The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V-1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V-0) containing at least five polypeptides of 100, 36, 23, 17, and 16 kDa. Other yeast genes have been identified that are required for V-ATPase assembly but whose protein products do not co-purify with the enzyme complex. One such gene, VMA12, encodes a 25-kDa protein (Vma12p) that is predicted to contain two membrane-spanning domains. Biochemical analysis has revealed that Vma12p behaves as an integral membrane protein with both the N and C termini oriented toward the cytosol, and this protein immunolocalizes to the endoplasmic reticulum (ER). In cells lacking Vma12p (vma12 Delta), the 100-kDa subunit of the V-0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t(1/2) similar to 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12 Delta cells. These data indicate that Vma12p functions in the ER after the insertion of V-0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V-0 subunits into a complex in the ER, and that assembly is required for the stability of the V-0 subunits and their transport as a complex out of this compartment.
