Components and assembly factors of the yeast vacuolar-type H+-translocating ATPase

The Vacuolar-type H+-translocating ATPase (V-ATPase) is a large multi-subunit complex found in all eukaryotic organisms including humans. The V-ATPase transports protons across lipid bilayers, and is required for life in many organisms. The most extensively characterized V-ATPase is that of the yeast Saccharomyces cerevisiae. Thirteen subunits and three assembly factors are required for the function of the yeast V-ATPase. The subunits of the V-ATPase are distributed between two subcomplexes: a peripheral membrane V1 subcomplex and an integral membrane V0 subcomplex. V-ATPase assembly factors are not subunits of the V-ATPase, but endoplasmic reticulum localized proteins required for its biogenesis. Recent Saccharomyces cerevisiae genome-wide screens have identified Vma9p and Pkr1p as proteins that are required for the efficient assembly and function of the V-ATPase.; Vma9p (subunit e) is a subunit of the V-ATPase that is small, hydrophobic and conserved. Vma9p is an integral membrane protein that is initially translated and inserted into the endoplasmic reticulum and subsequently localizes with the V-ATPase on the vacuole. In the endoplasmic reticulum, Vma9p participates in a V0-Vma21p assembly complex and requires all V0 subunits and assembly factors to do so. In turn, Vma9p is required for Vph1p and Vma6p but not the proteolipids to associate with the V0-Vma21p assembly complex. These findings demonstrate that Vma9p is a V0 subunit of the VATPase and suggest that it is a V0 subcomplex stator component.; Pkr1p is not a subunit of the Golgi or vacuolar localized V-ATPase, but an integral membrane protein of the endoplasmic reticulum. Like known V 0 subunits and assembly factors, cells that lack Pkr1p display lower levels and increased turnover of the V0 subunit Vph1p. Interestingly, pkr1Delta cells display intermediate vacuolar acidification defects compared to cells with no functional V-ATPase. Consistent with the phenotypic data, some functional V-ATPases can be isolated from pkr1Delta cells, but at a level significantly below that of wild-type cells. These results demonstrate that Pkr1p is an endoplasmic reticulum localized protein required for the efficient assembly of the VATPase. The unusual presence of residual V-ATPase complexes in pkr1Delta cells suggests that Pkr1p's V-ATPase assembly role is novel. This dissertation includes my co-authored materials.