In this study yeast mitochondria were used as a model system to apply, evaluate, and integrate different genomic approaches to define the proteins of an organelle. Liquid chromatography mass spectrometry applied to purified mitochondria identified 546 proteins. By expression analysis and comparison to other proteome studies, we demonstrate that the proteomic approach identifies primarily highly abundant proteins. By expanding our evaluation to other types of genomic approaches, including systematic deletion phenotype screening, expression profiling, subcellular localization studies, protein interaction analyses, and computational predictions, we show that an integration of approaches moves beyond the limitations of any single approach. We report the success of each approach by benchmarking it against a reference set of known mitochondrial proteins, and predict approximately 700 proteins associated with the mitochondrial organelle from the integration of 22 datasets. We show that a combination of complementary approaches like deletion phenotype screening and mass spectrometry can identify over 75% of the known mitochondrial proteome. These findings have implications for choosing optimal genome-wide approaches for the study of other cellular systems, including organelles and pathways in various species. Furthermore, our systematic identification of genes involved in mitochondrial function and biogenesis in yeast expands the candidate genes available for mapping Mendelian and complex mitochondrial disorders in humans.
References
[1]
Achleitner G, Gaigg B, Krasser A, Kainersdorfer E, Kohlwein SD, et al. (1999) Association between the endoplasmic reticulum and mitochondria of yeast facilitates interorganelle transport of phospholipids through membrane contact. Eur J Biochem 264: 545–553.
[2]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
[3]
Andreoli C, Prokisch H, Hortnagel K, Mueller JC, Munsterkotter M, et al. (2004) MitoP2: An integrated database on mitochondrial proteins in yeast and man. Nucleic Acids Res 32: D459–D462.
[4]
DeRisi JL, Iyer VR, Brown PO (1997) Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278: 680–686.
[5]
DeSouza L, Shen Y, Bognar AL (2000) Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in . Arch Biochem Biophys 376: 299–312.
[6]
DiMauro S, Schon EA (1998) Nuclear power and mitochondrial disease. Nat Genet 19: 214–215.
[7]
Dimmer KS, Fritz S, Fuchs F, Messerschmitt M, Weinbach N, et al. (2002) Genetic basis of mitochondrial function and morphology in . Mol Biol Cell 13: 847–853.
[8]
Drawid A, Gerstein M (2000) A Bayesian system integrating expression data with sequence patterns for localizing proteins: Comprehensive application to the yeast genome. J Mol Biol 301: 1059–1075.
[9]
Eng JK, McCormack AL, Yates JR (1994) An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J Am Soc Mass Spectrom 5: 976–989.
[10]
Ferguson PL, Smith RD (2003) Proteome analysis by mass spectrometry. Annu Rev Biophys Biomol Struct 32: 399–424.
[11]
Foury F (1997) Human genetic diseases: A cross-talk between man and yeast. Gene 195: 1–10.
[12]
Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, et al. (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415: 141–147.
[13]
Ghaemmaghami S, Huh WK, Bower K, Howson RW, Belle A, et al. (2003) Global analysis of protein expression in yeast. Nature 425: 737–741.
[14]
Glick BS, Pon LA (1995) Isolation of highly purified mitochondria from . Methods Enzymol 260: 213–223.
[15]
Ho Y, Gruhler A, Heilbut A, Bader GD, Moore L, et al. (2002) Systematic identification of protein complexes in by mass spectrometry. Nature 415: 180–183.
[16]
Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, et al. (2003) Global analysis of protein localization in budding yeast. Nature 425: 686–691.
[17]
Ito T, Chiba T, Ozawa R, Yoshida M, Hattori M, et al. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci U S A 98: 4569–4574.
[18]
Kumar A, Agarwal S, Heyman JA, Matson S, Heidtman M, et al. (2002) Subcellular localization of the yeast proteome. Genes Dev 16: 707–719.
[19]
Lascaris R, Bussemaker HJ, Boorsma A, Piper M, van der Spek H, et al. (2003) Hap4p overexpression in glucose-grown induces cells to enter a novel metabolic state. Genome Biol 4: R3.
[20]
Lipton MS, Pasa-Tolic L, Anderson GA, Anderson DJ, Auberry DL, et al. (2002) Global analysis of the proteome by using accurate mass tags. Proc Natl Acad Sci U S A 99: 11049–11054.
[21]
Marc P, Margeot A, Devaux F, Blugeon C, Corral-Debrinski M, et al. (2002) Genome-wide analysis of mRNAs targeted to yeast mitochondria. EMBO Rep 3: 159–164.
[22]
Mewes HW, Frishman D, Guldener U, Mannhaupt G, Mayer K, et al. (2002) MIPS: A database for genomes and protein sequences. Nucleic Acids Res 30: 31–34.
[23]
Nakai K, Horton P (1999) PSORT: A program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci 24: 34–36.
[24]
Ohlmeier S, Kastaniotis AJ, Hiltunen JK, Bergmann U (2003) The yeast mitochondrial proteome: A study of fermentative and respiratory growth. J Biol Chem 279: 3956–3979.
[25]
Patterson SD, Aebersold RH (2003) Proteomics: The first decade and beyond. Nat Genet 33: (Suppl)311–323.
[26]
Pflieger D, Le Caer JP, Lemaire C, Bernard BA, Dujardin G, et al. (2002) Systematic identification of mitochondrial proteins by LC-MS/MS. Anal Chem 74: 2400–2406.
[27]
Scharfe C, Zaccaria P, Hoertnagel K, Jaksch M, Klopstock T, et al. (2000) MITOP, the mitochondrial proteome database: 2000 update. Nucleic Acids Res 28: 155–158.
[28]
Shen Y, Zhao R, Belov ME, Conrads TP, Anderson GA, et al. (2001) Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics. Anal Chem 73: 1766–1775.
[29]
Sickmann A, Reinders J, Wagner Y, Joppich C, Zahedi R, et al. (2003) The proteome of mitochondria. Proc Natl Acad Sci U S A 100: 13207–13212.
[30]
Small I, Peeters N, Legeai F, Lurin C (2004) Predotar: A tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics. In press.
[31]
Smith RD, Anderson GA, Lipton MS, Pasa-Tolic L, Shen Y, et al. (2002) An accurate mass tag strategy for quantitative and high-throughput proteome measurements. Proteomics 2: 513–523.
[32]
Steinmetz LM, Scharfe C, Deutschbauer AM, Mokranjac D, Herman ZS, et al. (2002) Systematic screen for human disease genes in yeast. Nat Genet 31: 400–404.
[33]
Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, et al. (2000) A comprehensive analysis of protein–protein interactions in . Nature 403: 623–627.
[34]
von Mering C, Krause R, Snel B, Cornell M, Oliver SG, et al. (2002) Comparative assessment of large-scale data sets of protein–protein interactions. Nature 417: 399–403.
[35]
Wallace DC (1999) Mitochondrial diseases in man and mouse. Science 283: 1482–1488.
[36]
Washburn MP, Wolters D, Yates JR (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19: 242–247.
[37]
Washburn MP, Koller A, Oshiro G, Ulaszek RR, Plouffe D, et al. (2003) Protein pathway and complex clustering of correlated mRNA and protein expression analyses in . Proc Natl Acad Sci U S A 100: 3107–3112.
[38]
Westermann B, Neupert W (2003) ‘Omics' of the mitochondrion. Nat Biotechnol 21: 239–240.
[39]
Wu CC, MacCoss MJ, Howell KE, Yates JR (2003) A method for the comprehensive proteomic analysis of membrane proteins. Nat Biotechnol 21: 532–538.
[40]
Zischka H, Weber G, Weber PJ, Posch A, Braun RJ, et al. (2003) Improved proteome analysis of mitochondria by free-flow electrophoresis. Proteomics 3: 906–916.
