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 hyd rophobic component of the membrane F-o sector. Encoded by the mitochon drial aap1 gene, Y8 is a 48-amino-acid polypeptide having a central hy drophobic domain (CHD) spanning 19 residues. Site-directed mutagenesis was carried out on a nuclear code-equivalent gene encoding Y8, to int roduce either adjacent charged amino acids (positive or negative) or p roline residues into the CHD, or to alter the length of this domain by deletion or insertion of additional non-polar residues. We report a f unctional resilience of Y8 in tolerating the introduction of charged r esidues implanted within the CHD. Thus, expression of variants having adjacent positively charged amino acids (arginines) in Y8-deficient ce lls restored growth on the non-fermentable substrate ethanol, though i n some cases this was impaired compared to that conferred by the paren t Y8 construct. Introduction of adjacent negative charges (aspartate r esidues) was less well tolerated, but in all cases a measurable rate o f cell growth on ethanol was retained. These results underscore the in terpretation that it is not necessary for Y8 to maintain a transmembra ne stem in its role as an integral component of functional mtATPase. F urther, the impaired growth properties of cells expressing variants of Y8 having changes designed to perturb the structure (proline substitu tions) and length (insertions or deletions) of the CHD lead us to conc lude that the overall shape and dimensions of Y8 are important for its function in mtATPase.