Molecular genetic analysis of the central hydrophobic domain of subunit 8 of yeast mitochondrial ATP synthase

Subunit 8 (Y8) of yeast mitochondrial ATP synthase (mtATPase) is a hydrophobic component of the membrane F₀ sector. Encoded by the mitochondrial aap1 gene, Y8 is a 48-amino-acid polypeptide having a central hydrophobic domain (CHD) spanning 19 residues. Site-directed mutagenesis was carried out on a nuclear code-equivalent gene encoding Y8, to introduce either adjacent charged amino acids (positive or negative) or proline residues into the CHD, or to alter the length of this domain by deletion or insertion of additional non-polar residues. We report a functional resilience of Y8 in tolerating the introduction of charged residues implanted within the CHD. Thus, expression of variants having adjacent positively charged amino acids (arginines) in Y8-deficient cells restored growth on the non-fermentable substrate ethanol, though in some cases this was impaired compared to that conferred by the parent Y8 construct. Introduction of adjacent negative charges (aspartate residues) was less well tolerated, but in all cases a measurable rate of cell growth on ethanol was retained. These results underscore the interpretation that it is not necessary for Y8 to maintain a transmembrane stem in its role as an integral component of functional mtATPase. Further, the impaired growth properties of cells expressing variants of Y8 having changes designed to perturb the structure (proline substitutions) and length (insertions or deletions) of the CHD lead us to conclude that the overall shape and dimensions of Y8 are important for its function in mtATPase.