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 extrtransmembrane 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.