Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology, We have previously demonstrated that intact mtATPase complexes incorporating a Hex(6)-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, RI., Devenish, R, J., Nagley, P., and Prescott, M, (1996) Anal. Biochem. 238, 14-18), Strains were constructed in which Hex(6)-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex(6)-tagged subunits, The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex(6)-tagged subunit, As only the Hex(6)-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.