Computationally Driven, Quantitative Experiments Discover Genes Required for Mitochondrial Biogenesis David C. Hess1,ChadL.Myers1,2,3., Curtis Huttenhower1,2., Matthew A. Hibbs1,2., Alicia P. Hayes1, Jadine Paw4, John J. Clore1, Rosa M. Mendoza1, Bryan San Luis4, Corey Nislow4, Guri Giaever4, Michael Costanzo4, Olga G. Troyanskaya1,2"*, Amy A. Caudy1"* 1 Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, United States of America, 2 Department of Computer Science, Princeton University, Princeton, New Jersey, United States of America, 3 Department of Computer Science and Engineering, University of Minnesota, Minneapolis, Minnesota, United States of America, 4 Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada Abstract Mitochondria are central to many cellular processes including respiration, ion homeostasis, and apoptosis. Using computational predictions combined with traditional quantitative experiments, we have identified 100 proteins whose deficiency alters mitochondrial biogenesis and inheritance in Saccharomyces cerevisiae. In addition, we used computational predictions to perform targeted double-mutant analysis detecting another nine genes with synthetic defects in mitochondrial biogenesis. This represents an increase of about 25% over previously known participants. Nearly half of these newly characterized proteins are conserved in mammals, including several orthologs known to be involved in human disease. Mutations in many of these genes demonstrate statistically significant mitochondrial transmission phenotypes more subtle than could be detected by traditional genetic screens or high-throughput techniques, and 47 have not been previously localized to mitochondria. We further characterized a subset of these genes using growth profiling and dual immunofluorescence, which identified genes specifically required for aerobic respiration and an uncharacterized cytoplasmic protein required for normal mitochondrial motility. Our results demonstrate that by leveraging computational analysis to direct quantitative experimental assays, we have characterized mutants with subtle mitochondrial defects whose phenotypes were undetected by high-throughput methods. Citation: Hess DC, Myers CL, Huttenhower C, Hibbs MA, Hayes AP, et al. (2009) Computationally Driven, Quantitative Experiments Discover Genes Required for Mitochondrial Biogenesis. PLoS Genet 5(3): e1000407. doi:10.1371/journal.pgen.1000407 Editor: Stuart K. Kim, Stanford University Medical Center, United States of America Received November 13, 2008; Accepted February 5, 2009; Published March 20, 2009 Copyright: ß 2009 Hess et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported by NIH grant R01 GM071966, NSF CAREER award DBI-0546275, NSF grant IIS-0513552, and Google Research Award to OGT and NIH grant T32 HG003284 and NIGMS Center of Excellence grant P50 GM071508. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (OGT); [email protected] (AAC) . These authors contributed equally to this work. " These authors also contributed equally to this work. Introduction largely unproven, and consequently underutilized by the scientific community [2,3]. Here, we demonstrate that computational In order to understand molecular biology at a systems level, it is predictions can successfully drive the characterization of protein first necessary to learn the functions of genes by identifying their roles using traditional experiments. To test the approach, we participation in specific cellular pathways and processes. While systematically measured the mitochondrial transmission rates of a protein sequence and structural analyses can provide valuable tractable set of S. cerevisiae strains carrying deletions of genes insights into the biochemical roles of proteins, it has proven much predicted to be necessary for this biological process. more difficult to associate proteins with the pathways where they The mitochondrion is an organelle central to several key cellular perform these roles. Recently, high-throughput and whole-genome processes including respiration, ion homeostasis, and apoptosis. screens have been used to form basic hypotheses of protein Proper biogenesis and inheritance of mitochondria is critical for participation in biological processes. However, the results of these eukaryotes as 1 in 5,000 humans suffers from a mitochondrial studies are not individually reliable enough to functionally disease [4]. Saccharomyces has proven to be an invaluable system for associate proteins with pathways. Many computational approaches studying a variety of human diseases [5,6], including cancer [7], have been developed to integrate data from such high-throughput neurologic disorders [8], and mitochondrial diseases [9–11]. Yeast assays and to generate more reliable predictions [1], but protein is a particularly attractive model system for studying mitochondrial function cannot be confidently assigned without rigorous exper- biology due to its ability to survive without respiration, permitting imental validation targeted specifically to the predicted pathway or the characterization of mutants that impair mitochondrial process. Surprisingly few follow-up laboratory efforts have been function. The process of mitochondrial biogenesis and inheritance performed on the basis of computational predictions of protein [12] (hereafter, mitochondrial biogenesis) comprises a number of function, and as such, these computational approaches remain sub-processes that together ensure that new mitochondria are PLoS Genetics | www.plosgenetics.org 1 March 2009 | Volume 5 | Issue 3 | e1000407 New Genes Required for Mitochondrial Biogenesis Author Summary by directly measuring the ability of cells carrying deletions of candidate genes to propagate functioning mitochondria to Mitochondria are the proverbial powerhouses of the cell, daughter cells. We assayed our 193 strongest predictions with no running the fundamental biochemical processes that previous experimental literature evidence of phenotypes and produce energy from nutrients using oxygen. These interactions establishing a function in mitochondrial biogenesis. processes are conserved in all eukaryotes, from humans By these assays we experimentally discovered an additional 109 to model organisms such as baker’s yeast. In humans, proteins required for proper mitochondrial biogenesis at a level of mitochondrial dysfunction plays a role in a variety of rigor acceptable for function annotation. Further, we identified diseases, including diabetes, neuromuscular disorders, and more specific roles in mitochondrial biogenesis for several aging. In order to better understand fundamental mito- predicted genes through mitochondrial motility assays and chondrial biology, we studied genes involved in mito- measurements of respiratory growth rates. We also discovered chondrial biogenesis in the yeast S. cerevisiae, discovering genes with redundant mitochondrial biogenesis roles through over 100 proteins with novel roles in this process. These experiments assigned function to 5% of the genes whose targeted examination of double knockout phenotypes. This function was not known. In order to achieve this rapid rate demonstrates that using an ensemble of computational function of discovery, we developed a system incorporating highly prediction methods to target definitive, time-consuming experi- quantitative experimental assays and an integrated, ments to a tractably sized set of candidate proteins can result in the iterative process of computational protein function pre- rapid discovery of new functional roles for proteins. Our results diction. Beginning from relatively little prior knowledge, also show that most mutants resulting in severe respiratory defects we found that computational predictions achieved about have already been discovered. This is likely to be the case for 60% accuracy and rapidly guided our laboratory work mutant screens in many fundamental biological processes, because towards hundreds of promising candidate genes. Thus, in saturating screens have discovered mutations with strong pheno- addition to providing a more thorough understanding of types. However, even in a well-studied eukaryote like S. cerevisiae, mitochondrial biology, this study establishes a framework there are many processes that have not yet been fully for successfully integrating computation and experimenta- characterized by identifying all proteins required for its normal tion to drive biological discovery. A companion manuscript, function [25]. As such, most of the remaining undiscovered published in PLoS Computational Biology (doi:10.1371/ protein functions are only identifiable by rigorous, quantitative journal.pcbi.1000322), discusses observations and conclu- assays that can detect subtle phenotypes, such as those used by our sions important for the computational community. study. generated and segregated to a daughter cell. Mitochondrial Results biogenesis begins with the nuclear genes encoding mitochondrial We utilized an ensemble of computational gene function proteins being transcribed,