Evidence that there are two copies of subunit c" in V0 complexes in the vacuolar H+-ATPase.

The proton-translocating core of eukaryotic vacuolar H(+)-ATPase (V-ATPase), V(0) consists of a hexameric arrangement of transmembrane alpha-helices formed from the related polypeptides, subunit c and subunit c". The former is comprised of four transmembrane alpha-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V(0) contains a minor subunit c-related polypeptide, subunit c'. All three are required for activity of the proton pump in Saccharomyces cerevisiae. We have introduced cysteine residues in the N-terminal extension of subunit c" in a cysteine-free form. All mutant forms are active in the V-ATPase from S. cerevisiae. Oxidation of vacuolar membranes containing the cysteine-replaced forms gave a cross-linked product of 42000Da. Analysis of this species showed it to be a dimeric form of subunit c", and further studies confirmed there are two copies of subunit c" in the V-ATPases in which it is present. Co-expression of double cysteine-replaced forms of both subunit c and c" gave rise to only homotypic cross-linked forms. Also, subunit c oligomeric complexes are present in vacuolar membranes in the absence of subunit c", consistent with previous observations showing hexameric arrangements of subunit c in gap-junction-like membranes. In vitro studies showed subunit c" can bind to subunit c and itself. The extent of binding can be increased by removal of the N-terminal domain of subunit c". This domain may therefore function to limit the copy number of subunit c" in V(0). A deletion study shows that the domain is essential for the activity of subunit c". The results can be combined into a model of V(0) which contains two subunit c" protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V(0) in which subunit c" is present is subunit c(3):subunit c'(1):subunit c"(2). On the basis of the mutational and binding studies, it seems likely that two copies of subunit c" are next to each other.