Summary
The mitochondrial oxidative phosphorylation involves five multimeric complexes imbedded in the inner membrane: complex I (Nicotinamide Adenine Dinucleotide (NADH) quinone oxidoreductase), II (succinate dehydrogenase), III (ubiquinol cytochrome c oxido reductase or bc1 complex), IV (cytochrome c oxidase), and V (ATP synthase). These respiratory complexes are conserved from the yeast Saccharomyces cerevisiae to human with the exception of complex I, which is replaced by three NADH dehydrogenases in S. cerevisiae. Here, we provide several protocols allowing an exhaustive characterization of each yeast complex: this chapter describes procedures from mitochondria preparation to measurement of the activity of each complex and analysis of their subunit composition and provides information on the interactions between different complexes.
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References
Meisinger, C., Sommer, T., and Pfanner, N. (2000) Purification of Saccharomyces cerevisiae. Mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 287, 339–342.
Hatefi, Y. and Stiggall, D. (1978) Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol. 53, 21–27.
Bousquet, I., Dujardin, G., and Slonimski, P. (1991) ABC1, a novel yeast nuclear gene has a dual function in mitochondria: it suppresses a cytochrome b mRNA translation defect and is essential for the electron transfer in the bc1 complex. EMBO J. 10, 2023–2031.
Ryan, M. T., Voos, W., and Pfanner, N. (2001) Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215.
Schagger, H. and von Jagow, G. (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199, 223–231.
Sellem, C. H., Lemaire, C., Lorin, S., Dujardin, G., and Sainsard-Chanet, A. (2005) Interaction between the oxa1 and rmp1 genes modulates respiratory complex assembly and life span in Podospora anserina. Genetics 169, 1379–1389.
Pflieger, D., Le Caer, J. P., Lemaire, C., Bernard, B. A., Dujardin, G., and Rossier, J. (2002) Systematic identification of mitochondrial proteins by LC-MS/MS. Anal. Chem. 74, 2400–2406.
Sickmann, A., Reinders, J., Wagner, Y., Joppich, C., Zahedi, R., Meyer, H. E., et al. (2003) The proteome of Saccharomyces cerevisiae mitochondria. Proc. Natl. Acad. Sci. U.S.A. 100, 13207–13212.
Dujardin, G., Pajot, P., Groudinsky, O., and Slonimski, P. (1980) Long range control circuits within mitochondria and between nucleus and mitochondria. I. Methodology and phenomenology of suppressors. Mol. Gen. Genet. 179, 469–482.
Yonetani, T. (1965) Studies on cytochrome c peroxydase II – stoichiometry between enzyme, H\(_{2}\)O\(_{2}\), and ferricytochrome c and enzymic determination of extinction coefficients of cytochrome c. J. Biol. Chem. 240, 4509–4513.
Rieske, J. S. (1967) Preparation and properties of a respiratory chain iron-protein. Methods Enzymol. 10, 239–245.
Wallis, M., Groudinsky, O., Slonimski, P., and Dujardin, G. (1994) The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix. Eur. J. Biochem. 222, 27–32.
Claisse, M. L., Pere-Aubert, G. A., Clavilier, L. P., and Slonimski, P. P. (1970) Method for the determination of cytochrome concentrations in whole yeast cells. Eur. J. Biochem 16, 430–438.
Dutta, C. and Henry, H. L. (1990) Detection of hemoprotein peroxidase activity on polyvinylidene difluoride membrane. Anal. Biochem. 184, 96–99.
Vargas, C., McEwan, A. G., and Downie, J. A. (1993) Detection of c-type cytochromes using enhanced chemiluminescence. Anal. Biochem. 209, 323–326.
Brasseur, G., Coppee, J. Y., Colson, A. M., and Brivet-Chevillotte, P. (1995) Structure-function relationships of the mitochondrial bc1 complex in temperature-sensitive mutants of the cytochrome b gene, impaired in the catalytic center N. J. Biol. Chem. 270, 29356–29364.
Pajot, P., Wambier-Kluppel, M., Kotylak, Z., and Slonimski, P. P. (1976) Regulation of cytochrome oxidase formation by mutations in a mitochondrial gene for cytochrome b. In Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, T. H., Neupert, W., Sebald, W. and Werner, S., eds), Elsevier, North Holland, and Biochemical Press, Amsterdam, 443–451.
Somlo, M. (1968) Induction and repression of mitochondrial ATPase in yeast. Eur. J. Biochem. 5, 276–284.
Kassenbrock, C., Cao, W., and Douglas, M. (1993) Genetic and biochemical characterization of ISP6, a small mitochondrial outer membrane protein associated with the protein translocation complex. EMBO J. 12, 3023–3034.
Nijtmans, L., Henderson, N., and Holt, I. (2002) Blue native electrophoresis to study mitochondrial and other protein complexes. Methods 26, 327–334.
Cruciat, C., Brunner, S., Baumann, F., Neupert, W., and Stuart, R. (2000) The cytochrome bc1 and cytochrome c oxidase complexes associate to form a single supracomplex in yeast mitochondria. J. Biol. Chem. 275, 18093–18098.
Schagger, H. and Pfeiffer, K. (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19, 1777–1783.
Soubannier, V., Vaillier, J., Paumard, P., Coulary, B., Schaeffer, J., and Velours, J. (2002) In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize. J. Biol. Chem. 277, 10739–10745.
Lemaire, C., Guibet-Grandmougin, F., Angles, D., Dujardin, G., and Bonnefoy, N. (2004) A yeast mitochondrial membrane methyltransferase-like protein can compensate for oxa1 mutations. J. Biol. Chem. 279, 47464–47472.
Bonnefoy, N., Chalvet, F., Hamel, P., Slonimski, P., and Dujardin, G. (1994) OXA1, a Saccharomyces cerevisiae nuclear gene whose sequence is conserved from prokaryotes to eukaryotes controls cytochrome oxidase biogenesis. J. Mol. Biol. 239, 201–212.
Acknowledgments
We thank N. Lachacinski and S. Marsy for excellent technical assistance; Drs T.D. Fox and J. Velours for antibodies; and Dr. P. Hamel for critical reading of the manuscript and for looking over the English. This work was supported by grants from the “Association Franccaise contre les Myopathies” and from the C.N.R.S. program “Prot’eomique et G’enie des Prot’eines”.
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Lemaire, C., Dujardin, G. (2008). Preparation of Respiratory Chain Complexes from Saccharomyces cerevisiae Wild-Type and Mutant Mitochondria. In: Pflieger, D., Rossier, J. (eds) Organelle Proteomics. Methods in Molecular Biology™, vol 432. Humana Press. https://doi.org/10.1007/978-1-59745-028-7_5
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DOI: https://doi.org/10.1007/978-1-59745-028-7_5
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