Organelle Proteomics pp 65-81

Part of the Methods in Molecular Biology™ book series (MIMB, volume 432)

Preparation of Respiratory Chain Complexes from Saccharomyces cerevisiae Wild-Type and Mutant Mitochondria

Activity Measurement and Subunit Composition Analysis
  • Claire Lemaire
  • Geneviève Dujardin


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.

Key words

S. cerevisiae mitochondria respiratory complexes BN–PAGE respiratory activities 


  1. 1.
    Meisinger, C., Sommer, T., and Pfanner, N. (2000) Purification of Saccharomyces cerevisiae. Mitochondria devoid of microsomal and cytosolic contaminations. Anal. Biochem. 287, 339–342.CrossRefPubMedGoogle Scholar
  2. 2.
    Hatefi, Y. and Stiggall, D. (1978) Preparation and properties of succinate: ubiquinone oxidoreductase (complex II). Methods Enzymol. 53, 21–27.CrossRefPubMedGoogle Scholar
  3. 3.
    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.PubMedGoogle Scholar
  4. 4.
    Ryan, M. T., Voos, W., and Pfanner, N. (2001) Assaying protein import into mitochondria. Methods Cell Biol. 65, 189–215.CrossRefPubMedGoogle Scholar
  5. 5.
    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.CrossRefPubMedGoogle Scholar
  6. 6.
    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.CrossRefPubMedGoogle Scholar
  7. 7.
    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.CrossRefGoogle Scholar
  8. 8.
    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.CrossRefPubMedGoogle Scholar
  9. 9.
    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.CrossRefPubMedGoogle Scholar
  10. 10.
    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.PubMedGoogle Scholar
  11. 11.
    Rieske, J. S. (1967) Preparation and properties of a respiratory chain iron-protein. Methods Enzymol. 10, 239–245.CrossRefGoogle Scholar
  12. 12.
    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.CrossRefPubMedGoogle Scholar
  13. 13.
    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.CrossRefPubMedGoogle Scholar
  14. 14.
    Dutta, C. and Henry, H. L. (1990) Detection of hemoprotein peroxidase activity on polyvinylidene difluoride membrane. Anal. Biochem. 184, 96–99.CrossRefPubMedGoogle Scholar
  15. 15.
    Vargas, C., McEwan, A. G., and Downie, J. A. (1993) Detection of c-type cytochromes using enhanced chemiluminescence. Anal. Biochem. 209, 323–326.CrossRefPubMedGoogle Scholar
  16. 16.
    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.CrossRefPubMedGoogle Scholar
  17. 17.
    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.Google Scholar
  18. 18.
    Somlo, M. (1968) Induction and repression of mitochondrial ATPase in yeast. Eur. J. Biochem. 5, 276–284.CrossRefPubMedGoogle Scholar
  19. 19.
    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.PubMedGoogle Scholar
  20. 20.
    Nijtmans, L., Henderson, N., and Holt, I. (2002) Blue native electrophoresis to study mitochondrial and other protein complexes. Methods 26, 327–334.CrossRefPubMedGoogle Scholar
  21. 21.
    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.CrossRefPubMedGoogle Scholar
  22. 22.
    Schagger, H. and Pfeiffer, K. (2000) Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19, 1777–1783.CrossRefPubMedGoogle Scholar
  23. 23.
    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.CrossRefPubMedGoogle Scholar
  24. 24.
    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.CrossRefPubMedGoogle Scholar
  25. 25.
    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.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Claire Lemaire
    • 1
  • Geneviève Dujardin
    • 2
  1. 1.CNRS, Centre de Génétique MoléculaireGif sur Yvette
  2. 2.CNRS, Centre de Génétique MoléculaireAvenue de la TerrasseGif sur Yvette

Personalised recommendations