*2.7. Biochemical Investigation of Mitochondria*

Oxygen consumption measurements in mitochondria isolated from yeast cells grown in complete galactose medium were performed according to [32], using a Clark electrode (Heito, Paris, France). Freshly prepared mitochondria were added to 1 mL of respiration buffer (10 mM Tris-maleate pH 6.8, 0.65 M sorbitol, 0.3 mM EGTA, and 3 mM potassium phosphate) at 0.15 mg/mL in the reaction chamber maintained at 28 ◦C. The oxygen consumption was measured after successive additions of 4 mM NADH, 150 µM ADP, 4 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP). The rate of ATP synthesis was measured in the same conditions but with 750 µM ADP. Aliquots were withdrawn every 15 s from the reaction mix and supplemented with 3.5% (*w*/*v*) perchloric acid and 12.5 mM EDTA. After neutralization of the samples to pH 6.5 with KOH 0.3 M/MOPS, ATP was quantified by luciferin/luciferase assay (ATPLite kit from Perkin Elmer, Waltham, MA, USA) on a LKB bioluminometer. Oligomycin (3 µg/mL) was used to determine the amount of ATP produced by ATP synthase. ATPase activity was measured in non-osmotically protected mitochondria at pH 8.4 as described [33]. Blue native polyacrylamide gel electrophoresis (BN-PAGE) was performed as described [34]. For this, 200 µg of mitochondrial proteins were suspended in 50 µL of extraction buffer (30 mM HEPES, 150 mM potassium acetate, 12% glycerol, 2 mM 6-aminocaproic acid, 1 mM EGTA, 2% digitonin (Sigma), one protease inhibitor cocktail tablet (Roche) (pH 7.4) and incubated for 30 min on ice. After centrifugation (14,000 rpm, 4 ◦C, 30 min) the supernatant containing the solubilized complexes were supplemented with 2.25 µL of loading dye (5% Serva Blue G-250, 750 mM 6-aminocaproic acid) and run into NativePAGE 3–12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA). After transfer onto PVDF membrane the yeast ATP synthase complexes were detected with polyclonal antibodies against α-F<sup>1</sup> (Atp1), subunit *c* (Atp9) and subunit *a* (Atp6) used after 1:10,000, 1:5000, and 1:1000 dilutions respectively. The Atp1 antibodies were kindly provided by J. Velours. Anti-Atp9 antibodies were prepared by Eurogentec (Seraing, Belgium) with the synthetic peptide corresponding to the loop connecting the two transmembrane helices of Atp9 as an immunogen. The procedure used to in-gel visualize ATP synthase by its ATPase activity is described in [31].
