Abstract
The Saccharomyces cerevisiae vacuolar membrane proton-translocating ATPase (V-ATPase) can be divided into a peripheral membrane complex (V1) containing at least eight polypeptides of 69, 60, 54, 42, 32, 27, 14, and 13 kDa, and an integral membrane complex (V0) 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 (vma12Delta), the 100-kDa subunit of the V0 complex (which contains six to eight putative membrane-spanning domains) was rapidly degraded (t1/2 approximately 30 min). Protease protection assays revealed that the 100-kDa subunit was inserted/translocated correctly into the ER membrane of vma12Delta cells. These data indicate that Vma12p functions in the ER after the insertion of V0 subunits into the ER membrane. We propose that Vma12p functions directly in the assembly of the V0 subunits into a complex in the ER, and that assembly is required for the stability of the V0 subunits and their transport as a complex out of this compartment.
Highlights
The yeast V-ATPase is similar in both structure and subunit composition to the F1F0-ATP synthase of mitochondria
These results indicate that Vma12p functions in assembly of the V0 membrane complex after V0 subunits are inserted into the endoplasmic reticulum (ER) membrane
We have characterized Vma12p in an effort to elucidate its function in yeast cells
Summary
The yeast V-ATPase is similar in both structure and subunit composition to the F1F0-ATP synthase of mitochondria. In cells lacking a V0 subunit, no V-ATPase subunits are found associated with the vacuolar membrane; V1 subunits assemble into a stable complex in the cytosol (20 –21), whereas other V0 subunits are present at reduced levels [18]. Yeast cells lacking Vma12p (vma12⌬) rapidly degrade the 100-kDa V0 subunit (Vph1p) at a rate that is independent of vacuolar proteases or secretory traffic exiting the ER. Vph1p is localized to the ER membrane in vma12⌬ cells, where it is translocated and inserted normally These results indicate that Vma12p functions in assembly of the V0 membrane complex after V0 subunits are inserted into the ER membrane
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