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 cer evisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichio metry of these subunits by the application of hexahistidine affinity purifi cation technology, We have previously demonstrated that intact mtATPase com plexes incorporating a Hex(6)-tagged subunit can be isolated via Ni2+-nitri lotriacetic acid affinity chromatography (Bateson, RI., Devenish, R, J., Na gley, 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 re sulted in a mixed population of mtATPase complexes containing untagged wild -type and Hex(6)-tagged subunits, The stoichiometry of each subunit was the n assessed by determining whether or not the untagged wild-type subunit cou ld be recovered from Ni2+-nitrilotriacetic acid purifications as an integra l component of those complexes absorbed by virtue of the Hex(6)-tagged subu nit, As only the Hex(6)-tagged subunit was recovered from such purification s, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeas t is 1 in each case.