Vacuoles purified from Saccharomyces cerevisiae bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. Furthermore, the vacuolar H(+)-ATPase (V-ATPase) nucleotide binding subunits were no longer associated with vacuolar membranes yet were present at wild-type levels in yeast whole-cell extracts. The VPH1 gene was cloned by screening a lambda gt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vph1-1 mutation. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is required for vacuolar H(+)-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases. VPH1 encodes a predicted polypeptide of 840 amino acid residues (95.6 kDa) with putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with V-ATPase activity. Vph1p has 42% identity to the 116 kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, 42% identity to the TJ6 mouse immune suppressor factor, 42% identity to the Caenorhabditis elegans proton pump homologue and 54% identity to the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.