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), V₀ consists of a hexameric arrangement of transmembrane α-helices formed from the related polypeptides, subunit c and subunit c′′. The former is comprised of four transmembrane α-helices, whilst the latter has an extra transmembrane domain at its N-terminus. In addition, the fungal form of V₀ 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 V₀. A deletion study shows that the domain is essential for the activity of subunit c′′. The results can be combined into a model of V₀ which contains two subunit c′′ protomers with the extra transmembrane domain located toward the central pore. Thus the predicted stoichiometry of V₀ in which subunit c′′ is present is subunit c₃: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.