The vacuolar-type H{dollar}sp+{dollar}-translocating ATPase (V-ATPase) is a multisubunit enzyme found in all eukaryotic cells. V-ATPases acidify the lumen of many organelles, play a major role in a wide variety of cellular processes, and play a crucial role in the function of many specialized cells. The Saccharomyces cerevisiae V-ATPase can be divided into a peripheral membrane complex (V{dollar}sb1{dollar}) containing at least 69, 60, 54, 42, 32, 27, 14, and 13 kD polypeptides and an integral membrane complex (V{dollar}sb0{dollar}) containing at least 100, 36, 22.6, 17.0, and 16.3 kD polypeptides.; The yeast VMA12 gene encodes a 25 kD protein that is required for V-ATPase assembly but that does not co-purify with the V-ATPase enzyme. Vma12p was characterized in order to understand its role in the V-ATPase assembly process. Vma12p was found to be an integral membrane protein of the endoplasmic reticulum (ER). The absence of Vma12p caused the 100 kD V-ATPase subunit Vph1p to become unstable but did not affect its translocation across and insertion into the ER membrane. These results indicate that Vma12p is a V-ATPase assembly factor/chaperone that functions directly in the assembly of the V{dollar}sb0{dollar} subunits into a complex in the ER. V{dollar}sb0{dollar} assembly is required for the stability of V{dollar}sb0{dollar} subunits as well as their transport as a complex out of the ER. Vma12p may also function in the loading of V{dollar}sb0{dollar} complexes into ER-derived secretory vesicles.; The VPH1 gene encodes a topologically complex 100 kD V-ATPase subunit whose precise function is unknown. Vph1p can be divided into a hydrophilic N-terminus, and a hydrophobic C-terminus which contains either 6 or 7 transmembrane domains. In this analysis, Vph1p was shown to adopt a topology with its N- and C-termini in the cytosol. Stv1p, a Golgi-localized, organelle-specific isoform of Vph1p, was found to be unstable in cells lacking V{dollar}sb0{dollar} subunits or assembly factors. Vma12p, and most likely assembly factors Vma21p and Vma22p, assembles Stv1p-containing Golgi V-ATPases in addition to assembling Vph1p-containing vacuolar V-ATPases.
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