3.3.1. Influence of the *aF145S* Mutation on Yeast Respiratory Growth

The *a*F145S mutant grew well from fermentable substrates like glucose (Figure 3A), where ATP synthase is not required. Mitochondrial-dependent growth on glycerol was also normal at both 28 ◦C (the optimal temperature for mitochondrial function in yeast), and at 36 ◦C (Figure 3A). However, in the presence of increasing concentrations of oligomycin, a chemical inhibitor of ATP synthase [43], respiratory growth of the *a*F145S mutant was less efficient compared to the WT (Figure 3A). An increased sensitivity to oligomycin is usually observed in yeast ATP synthase defective mutants because less of this drug is needed to reach the threshold of ATP synthase activity (20%) below which respiratory growth of yeast becomes obviously compromised [13,44]. The increased sensitivity to oligomycin of the *aF145S* mutant was further characterized by spreading the cells as a dense layer on glycerol medium and then exposed to a drop of oligomycin deposited on a sterile disk of paper (Figure 3B). Oligomycin diffuses in the growth medium, which results in the establishment of a continuous gradient around

the disk. Growth is inhibited until a certain drug concentration. The halos of growth inhibition had a much higher diameter for the mutant vs. wild type yeast (Figure 3B), consistent with the growth tests shown in Figure 3A. These in vivo observations provide a strong indication that the *aF145S* mutation has detrimental consequences on ATP synthase.

**Figure 3.** Consequences of an equivalent of the m.8909T>C variant (*aF145S*) in yeast. (**A**) Cells from the WT (MR6) and two genetically independent clones (denoted as 1 and 2) of the *aF145S* mutant strain (RKY108) grown in glucose were serially diluted and spotted on plates containing glucose, glycerol, and glycerol supplemented with indicated concentrations of oligomycin (Oligo). The plates were scanned after the indicated days of incubation. Representative data from two WT and two independent mutant clones and at least two repeats of each are shown. A mm scale is shown on the upper left image. (**B**) On the left panel, cells from WT and *aF145S* mutant strains were spread as dense layers onto rich glycerol solid media and then exposed to sterile filters spotted with 4 nmol oligomycin (Oligo) and DMSO as a negative control (solvent). The plates were scanned after 3 days of incubation at 28 ◦C and 36 ◦C. The shown scale is 1 cm. The diameters of the halos of growth inhibition (in % of WT) are reported in the shown histograms. \*\*\*\* indicates a *p-*value < 0.0001. (**C**) Assembly/stability of ATP synthase. Protein extracts were prepared from mitochondria isolated from WT and *aF145S* strains grown at 36 ◦C. Samples with a same content in porin were solubilized with 2% digitonin and resolved by Blue native–polyacrylamide gel electrophoresis (BN-PAGE, 200 µg proteins per lane). Dimers (V<sup>2</sup> ) and monomers (V<sup>1</sup> ) of F1F<sup>O</sup> complexes and free F<sup>1</sup> were in-gel visualized by their ATPase activity. The protein complexes were transferred onto nitrocellulose and probed with antibodies against subunit *c* (Atp9) and *a* (Atp6). On the left margin is a molecular weight ladder. (**D**) Western blot (WB) of mitochondrial proteins resolved by SDS-PAGE with antibodies against subunit *a* (Atp6) and Porin. The levels of Atp6 are normalized to Porin. The shown plates and gels have been cropped to eliminate samples not linked to this study and that were intercalated between those of interest (WT and *aF145S*). Representative data from at least two repeats are shown.

3.3.2. Influence of the *aF145S* Mutation on Mitochondrial Respiration and ATP Synthesis

The impact of the *aF145S* mutation on mitochondrial oxygen consumption and ATP synthesis was investigated in mitochondria extracted from cells grown at 36 ◦C in a rich galactose medium. Oxygen consumption was measured with NADH as an electron donor, alone (basal or State 4 respiration, which is induced only by the passive permeability to protons of the inner membrane), and after successive additions of ADP (State 3 or phosphorylating conditions, where respiration is normally twice

stimulated vs. State 4) and the uncoupler CCCP (thus without any membrane potential, which further stimulates 2-fold the rate of respiration vs. State 3) (Table 5). State 4 respiration was not increased in the *aF145S* vs. *WT* mitochondria, indicating that the inner membrane had a normal passive permeability to protons and that there were no proton leak through the membrane domain (FO) of ATP synthase. At State 3 as well as in the presence of CCCP, the rate of oxygen consumption was decreased by 20–30% in the mutant vs. WT mitochondrial samples, and the rate of ATP synthesis (at State 3) was diminished in similar proportions (Table 5). These data indicate that the *aF145S* mutation slows down the rate of ATP production with no loss in the yield in ATP per electron transferred to oxygen. A decreased capacity to transfer electrons to oxygen is usually observed in yeast ATP synthase defective mutants, except in those with FO-mediated proton leaks [10–12,30,45], from which it was argued that the proton translocation activity of ATP synthase modulates biogenesis of the respiratory system presumably as a mean to co-regulate mitochondrial electron transfer and ATP synthesis activities [46].


**Table 5.** Mitochondrial respiration and ATP synthesis.

Mitochondria were isolated from cell strains grown for 5–6 generations in YPGALA medium (rich galactose) at 36 ◦C. Reaction mixes contained 0.15 mg/mL of mitochondrial proteins, 4 mM NADH, 150 (for respiration assays) or 750 (for ATP synthesis) µM ADP, 4 µM CCCP, 3 µg/mL oligomycin (*oligo*). Respiratory and ATP synthesis activities were measured using freshly isolated, osmotically protected, mitochondria buffered at pH 6.8. The reported values are averages of two biological replicates and three technical replicates for each assay. Statistical significance of the data was tested using unpaired *t*-test (\* indicates a *p*-value < 0.05).
