Genetic basis of mitochondrial function and morphology in Saccharomyces. Kai Stefan Dimmer, Stefan Fritz, Florian Fuchs, Marlies Messerschmitt, Nadja

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1 MBC in Press, published on February 4, 2002 as /mbc Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae Kai Stefan Dimmer, Stefan Fritz, Florian Fuchs, Marlies Messerschmitt, Nadja Weinbach, Walter Neupert and Benedikt Westermann* Institut für Physiologische Chemie der Universität München Butenandtstr. 5 D München Germany * Corresponding author phone: fax: benedikt.westermann@bio.med.uni-muenchen.de Running title: Mitochondrial biogenesis in yeast Key words: genome-wide screen; mitochondria; organelle inheritance; oxidative phosphorylation; yeast

2 ABSTRACT The understanding of the processes underlying organellar function and inheritance requires the identification and characterization of the molecular components involved. We pursued a genomic approach to define the complements of genes required for respiratory growth and inheritance of mitochondria with normal morphology in yeast. With the systematic screening of a deletion mutant library covering the non-essential genes of Saccharomyces cerevisiae the numbers of genes known to be required for respiratory function and establishment of wild type-like mitochondrial structure have been more than doubled. In addition to the identification of novel components, the systematic screen revealed unprecedented mitochondrial phenotypes that have never been observed by conventional screens. These data provide a comprehensive picture of the cellular processes and molecular components required for mitochondrial function and structure in a simple eukaryotic cell. 2

3 INTRODUCTION Mitochondria are essential organelles of eukaryotic cells. They carry out a variety of metabolic processes including reactions of the tricarboxylic acid cycle, iron/sulfur cluster assembly, and biosynthesis of many cellular metabolites (Scheffler, 2000). Their most prominent function, however, is to supply the cell with energy generated by oxidative phosphorylation (Saraste, 1999). The cellular role of mitochondria is reflected by their structure. They are complex double membrane-bounded organelles with a characteristic morphology and intracellular distribution. Inheritance and morphogenesis depend on active transport along the cytoskeleton and continuous membrane fission and fusion events (Bereiter-Hahn and Vöth, 1994; Yaffe, 1999; Griparic and van der Bliek, 2001). The understanding of the processes underlying mitochondrial function and inheritance requires the identification and characterization of the molecular components involved. During the past few decades many of the proteins required for respiratory growth (Tzagoloff and Dieckmann, 1990; Contamine and Picard, 2000) and establishment and maintenance of mitochondrial structure (Hermann and Shaw, 1998; Jensen et al., 2000; Boldogh et al., 2001b) have been identified in yeast. The advent of the post-genomic era allows us to conduct systematic genome-wide screens to define whole complements of genes associated with particular functions (Winzeler et al., 1999; Vidan and Snyder, 2001). The fact that Saccharomyces cerevisiae is a facultative anaerobic yeast capable of satisfying its energy requirements with ATP generated by fermentation is the reason why only relatively few mitochondrial proteins are essential for cell viability. These are restricted to a handful of factors essential for import, processing and folding of 3

4 precursor proteins, iron/sulfur cluster assembly and flavin mononucleotide synthesis. This makes budding yeast an ideal organism for dissecting the molecular processes required for maintenance of respiratory-competent mitochondria. Its mitochondrial genome encodes eight proteins which are all essential for oxidative phosphorylation. Despite the capacity of mitochondria to encode and synthesize proteins, a vast array of genes located in the nucleus is required for respiratory competence. Mutants in these genes are commonly referred to as nuclear petite or pet mutants (Tzagoloff and Dieckmann, 1990). The Munich Information Center for Protein Sequences, MIPS, (Mewes et al., 2000) currently lists 171 yeast ORFs which are required for respiratory growth ( Mitochondria are amazingly dynamic organelles. Their morphology and distribution reflects the energy requirements of the cell (Bereiter-Hahn, 1990; Warren and Wickner, 1996; Hermann and Shaw, 1998; Yaffe, 1999). In S. cerevisiae, mitochondria form a branched tubular network below the cell cortex (Hoffmann and Avers, 1973; Stevens, 1981) the continuity of which is maintained by active actindependent transport and balanced membrane fusion and fission events (Nunnari et al., 1997; Hermann and Shaw, 1998). Morphological screens of randomly mutagenized yeast samples employing mitochondria-specific fluorescent dyes have proven to be very successful for the identification of some key components required for maintenance of this complex structure (McConnell et al., 1990; Burgess et al., 1994; Hermann et al., 1997; Sesaki and Jensen, 1999). Here, we report on the systematic screening of a yeast deletion mutant library covering the non-essential genes to define the sets of genes involved in mitochondrial structure and function. A total number of 341 open reading frames (ORFs) was 4

5 identified to be required for respiratory growth, 38 of which encode unknown proteins. Mutants with aberrant mitochondrial morphology include 5 known genes that were not previously implicated in mitochondrial morphogenesis and 10 genes encoding novel components. These data provide a comprehensive picture of the cellular processes and molecular components required for respiratory growth and inheritance of mitochondria with a normal morphology in yeast. 5

6 MATERIALS AND METHODS Yeast genetic methods Cultivation of yeast was according to standard procedures (Sherman et al., 1986). The homozygous diploid knock out library was constructed by an international consortium of yeast laboratories (Winzeler et al., 1999). It was obtained from Research Genetics (Huntsville, AL). A list of strains present in the library is available at the company's website (ftp://ftp.resgen.com/pub/deletions/homo_diploids_ txt) or from the authors upon request. Screening for pet mutants After completely thawing 96-well plates, cells were transferred to yeast extract/peptone/glucose (YPD) and yeast extract/peptone/glycerol (YPG) plates using a sterile pinning tool. Plates were incubated at 30 C for 2 (YPD) or 3 (YPG) days before the growth behavior was examined. To obtain an estimate of the saturation of the genome-wide screen, we compared our results to the published information about yeast mitochondrial ribosomal proteins. 71 nuclear genes encoding mitochondrial ribosomal proteins are known, according to the Yeast Proteome Database (Costanzo et al., 2001). Deletions of 62 of these genes are present in the library, and the observed growth phenotypes of 61 of these mutants correspond to published phenotypes (for detailed information see supplemental table 1). From these numbers we estimate that the knock out library is 80-90% saturating for all non-essential yeast ORFs. In addition, some genes encoding very small proteins of less than 100 amino acid residues which are only partially covered by the library and 6

7 genes encoding proteins that perform redundant functions might have been missed during the screen. Screening for mdm mutants Logarithmically growing yeast cultures in YPD medium were stained with 0.1 µm rhodamine B hexyl ester (Molecular Probes, Eugene, OR) and inspected by standard fluorescence microscopy (Prokisch et al., 2000). Screening was repeated for strains that showed aberrant mitochondrial morphology or that did not stain well due to a lack of mitochondrial membrane potential. The second round of screening was performed by a different individual and included a number of wild type strains as a control. 268 mutant strains that reproducibly did not exhibit wild type mitochondria were transformed with plasmid pvt100u-mtgfp expressing mitochondria-targeted GFP (Westermann and Neupert, 2000). All transformants were screened at least two more times for mutants with aberrant mitochondrial morphology. All rounds of screening were performed without reference to strain identity. For all clearly identified mutants correct gene disruption was confirmed by PCR, and the mitochondrial phenotype was confirmed in a haploid genetic background (haploid deletants were obtained from EUROSCARF; Frankfurt, Germany; Strains that for different reasons failed to give a clear result are listed in supplemental table 2. 7

8 RESULTS Genes needed for respiratory growth To define the molecular basis of respiratory competence we systematically screened a library containing deletion mutants of 4794 non-essential yeast genes for strains that are respiratory deficient. Yeast knock out strains were plated on media containing the fermentable carbon source glucose or the non-fermentable carbon source glycerol and scored for mutants unable to grow on glycerol. A total number of 341 ORFs were identified to be required for respiratory growth (Fig. 1; supplemental table 3). More than half of the identified pet genes encode known mitochondrial proteins, the majority of which is devoted to replication, transcription and translation of the mitochondrial genome or assembly of the respiratory chain. A large fraction of the pet genes encoding known non-mitochondrial proteins is associated either with vacuolar functions or encodes nuclear transcription factors. The remainder is associated with a variety of different cellular functions, and failure to grow on glycerol-containing medium might be due to cumulative effects of a compromised general cell physiology. 17% of the pet mutants contain deletions in ORFs of unknown function. We classified 22 of these as questionable ORFs because they overlap with other genes that encode known or conserved proteins. 38 ORFs encode so far unknown proteins that presumably play important roles in the maintenance of respiratory-competent mitochondria. Genes needed for mitochondrial distribution and morphology To obtain a more complete understanding of the molecular machinery determining mitochondrial behavior we conducted a systematic genome-wide screen for genes 8

9 important for mitochondrial distribution and morphology (MDM) (McConnell et al., 1990) yeast knock out strains were stained with mitochondria-specific probes and screened for mutants with aberrant mitochondrial morphology. The isolated mdm mutants were grouped into three classes. Class I mutant genes encode proteins that are essential for establishing wild type mitochondrial morphology. For these mutants, cells with wild type-like mitochondria were never observed (see below). Mitochondria of class II and class III mutants were often fragmented or aggregated, however, a certain subfraction showed wild type-like morphology (supplemental table 4). Thus, these genes are considered as not being essential for establishment of normal mitochondrial structure. Class II mutants are respiratory-competent. This class includes strains that appear to exhibit mitochondrial morphology defects only under certain conditions, such as clu1, mdm20, ptc1 and yme1 (Campbell et al., 1994; Hermann et al., 1997; Fields et al., 1998; Roeder et al., 1998). Interestingly, three genes involved in biosynthesis of ergosterol, ERG6, ERG24 and ERG28, fall into this class. This is consistent with a role of ergosterol in membrane fusion, that was recently demonstrated by a similar approach aimed at the identification of vacuolar inheritance components (Kato and Wickner, 2001). Class III mutants display a pet phenotype in addition to their mitochondrial morphology defect. It is known for a long time that loss of respiratory function often results in the loss of inner membrane cristae (Pon and Schatz, 1991). Since processes of mitochondrial morphogenesis are intimately linked to connection of the mitochondrial outer and inner membranes (Aiken Hobbs et al., 2001; Fritz et al., 2001) it is 9

10 conceivable that changes in the structure of the inner membrane may affect the global structure of the organelle (Wong et al., 2000). Among the class I mutants are eight genes that are known to encode key components of the machinery of mitochondrial morphology. In addition, we identified ten previously uncharacterized genes as being essential for the establishment of normal mitochondrial morphology (Table 1). Four of these genes, MDM31, MDM32, MDM37 and MDM38, encode proteins of unknown function that are predicted to be located in the mitochondrial inner membrane. Mutants of the closely related mdm31 and mdm32 genes contain compact mitochondrial aggregates (Fig. 2C, D). In contrast, mdm37 mutant cells harbor mainly fragmented mitochondria with very few short tubules (Fig. 2I) resembling mutants defective in mitochondrial fusion (Hermann et al., 1998; Rapaport et al., 1998; Fritz et al., 2001; Sesaki and Jensen, 2001). Mitochondria of cells lacking the evolutionarily conserved Mdm38 protein appear enlarged with very few branches and often form rings or lariat-like structures, a phenotype that was never reported before (Fig. 2J). Two genes, MDM33 and MDM39, encode predicted membrane proteins lacking a clear mitochondrial presequence. Most mdm33 mutant cells exhibit giant ring-like mitochondrial structures, an unprecedented phenotype (Fig. 2E), whereas the mdm39 mutant shows fragmented mitochondria (Fig. 2K). Four components, Mdm30p, Mdm34p, Mdm35p and Mdm36p, do not have predicted presequences or transmembrane domains. The mdm30 mutant displays many fragmented or aggregated mitochondria with only very few short tubules (Fig. 2B). Mutants with deletions in the mdm34 gene (Fig. 2F) or the overlapping questionable ORF ygl218w (not shown) have spherical mitochondria, similar to mdm10, mdm12, and 10

11 mmm1 mutants (Burgess et al., 1994; Sogo and Yaffe, 1994; Berger et al., 1997), and sometimes single tubules extending through the cell. Mitochondria of cells lacking the small, highly conserved Mdm35 protein are spherical (Fig. 2G), whereas the organelles of the mdm36 mutant are mostly aggregated at one side of the cell (Fig. 2H). Five genes were identified that encode known proteins which were previously not implicated in mitochondrial morphogenesis (Table 1). The tom7 mutant displays aggregated, often fenestrated mitochondria which are unevenly distributed in the cell (Fig. 2P). Tom7p is a component of the general preprotein import complex of mitochondria and plays an important role in the insertion of proteins into the outer membrane (Hönlinger et al., 1996). We propose that Tom7p is required for the insertion of one or several morphogenesis factor(s) into the outer membrane. A reduced import of this factor(s) would consequently lead to an abnormal mitochondrial morphology. Deletion mutants of num1 (Fig. 2N) and the overlapping questionable ORF ydr149c (not shown) display highly aggregated mitochondria. Num1p is a cell cortex-associated protein involved in nuclear migration (Kormanec et al., 1991; Heil-Chapdelaine et al., 2000). Our data assign to Num1p an additional function in positioning of mitochondria. Arg82p, a nuclear inositol 1,4,5-trisphosphate kinase, Mot2p, a global transcriptional repressor, and Ref2p, an RNA processing factor, each influence the expression of a large number of different genes (Cade and Errede, 1994; Dubois and Messenguy, 1994; Irie et al., 1994; Russnak et al., 1995; Odom et al., 2000). The pronounced mitochondrial phenotypes of these mutants (Fig. 2L, M, O) can be sufficiently explained by the assumption that expression of at least one crucial protein is affected. 11

12 DISCUSSION With the completion of a comprehensive genome-wide screen the number of genes implicated in mitochondrial function and morphology has been more than doubled. The systematic approach enabled the identification of a number of components that have been missed by classical approaches that typically were based on the screening of collections of temperature-sensitive mutants generated by random mutagenesis (Hermann and Shaw, 1998). Most notably, the systematic screening of a deletion mutant library revealed a number of mdm mutants lacking an obvious growth defect (e.g. mdm30, mdm33, mdm35, mdm36, mdm38, mdm39), that presumably could not be easily identified by conventional genetic screens. This illustrates the power of the genomic approach that should turn out to be equally fruitful also for the study of many other cellular processes. What might be the cellular roles of the newly discovered MDM genes? Only in two cases the predicted protein sequence is suggestive of a function. MDM30 encodes a protein of unknown function that contains an F-box, a motif involved in targeting of proteins to ubiquitin-dependent proteolysis (Patton et al., 1998). Furthermore, highthroughput two-hybrid analysis (Uetz et al., 2000) identified Mdm30p as a possible interaction partner of Cdc53p and Skp1p, two core components of the SCF (Skp1pcullin-F-box) complexes which target proteins for ubiquitin-dependent degradation (Skowyra et al., 1997). As it is known that protein ubiquitination is important for mitochondrial inheritance (Fisk and Yaffe, 1999) we propose that Mdm30p is a novel factor involved in this process. MDM38 encodes a protein that shares homology with a mitochondrial protein of unknown function of Drosophila, the CG4589 gene product 12

13 (Caggese et al., 1999). This protein is a calcium-binding protein that contains two EF hand calcium binding domains. It will be interesting to see whether Mdm38p plays a role in calcium homeostasis of mitochondria and thereby influences organellar morphology. During the past decade, several genetic and morphological screens have revealed a number of important components involved in mitochondrial inheritance, yet many processes determining mitochondrial behavior are not well understood. These include the machinery connecting mitochondria to the actin cytoskeleton (Simon et al., 1995; Boldogh et al., 1998; Boldogh et al., 2001a), proteins cooperating with Fzo1p (Hermann et al., 1998; Rapaport et al., 1998; Fritz et al., 2001) or Ugo1p (Sesaki and Jensen, 2001) in mediating mitochondrial membrane fusion, and components shaping the internal structure of mitochondria (Wong et al., 2000). The proteins encoded by the majority of the newly identified MDM genes do not share homology to other known proteins. Their varied and striking mutant phenotypes suggest that they are important players in a number of different processes contributing to the morphogenesis of mitochondria. The relatively small number of newly identified components suggests that the screen was rather specific. It is important to note, however, that our results were obtained with null alleles. Thus, it cannot be excluded that some of the newly identified genes perform primary functions not directly related to mitochondrial morphogenesis, and that the mitochondrial phenotypes might be due to indirect effects accumulating in the deletion strains. It will be a major challenge for the future to establish the molecular functions of the newly discovered proteins and to unravel the interactions among the components of mitochondrial behavior. These studies will certainly improve our 13

14 understanding of the mechanisms that shape this complex double membrane-bounded organelle. The results of the screen suggest that most of the proteins constituting the core machinery of mitochondrial morphogenesis uniquely affect this organelle. With the exception of the dynamin-related proteins Dnm1p and Mgm1p, none of the key components of mitochondrial inheritance shares homology with any other known protein involved in membrane trafficking events of other organelles. Vice versa, besides Num1p, none of the components involved in biogenesis of other organelles was found to be essential for normal mitochondrial structure. It appears that Nature invented an entirely new machinery of organelle maintenance after the endosymbiotic ancestors of mitochondria entered the eukaryotic cell. 14

15 ACKNOWLEDGEMENTS The first five authors contributed equally to this work and are listed in alphabetical order. We thank Gabriele Ludwig for excellent technical assistance and Johannes Herrmann and William Wickner for many helpful discussions. This work was supported by the Deutsche Forschungsgemeinschaft through grants WE 2174/2-1 (to B.W.), Sonderforschungsbereich 413 Teilprojekt B3 (to B.W. and W.N.) and by the BMBF through grant MITOP (to W.N.). 15

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26 Table 1. Genes essential for maintenance of normal mitochondrial morphology in yeast. Gene name Pre- Mitochondrial (standard/systematic) TM sequence morphology Reference DNM1/YLL001W - - net-like Otsuga et al., 1998; Bleazard et al., 1999; Sesaki and Jensen, 1999 FIS1/YIL065C OM - net-like Mozdy et al., 2000 FZO1/YBR179C OM - fragmented Hermann et al., 1998; Rapaport et al., 1998 MDM10/YAL010C OM - giant spherical Sogo and Yaffe, 1994 MDM12/YOL009C OM - giant spherical Berger et al., 1997 MDV1/YJL112W - - net-like Fekkes et al., 2000; Tieu and Nunnari, 2000; Cerveny et al., 2001 MGM1/YOR211C? e fragmented/ aggregated Guan et al., 1993; Shepard and Yaffe, 1999; Wong et al., 2000 MMM1/YLL006W OM - giant spherical Burgess et al., 1994 MDM30/YLR368W - - see Fig. 2B this study MDM31/YHR194W p see Fig. 2C this study MDM32/YOR147W p see Fig. 2D this study MDM33/YDR393W p see Fig. 2E this study MDM34/YGL219C - - see Fig. 2F this study MDM35/YKL053C-A - - see Fig. 2G this study MDM36/YPR083W - - see Fig. 2H this study MDM37/YGR101W p see Fig. 2I this study MDM38/YOL027C p see Fig. 2J this study MDM39/YGL020C p - see Fig. 2K this study ARG82/YDR173C - - see Fig. 2L this study MOT2/YER068W - - see Fig. 2M this study NUM1/YDR150W - - see Fig. 2N this study REF2/YDR195W - - see Fig. 2O this study TOM7/YNL070W OM - see Fig. 2P this study The upper part lists known components of mitochondrial morphology that were rediscovered during the screen. The middle part lists novel components of previously unknown function, the bottom part lists known genes that were not linked to mitochondrial morphogenesis before. TM, transmembrane protein. OM, located in the outer membrane. p, transmembrane domain(s) predicted (Costanzo et al., 2001). Presequence: e, experimentally determined; numbers indicate the likelihood of 26

27 mitochondrial import as predicted by the MITOPROT program (Claros and Vincens, 1996) (maximum score is 1). 27

28 FIGURE LEGENDS Figure 1. Distribution of functional classes of known and newly identified yeast pet genes using criteria from the Yeast Proteome Database (Costanzo et al., 2001). Numbers indicate the number of pet genes falling into each functional class. An annotated list of all identified pet genes catalogued according to their cellular function can be found in supplemental table 3. mt, mitochondrial. Figure 2. Mitochondrial morphology of newly identified mdm mutants. Strains expressing mitochondria-targeted GFP were grown in glucose-containing YPD medium at 30 C to logarithmic growth phase and subjected to fluorescence microscopy. Left, mitochondrial morphology of representative cells; right, overlay with the corresponding phase contrast image. WT, wild type. Bar, 5 µm. 28

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31 Supplementary Material Supplemental table 1. List of genes encoding mitochondrial ribosomal proteins in yeast. The published growth phenotypes of the deletion mutants as indicated in the Yeast Proteome Database (Costanzo et al., 2001) are compared to the observed growth behavior of the strains of the homozygous diploid yeast knock out library (Winzeler et al., 1999). Mutants not present in the library are marked with three dashes. pet, petite phenotype (respiratory deficiency). Nuclear genes encoding mitochondrial ribosomal proteins in yeast Gene name Published phenotype Growth phenotype (systematic/standard)(ypd database) observed YBL038W/MRPL16 pet pet YBL090W/MRP21 pet pet YBR122C/MRPL36 viable --- YBR146W/MRPS9 pet normal growth YBR251W/MRPS5 viable pet YBR268W/MRPL37 viable pet YBR282W/MRPL27 pet pet YCR003W/MRPL32 slow growth pet YCR046C/IMG1 pet pet YDL045W-A/MRP10 pet pet YDL202W/MRPL11 pet pet YDR041W/RSM10 lethal --- YDR115W pet pet YDR116C defect on glycerol very slow on glycerol YDR175C/RSM24 pet pet YDR237W/MRPL7 viable pet YDR322W/MRPL35 slow growth pet YDR337W/MRPS28 pet pet YDR347W/MRP1 pet pet YDR405W/MRP20 pet pet YDR462W/MRPL28 viable pet YEL050C/RML2 pet pet YER050C/RSM18 pet pet YFR049W/YMR31 viable normal growth YGL068W lethal --- YGL129C/RSM23 pet pet YGR076C/MRPL25 pet pet YGR084C/MRP13 normal growth normal growth YGR215W/RSM27 viable pet YGR220C/MRPL9 viable pet YHL004W/MRP4 pet ---

32 YHR075C/PPE1 no growth defect normal growth YHR147C/MRPL6 pet pet YIL093C/RSM25 pet pet YJL063C/MRPL8 pet pet YJL096W/MRPL49 pet pet YJR101W/RSM26 pet --- YJR113C/RSM7 viable pet YKL003C/MRP17 pet pet YKL138C/MRPL31 pet pet YKL142W/MRP8 normal growth normal growth YKL155C/RSM22 pet pet YKL167C/MRP49 cold-sensitive pet respiratory growth YKL170W/MRPL38 viable pet YKR006C/MRPL13 reduced pet respiratory growth YKR085C/MRPL20 pet pet YLR312W-A/MRPL15 viable pet YLR439W/MRPL4 pet pet YML009C/MRPL39 viable normal growth YML025C lethal --- YMR024W/MRPL3 viable normal growth YMR188C pet pet YMR193W/MRPL24 viable pet YMR225C/MRPL44 viable normal growth YMR286W/MRPL33 pet pet YNL005C/MRP7 pet pet YNL081C slow on glycerol pet YNL137C/NAM9 pet --- YNL185C/MRPL19 viable --- YNL252C/MRPL17 pet pet YNL284C/MRPL10 viable pet YNL306W/MRPS18 lethal --- YNR036C viable pet YNR037C/RSM19 pet pet YOR150W/MRPL23 pet pet YOR158W/PET123 pet pet YPL013C viable pet YPL118W/MRP51 pet pet YPL173W/MRPL40 viable pet YPL183W-A viable pet YPR166C/MRP2 pet pet 2

33 Supplemental table 2. List of genes that failed to be analyzed for mitochondrial morphology defects. 19 mutants are listed that could not be analyzed for mitochondrial morphology defects because strains failed to grow in liquid media, or transformants with a plasmid expressing mitochondria-targeted GFP could not be obtained. The systematic gene name is indicated together with the standard gene name and a brief description of the protein's function (Costanzo et al., 2001). Strains that failed to be analyzed for mitochondrial morphology defects YDR115W, probable component of the mitochondrial ribosome YDR347W/MRP1, mitochondrial ribosomal protein of the small subunit YDR470C/UGO1, protein of the mitochondrial outer membrane required for mitochondrial fusion YER014W/HEM14, protoporphyrinogen oxidase YGL070C/RPB9, RNA polymerase II, non-essential subunit, not shared YGR262C/BUD32, may be involved in polar bud site selection in diploid cells YJR122W/CAF17, component of the CCR4 transcription complex YKL169C, unknown function; questionable ORF YLR304C/ACO1, aconitate hydratase (aconitase) YLR382C/NAM2, leucyl-trna synthetase, mitochondrial YNL064C/YDJ1, protein involved in protein import into mitochondria and ER YNL080C, unknown function YOR241W/MET7, involved in methionine biosynthesis and maintenance of mitochondrial genome YOR305W, unknown function YPL050C/MNN9, required for complex N-glycosylation YPL148C/PPT2, acyl carrier-protein synthase YPL183W-A, possible mitochondrial ribosomal protein YPR036W/VMA13, vacuolar H(+)-ATPase (V-ATPase) 54 kda subunit of V1 sector YPR072W/NOT5, negatively regulates transcription of TATA-less promoters 3

34 Supplemental table 3. List of pet genes of Saccharomyces cerevisiae identified in a genome-wide screen for respiratory-deficient mutants. Genes were catalogued according to their cellular functions using criteria from the Yeast Proteome Database (Costanzo et al., 2001). The left column gives the systematic gene name, the right column indicates the standard gene name together with a brief description of the protein's function. For proteins of unknown function, the likelihood of import into mitochondria as predicted by the MITOPROT program (Claros and Vincens, 1996) is indicated (maximum score is 1). At the end of the table, 64 mutants are listed which are respiratory-competent but showed severely impaired growth on media containing the non-fermentable carbon source glycerol. pet genes of S. cerevisiae KNOWN MITOCHONDRIAL PROTEINS Mitochondrial DNA metabolism YCR028C-A RIM1, binds single-stranded DNA, required for DNA replication in mitochondria YDR296W MHR1, involved in repair, recombination and maintenance of mitochondrial DNA YHR120W MSH1, involved in mitochondrial DNA repair YJR144W MGM101, mitochondrial genome maintenance protein YML061C PIF1, single-stranded DNA-dependent ATPase and 5'-3' DNA helicase YMR072W ABF2, DNA-binding protein required for maintenance, transmission and recombination of mitochondrial genome YOL095C HMI1, mitochondrial DNA helicase involved in maintenance of integrity of mitochondrial genome YOR330C MIP1, mitochondrial DNA-directed DNA polymerase Mitochondrial RNA synthesis YDL044C MTF2, mitochondrial protein involved in mrna splicing and protein synthesis YDR194C MSS116, mitochondrial RNA helicase of the DEAD box family, required for splicing of group II introns of COX1 and COB YDR332W Member of the DEAD-box family of predicted RNA helicases, mitochondrial localization predicted YFL036W RPO41, RNA polymerase, mitochondrial YHL038C CBP2, required for splicing of the COB ai5 intron and for efficient splicing of 21S mitochondrial rrna (LSU) intron YIR021W MRS1, protein involved in mitochondrial RNA splicing of COB mrna YJL209W CBP1, required for COB mrna stability or 5' processing YKL208W CBT1, required for 3' end processing of the mitochondrial COB mrna YLR067C PET309, required for stability and translation of COX1 mrna 4

35 YMR228W YMR287C YPL029W YPR134W MTF1, mitochondrial RNA polymerase specificity factor MSU1, component of a mitochondrial 3'-5' exonuclease complex that is essential for mitochondrial biogenesis SUV3, mitochondrial RNA helicase MSS18, involved in splicing a15beta intron of the mitochondrial COX1 transcript Mitochondrial protein synthesis Mitochondrial ribosomal subunits YBL038W MRPL16, mitochondrial ribosomal protein YBL090W MRP21, mitochondrial ribosomal protein YBR251W MRPS5, mitochondrial ribosomal protein YBR268W MRPL37, mitochondrial ribosmal protein YBR282W MRPL27, mitochondrial ribosomal protein YCR003W MRPL32, mitochondrial ribosomal protein YCR046C IMG1, putative mitochondrial ribosomal protein YDL045W-A MRP10, mitochondrial ribosomal protein YDL202W MRPL11, mitochondrial ribosomal protein YDR115W Mitochondrial ribosomal protein YDR175C RSM24, mitochondrial ribosomal protein YDR237W MRPL7, mitochondrial ribosomal protein YDR322W MRPL35, mitochondrial ribosomal subunit YDR337W MRPS28, mitochondrial ribosomal protein YDR347W MRP1, mitochondrial ribosomal protein YDR405W MRP20, mitochondrial ribosomal protein YDR462W MRPL28, mitochondrial ribosomal protein YEL050C RML2, mitochondrial ribosomal protein L2 of the large subunit YER050C RSM18, component of the mitochondrial ribosomal small subunit YGL129C RSM23, mitochondrial ribosomal protein YGR076C MRPL25, mitochondrial ribosomal protein YGR215W RSM27, mitochondrial ribosomal protein YGR220C MRPL9, mitochondrial ribosomal protein YHR147C MRPL6, mitochondrial ribosomal protein YIL093C RSM53, mitochondrial ribosome small subunit YJL063C MRPL8, mitochondrial ribosomal protein of the large subunit YJL096W MRPL49, mitochondrial ribosomal protein of the large subunit YJR113C RSM7, probable mitochondrial ribosomal YKL003C MRP17, mitochondrial ribosomal protein YKL138C MRPL31, mitochondrial ribosomal protein YKL155C RSM22, mitochondrial ribosomal protein YKL167C MRP49, mitochondrial ribosomal protein of the large subunit YKL170W MRPL38, mitochondrial ribosomal protein YKR006C MRPL13, mitochondrial ribosomal protein YKR085C MRPL20, mitochondrial ribosomal protein YLR312W-A MRPL15, mitochondrial ribosomal protein YLR439W MRPL4, mitochondrial ribosomal protein of the large subunit YMR188C Weak similarity to prokaryotic 30S ribosomal protein YMR193W MRPL24, mitochondrial ribosomal protein of the large subunit YMR286W MRPL33, mitochondrial ribosomal protein of the large subunit YNL005C MRP7, mitochondrial ribosomal protein YNL081C Putative mitochondrial ribosomal protein of the small subunit YNL252C MRPL17, mitochondrial ribosomal protein YNL284C MRPL10, mitochondrial ribosomal protein of the large subunit YNR036C Putative mitochondrial ribosomal protein YNR037C RSM19, putative mitochondrial ribosomal protein YOR150W MRPL23, Mitochondrial ribosomal protein YOR158W PET123, mitochondrial ribosomal protein YPL013C Similarity to Neurospora crassa mitochondrial ribosomal protein S24 YPL118W MRP51, mitochondrial ribosomal protein of the small subunit 5

36 YPL173W MRPL40, mitochondrial ribosomal protein of the large subunit YPL183W-A Possible mitochondrial ribosomal protein YPR166C MRP2, mitochondrial ribosomal protein Mitochondrial trna synthetases YCR024C Asparaginyl-tRNA synthetase, mitochondrial YDR268W MSW1, Trp-tRNA synthetase, mitochondrial YER087W Similarity to E. coli prolyl-trna synthetase YGR171C MSM1, Met-tRNA synthetase, mitochondrial YHR011W DIA4, trna synthetase, may be involved in mitochondrial function YHR091C MSR1, arginyl-trna synthetase of mitochondria YLR382C NAM2, leucyl-trna synthetase, mitochondrial, YNL073W MSK1, lysyl-trna synthetase, mitochondrial YOL033W MSE1, glutamyl-trna synthetase, mitochondrial YPL040C ISM1, isoleucyl-trna synthetase, mitochondrial YPL097W MSY1, tyrosyl-trna synthetase, mitochondrial YPL104W MSD1, aspartyl-trna synthetase, mitochondrial YPR047W MSF1, Phe-tRNA synthetase, mitochondrial Other YBL080C PET112, may have a general role in mitochondrial translation YDL069C CBS1, translational activator of COB mrna YDL107W MSS2, protein involved in Cox2 expression YDR197W CBS2, translational activator for cyt b YER153C PET122, translational activator required for mitochondrial translation of COX3 YGL143C MRF1, mitochondrial peptide chain release factor YGR222W PET54, specific translational activator for COX3 YHR038W FIL1, mitochondrial ribosome recycling factor YJL023C PET130, required for mitochondrial protein synthesis YJL102W MEF2, mitochondrial translation elongation factor YLR069C MEF1, mitochondrial translation elongation factor G YLR203C MSS51, mitochondrial protein required for respiratory growth and translation of COX1 mrna YMR064W AEP1, required for accumulation of transcript of ATP9/OLI1 YMR257C PET111, required for mitochondrial translation of COX2 mrna YMR282C AEP2, required for the expression of Atp9p YNR045W PET494, translational activator required for mitochondrial translation of COX3 YOL023W IFM1, mitochondrial translation initiation factor 2 YOR187W TUF1, translation elongation factor Tu, mitochondrial YOR201C PET56, ribose methyltransferase for mitochondrial 21S rrna Respiratory chain Succinate dehydrogenase complex (complex II) YKL148C SDH1, succinate dehydrogenase (ubiquinone) flavoprotein (Fp) subunit Cytochrome bc1 complex (Ubiquinol-cytochrome c reductase complex, complex III) YBL045C COR1, ubiquinol cytochrome c reductase core protein 1 YDR529C QCR7, ubiquinol cytochrome c reductase subunit 7 YEL024W RIP1, ubiquinol cytochrome c reductase iron-sulfur protein YJL166W QCR8, ubiquinol cytochrome c reductase subunit 8 YOR065W CYT1, cytochrome c1 YPR191W QCR2, ubiquinol cytochrome c reductase core protein 2 Cytochrome c oxidase (complex IV) YDL067C COX9, cytochrome c oxidase subunit VIIA YHR051W COX6, cytochrome c oxidase subunit VI YLR038C COX12, cytochrome-c oxidase, subunit VIb YMR256C COX7, cytochrome c oxidase, subunit VII YNL052W COX5A, cytochrome c oxidase subunit Va F0/F1 ATP synthase (complex V) YBL099W ATP1, alpha subunit of F1-ATP synthase YDR298C ATP5, subunit 5 of F0-ATP synthase, oligomycin sensitivity-conferring subunit 6

37 YDR377W ATP17, ATP synthase subunit f YJR121W ATP2, beta subunit of F1-ATP synthase YKL016C ATP7, ATP synthase subunit d YLR295C ATP14, ATP synthase subunit h YML081C-A ATP18, ATP synthase subunit i (or subunit j) YPL078C ATP4, subunit 4 of F0-ATP synthase YPL271W ATP15, epsilon subunit of F1-ATP synthase Assembly factors YAL039C CYC3, holocytochrome c synthase (cytochrome c heme lyase) YBR037C SCO1, role in copper transport or insertion of copper into cytochrome oxidase YDR079W PET100, required for assembly of cytochrome c oxidase YDR231C COX20, involved in maturation of Cox2p and its assembly into COX YDR375C BCS1, required for expression of functional Rieske iron-sulfur protein YER058W PET117, involved in assembly of cytochrome oxidase YER141W COX15, required for cytochrome oxidase assembly YGR062C COX18, required for activity of mitochondrial cytochrome oxidase YGR112W SHY1, mitochondrial protein required for respiration, assembly of cytochrome c oxidase complex YGR174C CBP4, ubiquinol-cytochrome c reductase assembly factor YJL180C ATP12, F1-ATP synthase assembly protein YJR034W PET191, involved in assembly of cytochrome oxidase YKL087C CYT2, holocytochrome-c1 synthase (CC1HL) YLR393W ATP10, required for F1-F0 ATP synthase assembly YML129C COX14, required for assembly of cytochrome oxidase YNL315C ATP11, F1-ATP synthase assembly protein YPL215W CBP3, required for assembly of cytochrome bc1 complex Mitochondrial enzymes YAL044C GCV3, glycine decarboxylase hydrogen carrier protein H subunit YBR003W COQ1, hexaprenyl pyrophosphate synthetase YDR148C KGD2, 2-oxoglutarate dehydrogenase complex E2 component YDR204W COQ4, involved in biosynthesis of coenzyme Q YDR226W ADK1, adenylate kinase (GTP:AMP phosphotransferase), cytoplasmic and mitochondrial YER014W HEM14, protoporphyrinogen oxidase YER061C CEM1, beta-ketoacyl-acp synthase, mitochondrial YFL018C LPD1, dihydrolipoamide dehydrogenase, (E3) component of pyruvate dehydrogenase complex YGR255C COQ6, monooxygenase required for coenzyme Q (ubiquinone) biosynthesis YHR008C SOD2, manganese superoxide dismutase, mitochondrial YIL125W KGD1, alpha-ketoglutarate dehydrogenase YLR304C ACO1, aconitase YMR267W PPA2, inorganic pyrophosphatase, mitochondrial YNR041C COQ2, para-hydroxybenzoate-polyprenyltransferase YPL132W COX11, required for heme A synthesis YPL172C COX10, farnesyl transferase required for heme A synthesis YPL262W FUM1, fumarate hydratase Lipids biosynthesis, mitochondrial YKL055C OAR1, 3-oxoacyl-[acyl-carrier-protein] reductase (mitochondrial type II fatty acid synthase) YOR196C LIP5, lipoic acid synthase (mitochondrial matrix) YOR221C MCT1, malonyl CoA:acyl carrier protein transferase (mitochondrial type II fatty acid synthase) YPL148C PPT2, acyl carrier-protein synthase, phosphopantetheine protein transferase for Acp1p (maybe mitochondrial) 7