Figure 4
Consequences of an equivalent of the G8969>A mutation (atp6-S175N) in yeast.
(A) WT and atp6-S175N mutant yeast cells grown in glucose were serially diluted and spotted onto plates containing glucose or glycerol; photographs were taken after 4 days of incubation. (B) Oxygen consumption (state 3) and ATP synthesis rates measured with NADH as an electron donor, expressed in % of the WT (see Supplementary Table S4 for details). (C) Variations in mitochondrial membrane potential (ΔΨ). These experiments were performed using Rhodamine 123, a fluorescent cationic dye. Increasing ΔΨ is followed by the uptake of the dye inside the matrix space and concomitant fluorescence quenching. The tracings in the upper panel reveal the capacity of externally added ADP to induce ΔΨ consumption after energization of the mitochondrial inner membrane by the respiratory chain (WT in blue, the mutant in red). The tracings in the bottom directly reflect the proton-pumping activity of ATP synthase using externally added ATP, after inhibition of the respiratory chain EtOH, ethanol; Oligo, oligomycin; Mito, mitochondria. (D) BN-PAGE analysis of mitochondrial protein extracts solubilized with 2% digitonin. ATP synthase complexes (V2, dimeric; V1, monomeric) and free F1 particles were visualized in-gel by their ATPase activity and by western blotting after transfer onto nitrocellulose using antibodies against subunits Atp1p (α-F1) and Atp6p (subunit-6).
