (EC 2.7.11.1) spcc794.01c Probable glucose-6-phosphate 1-dehydrogenase C794.01c (G6PD) (EC 1.1.1.49) spcc830.11c Adenylate kinase isoenzyme 6 homolog (AK6) (EC 2.7.4.3) (Dual activity adenylate kinase/ATPase) (AK/ATPase) spcpb16a4.05c Uncharacterized urease accessory protein ureG-like spi1 GTP-binding nuclear protein spi1 spp27 Upstream activation factor subunit spp27 (Upstream activation factor 27 KDa subunit) (p27) (Upstream activation factor 30 KDa subunit) (p30) (Upstream activation factor subunit uaf30) spt7 Transcriptional activator spt7 srp1 Pre-mRNA-splicing factor srp1 srp14 Signal recognition particle subunit srp14 (Signal recognition particle 14 kDa protein homolog) srp2 Pre-mRNA-splicing factor srp2 ssa1 Probable heat shock protein ssa1 ssa2 Probable heat shock protein ssa2 ssp1 Heat shock 70 kDa protein, mitochondrial sti1 Heat shock protein sti1 homolog stt4 Phosphatidylinositol 4-kinase stt4 (PI4-kinase) (PtdIns-4-kinase) (EC 2.7.1.67) sty1 Mitogen-activated protein kinase sty1 (MAP kinase sty1) (EC 2.7.11.24) (MAP kinase spc1) sua1 Sulfate adenylyltransferase (EC 2.7.7.4) (ATP-sulfurylase) (Sulfate adenylate transferase) (SAT) sum1 Eukaryotic translation initiation factor 3 subunit I (eIF3i) (Eukaryotic translation initiation factor 3 39 kDa subunit homolog) (eIF-3 39 kDa subunit homolog) (eIF3 p39) (Suppressor of uncontrolled mitosis 1) sup35 Eukaryotic peptide chain release factor GTP-binding subunit (ERF-3) (ERF3) (ERF2) (Polypeptide release factor 3) (Translation release factor 3) svf2 Survival factor 2 swo1 Heat shock protein 90 homolog tal1 Transaldolase (EC 2.2.1.2) tam10 Uncharacterized protein tam10 (Transcripts altered in meiosis protein 10) tbp1 26S protease regulatory subunit 6A tcg1 Single-stranded TG1-3 DNA-binding protein (Meiotically up-regulated gene 187 protein) tdh1 Glyceraldehyde-3-phosphate dehydrogenase 1 (GAPDH 1) (EC 1.2.1.12) tea1 Tip elongation aberrant protein 1 (Altered polarity protein 8) (Cell polarity protein tea1) tef101 Elongation factor 1-alpha-A (EF-1-alpha-A)

190

Prey Gene Description tef3 Elongation factor 3 (EF-3) tef3 Elongation factor 1-gamma (EF-1-gamma) (eEF-1B gamma) tef5 Elongation factor 1-beta (EF-1-beta) thrc Threonine synthase (TS) (EC 4.2.3.1) ths1 Threonine--tRNA ligase, cytoplasmic (EC 6.1.1.3) (Threonyl-tRNA synthetase) (ThrRS) tif1 ATP-dependent RNA helicase eIF4A (EC 3.6.4.13) (Eukaryotic initiation factor 4A) (eIF-4A) (Translation initiation factor 1) tif11 Eukaryotic translation initiation factor 1A (eIF-1A) (Eukaryotic translation initiation factor 4C) (eIF-4C) tif211 Eukaryotic translation initiation factor 2 subunit alpha (eIF-2-alpha) tif223 Probable translation initiation factor eIF-2B subunit gamma (eIF-2B GDP-GTP exchange factor subunit gamma) tif225 Probable translation initiation factor eIF-2B subunit epsilon (eIF-2B GDP-GTP exchange factor subunit epsilon) tif32 Eukaryotic translation initiation factor 3 subunit A (eIF3a) (Eukaryotic translation initiation factor 3 110 kDa subunit) (eIF3 p110) (Translation initiation factor eIF3, p110 subunit) tif35 Eukaryotic translation initiation factor 3 subunit G (eIF3g) (Eukaryotic translation initiation factor 3 RNA-binding subunit) (eIF-3 RNA-binding subunit) (Translation initiation factor eIF3 p33 subunit homolog) (eIF3 p33 homolog) tif471 Eukaryotic translation initiation factor 4 gamma (eIF-4-gamma) (eIF-4G) tif5 Probable eukaryotic translation initiation factor 5 (eIF-5) tif51b Eukaryotic translation initiation factor 5A-2 (eIF-5A-2) tif6 Eukaryotic translation initiation factor 6 (eIF-6) tim13 Mitochondrial import inner membrane translocase subunit tim13 tip1 Tip elongation protein 1 (Protein noc1) tom70 Probable mitochondrial import receptor subunit tom70 (Translocase of outer membrane 40 kDa subunit) tpi1 Triosephosphate isomerase (TIM) (EC 5.3.1.1) (Triose-phosphate isomerase) tpr1 Tetratricopeptide repeat protein 1 tpx1 Peroxiredoxin tpx1 (EC 1.11.1.15) (Peroxiredoxin tsa1) (Thioredoxin peroxidase) trp1 Multifunctional tryptophan biosynthesis protein [Includes: Anthranilate synthase component 2 (AS) (EC 4.1.3.27) (Anthranilate synthase, glutamine amidotransferase component); Indole-3-glycerol phosphate synthase (IGPS) (EC 4.1.1.48); N-(5'-phosphoribosyl)anthranilate isomerase (PRAI) (EC 5.3.1.24)] trp2 Tryptophan synthase (EC 4.2.1.20) trp3 Probable anthranilate synthase component 1 (EC 4.1.3.27) (Anthranilate synthase component I) trr1 Thioredoxin reductase (EC 1.8.1.9) (Caffeine resistance protein 4) trx1 Thioredoxin-1 (TR-1) (Trx-1) trx2 Thioredoxin-2, mitochondrial (Trx-2) tsc1 Tuberous sclerosis 1 protein homolog tuf1 Elongation factor Tu, mitochondrial tup11 Transcriptional repressor tup11 tup12 Transcriptional repressor tup12 tys1 Tyrosine--tRNA ligase, cytoplasmic (EC 6.1.1.1) (Tyrosyl-tRNA synthetase) (TyrRS) uap56 ATP-dependent RNA helicase uap56 (EC 3.6.4.13) ubc4 Ubiquitin-conjugating enzyme E2 4 (EC 2.3.2.23) (E2 ubiquitin-conjugating enzyme 4) (Ubiquitin carrier protein 4) (Ubiquitin-protein ligase 4) ubp16 Probable ubiquitin carboxyl-terminal hydrolase 16 (EC 3.4.19.12) (Deubiquitinating enzyme 16) (Ubiquitin thioesterase 16) (Ubiquitin-specific- processing protease 16) ubr11 E3 ubiquitin-protein ligase ubr11 (EC 6.3.2.-) (N-end-recognizing protein 11) (N- recognin-11)

191

Prey Gene Description uep1 Ubiquitin-60S ribosomal protein L40 [Cleaved into: Ubiquitin; 60S ribosomal protein L40 (CEP52)] ura1 Protein ura1 [Includes: Glutamine-dependent carbamoyl-phosphate synthase (EC 6.3.5.5); Aspartate carbamoyltransferase (EC 2.1.3.2)] ura7 CTP synthase (EC 6.3.4.2) (CTP synthetase) (UTP--ammonia ligase) urg1 Uracil-regulated protein 1 (EC 3.5.4.-) urg2 Putative uracil phosphoribosyltransferase urg2 (UPRTase urg2) (EC 2.4.2.9) (UMP pyrophosphorylase urg2) urg3 Protein urg3 utp10 U3 small nucleolar RNA-associated protein 10 (U3 snoRNA-associated protein 10) (U3 protein 10 required for transcription) utp23 rRNA-processing protein utp23 (U three protein 23) utp4 U3 small nucleolar RNA-associated protein 4 (U3 snoRNA-associated protein 4) (U3 protein 4 required for transcription) utp5 U3 small nucleolar RNA-associated protein 5 (U3 snoRNA-associated protein 5) (U3 protein 5 required for transcription) utp6 U3 small nucleolar RNA-associated protein 6 (U3 snoRNA-associated protein 6) vas2 Valine--tRNA ligase (EC 6.1.1.9) (Valyl-tRNA synthetase) (ValRS) vgl1 Vigilin 1 (KH domain-containing protein vgl1) vip1 Protein vip1 vma1 V-type proton ATPase catalytic subunit A (V-ATPase subunit A) (EC 3.6.3.14) (V- ATPase 67 kDa subunit) (Vacuolar proton pump subunit alpha) vma2 V-type proton ATPase subunit B (V-ATPase subunit B) (V-ATPase 57 kDa subunit) (Vacuolar proton pump subunit B) vma4 V-type proton ATPase subunit E (V-ATPase subunit E) (Vacuolar proton pump subunit E) vps1 Vacuolar protein sorting-associated protein 1 vtc4 Vacuolar transporter chaperone 4 win1 MAP kinase kinase kinase win1 (EC 2.7.11.25) ynd1 Golgi apyrase (EC 3.6.1.5) (ATP-diphosphatase) (ATP-diphosphohydrolase) (Adenosine diphosphatase) (ADPase) (Golgi nucleoside diphosphatase) ypt1 GTP-binding protein ypt1 ypt5 GTP-binding protein ypt5 zuo1 Zuotin (DnaJ-related protein zuo1) (J protein zuo1) (Ribosome-associated complex subunit zuo1)

Table A4. Difference in prey genes identified in EMM and YES

Genes exclusive to cells Genes exclusive to cells grown in YES grown in EMM aap1 ade3 adn1 ade9 alm1 aif1 alp11 air1 anc1 alg5 arg1 alp7 arg12 alr1 aru1 arb1 asn1 bag101 atg1 bms1 atg13 caf1 atg4 cdb4 atp16 cdc15 bio2 cdc28

192

Genes exclusive to cells Genes exclusive to cells grown in YES grown in EMM brr6 cdc42 cal1 cip2 cam2 cit1 cbf12 cmb1 cdc7 cmc2 cdc8 cog8 cft1 cpy1 chs1 crn1 cnd2 dbp3 csk1 dot2 cut7 dug1 cwf24 end4 cyp4 eno102 cyp9 fbp1 dbp5 for3 dcr1 gal1 dur3-3 gas5 erg11 gcn2 erg2 gef2 erm1 gld1 fim1 grn1 fkh1 grx4 fma2 his2 fmo1 idh2 fps1 idp1 frg1 ker1 frs1 kes1 frs2 kgd1 gas2 kri1 gcs1 lcf1 gdh2 lcf2 get3 lia1 gln1 lsd90 gly1 lvs1 gpx1 mcs2 hap3 MDH1 hcs1 met6 hmt1 mis15 hmt2 mug70 hst2 nab2 hta1 nhp2 int6 nog2 isp4 nop10 kap114 nop9 kap123 not1 ksg1 ntp1 las1 nup211 leu3 nup60 lsm4 obp1 lys3 pac1 mak3 pan1 meu27 pdf1 mnn9 pex14 mrpl40 pmd1

193

Genes exclusive to cells Genes exclusive to cells grown in YES grown in EMM msd1 ppk19 mug157 ppk25 mug30 pre6 mug4 prp43 mug82 pxr1 mug87 ret2 myo1 rex4 myo2 rgf2 nro1 rki1 oca8 rnp24 ogm4 rpa1 oxa101 rpa12 paa1 rpa2 pam1 rpc19 pam16 rpc40 par2 rtn1 pda1 sec14 phf2 sec231 pho1 sec72 pht1 sfc4 pmm1 shq1 pob3 snf22 ppa2 sod1 ppk30 sod2 prp1 sof1 prp39 spac11d3.11c psi1 spac144.05 qcr2 spac16e8.13 qcr6 spac17h9.07 qcr9 spac186.04c rae1 spac18g6.01c rfc2 spac23a1.17 rhb1 spac3a11.10c rhp7 spac3g9.11c rib2 spac3h8.01 rli1 spac4d7.02c rpa43 spac4h3.07c rpl1001 spac57a7.01 rpl15 spac8c9.04 rpl1601 spbc1198.01 rpl1702 spbc1198.05 rpl2402 spbc14c8.04 rpl3201 spbc1703.07 rpl3202 spbc1711.05 rpl3801 spbc17d11.09 rpl3802 spbc215.06c rpl39 spbc215.10 rpl402 spbc215.11c rpl42 spbc21b10.08c rpl4301 spbc25b2.10 rpl901 spbc29a3.06 rpn7 spbc29a3.16 rpp201 spbc2g5.02c rpp203 spbc354.10

194

Genes exclusive to cells Genes exclusive to cells grown in YES grown in EMM rps1001 spbc365.09c rps1501 spbc428.11 rps1801 spbc4c3.03 rps2401 spbc577.10 rps2601 spbc713.13c rps402 spbc725.01 rps502 spbc776.03 rqh1 spbc8d2.16c rtp1 spbp8b7.05c sbp1 spbp8b7.26 sce3 spcc126.12 sdo1 spcc1827.06c sec17 spcc417.13 sec27 spcc4g3.17 sen34 spcc63.06 sfc3 spcc663.09c skp1 spcc663.13c snd1 spcc663.18 snu13 spcc830.11c spac1039.02 spt7 spac105.02c srp14 spac11e3.14 tea1 spac12g12.07c tif11 spac1635.01 tim13 spac17g6.01 tip1 spac18b11.02c tom70 spac1f8.04c tpr1 spac212.06c trx1 spac222.01 trx2 spac22h10.11c tsc1 spac24b11.07c ubc4 spac24b11.12c ubp16 spac24h6.13 ubr11 spac25b8.12c utp10 spac25b8.17 utp23 spac25g10.08 utp4 spac27e2.03c utp5 spac30.10c vma4 spac31g5.05c spac3a12.13c spac3g6.03c spac3h1.02c spac4a8.14 spac4g9.12 spac4h3.03c spac4h3.04c spac521.04c spac56f8.03 spac57a7.13 spac589.06c spac607.08c spac644.13c spac6c3.08 spac6g10.04c

195

Genes exclusive to cells Genes exclusive to cells grown in YES grown in EMM spac955.02c spacunk4.10 spap27g11.14c spapb1a10.13 spapb1e7.01c spapj696.02 spbc119.03 spbc15c4.03 spbc1677.03c spbc1683.04 spbc16g5.05c spbc1703.03c spbc19f5.04 spbc19g7.10c spbc21c3.15c spbc26h8.11c spbc30d10.05c spbc342.04 spbc3e7.07c spbc4.03c spbc543.02c spbc713.03 spbc713.09 spbc725.08 spbc800.10c spbc83.16c spbc839.16 spbc902.04 spbcpt2r1.02 spbp23a10.11c spbp4g3.01 spbpj4664.04 spcc1223.09 spcc1259.09c spcc1322.09 spcc1393.09c spcc1494.08c spcc1620.08 spcc1672.09 spcc16a11.16c spcc297.01 spcc320.06 spcc330.03c spcc417.14c spcc4g3.03 spcc70.03c spcc794.11c spcc825.01 spcc965.12 spmit.02 spp27 srp54 ssp2 sui1

196

Genes exclusive to cells Genes exclusive to cells grown in YES grown in EMM sum2 sup45 swi2 tef102 thi5 tif212 tif213 tif224 tif412 tol1 trp4 tsr1 tvp15 ubi3 ubp6 utp13 vma13 vma8 vps16 vtc2 vti1 wbp1 win1 wis2 wpl1 wrs1 ypt1 ypt2 ypt3

197

Figure A1. Co-immunoprecipitation assays with His-Atg101 and GST-Rpn6 to confirm protein-protein interactions from proteomic mass spec in vitro. Top panel shows the Coomassie stained gel, bottom panel shows the western blot for the detection of His-Atg101. Gels were loaded to optimize visualization of the co-expressed constructs and pull down samples.

198