3.3.3. Influence of the *aF145S* Mutation on ATP Synthase Assembly/Stability

The ATP synthase organizes into a membrane-extrinsic domain (F1) and a domain (FO) largely anchored in the inner membrane [47–49]. The subunit *a* and a ring of identical subunits *c* move protons through the FO. As a result of this, the *c*-ring rotates and provokes conformational changes in the F<sup>1</sup> that promote ATP synthesis. In current models, the assembly of ATP synthase starts with formation of F1, followed by its association to the *c*-ring and peripheral stalk subunits that prevent rotation of the catalytic subdomain (αβ3) of F1. The process ends with incorporation of subunit *a* [50]. When incorporation of subunit *a* is compromised, the F<sup>1</sup> and the *c*-ring easily dissociate during BN-PAGE analysis of mitochondrial proteins, as was observed in a yeast strain lacking the *ATP6* gene [15,30,51]. In addition to fully assembled monomers and dimers of ATP synthase, free F<sup>1</sup> and *c*-ring were detected in mitochondrial samples from the *aF145S* mutant whereas these particles were absent in those from *WT* yeast, as revealed by Western blot with subunit *c* (Atp9) antibodies and by the in-gel F1-mediated ATP hydrolytic activity (Figure 3C). The steady state levels of the mutated subunit *a* were estimated by Western blot (WB) with Atp6 antibodies of mitochondrial proteins resolved in denaturing gels. They were significantly reduced in the mutant vs. *WT* (Figure 3D). Taken together these data show that the *aF145S* change partially compromises a stable incorporation of subunit *a* within ATP synthase.
