PMC

The diagram shows a linear correlation between the ATP synthesis rate (expressed as % of the wild type activity) and cytochrome c oxidase activity (expressed as % of the wild type activity) in Δatp6, atp6-L183P (T8993C), atp6-L183R (T8993G), atp6-L247P (T9176C) and atp6-L247R (T9176G) strains.

Panel A: Freshly grown cells of wild type yeast (MR6) and atp6-L247P mutant (RKY38) were serially diluted and 5 μl of each dilution were spotted onto YPGA (glucose) and N3 (glycerol) plates. The plates were incubated at 28°C and photographed after three (YPGA) or six (N3) days.

Energization of the mitochondrial inner membrane was monitored by rhodamine 123 fluorescence quenching with intact mitochondria from wild type yeast (MR6) and atp6-L247P mutant (RKY38). The additions were 0.5 μg/ml rhodamine 123, 0.15 mg/ml mitochondrial proteins (Mito), 10 μl of ethanol (EtOH), 6 μg/ml oligomycin (oligo), 0.2 mM potassium cyanide (KCN), 1 mM ATP, and 3 μM CCCP. Data are representative of at least 3 experiments.

Proteins encoded by the mtDNA were in vivo labeled with [³⁵S]-(methionine + cysteine) for 20 min. at 28°C in the presence of cycloheximide to inhibit cytosolic protein synthesis. Turnover of newly synthesized proteins was evaluated by the addition of excess cold (methionine + cysteine) and incubation for the indicated periods of time. After the labeling reactions, total protein extracts were prepared from the cells (0.2 OD at 650 nm) and loaded on a 12.5% polyacrylamide-4 M urea gel containing 25% glycerol. After electrophoresis the gel was dried and radioactive proteins visualized with a phosphoImager.

Panels A, B: BN-PAGE analyses of mitochondrial proteins from the yeast T9176C (atp6-L247P). For the analysis of complexes III and IV (panel A) the mitochondria (100 μg) were solubilized with 10 grams of digitonin per gram of proteins while 2 grams of digitonin per gram of proteins were used for the analysis of the ATP synthase (Panel B). After their electrophoretic separation the digitonin-extracted proteins (50 μg) were transferred to a nitrocellulose membrane and hybrized with the indicated antibodies. For the blots shown in panel B, between two hybridizations, the membrane was stripped to completely remove the previously hybridized antibodies. Panel C, SDS-PAGE analysis. Total mitochondrial proteins (10 μg) of atp6-L247P mutant and wild type strain MR6 were separated in SDS-PAGE, transferred onto a nitrocellulose membrane and probed with the indicated antibodies. Data are representative of at least 3 experiments.

Panel A: Freshly grown cells of wild type yeast (MR6), atp6-L247P mutant (RKY38), and a strain lacking the leader peptide of Atp6p (RKY48, atp6-Δleader) [] were serially diluted and 5 μl of each dilution spotted onto glucose (YPGA) and glycerol (N3) plates, and onto glycerol plates containing the indicated concentrations of oligomycin (Oligo). The plates were incubated at 28°C and photographed after 4 days. Panel B: Growth of yeast atp11 mutants on EG ± oligomycin. Wild type and atp11 mutant strains described in [] were grown overnight on rich glucose plates (2% glucose, 2% bactopeptone, 1% yeast extract), replica-plated to non-fermentable media (3% glycerol, 2% ethanol, 2% bactopeptone, 1% yeast extract) that was either untreated (EG) or supplemented with oligomycin to 2% (EGO), and incubated at 30°C for 48 h. From top to bottom, the strains analyzed were W303-1A, W303ΔATP11, and W303ΔATP11 transformants harboring plasmids for Atp11(R183)p, Atp11(Δ40–111)p, Atp11(Δ40–75)p, or Atp11(A300)p. The amount of oligomycin-sensitive ATPase activity measured in mitochondria isolated from each strain is shown on the left. For stylistic reasons, two separate regions of the same photograph were fused (white dotted line) to create the digital image of the EG and EGO plates shown in the figure.