Organelle Proteomics

Volume 432 of the series Methods in Molecular Biology™ pp 65-81

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

Activity Measurement and Subunit Composition Analysis
  • Claire LemaireAffiliated withCNRS, Centre de Génétique Moléculaire
  • , Geneviève DujardinAffiliated withCNRS, Centre de Génétique Moléculaire, Avenue de la Terrasse

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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