ABSTRACT
Vacuoles purified from Saccharomyces cerevisiae bearing the vphl-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 λgtl 1 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vphl-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 Vphlp is a vacuolar integral membrane protein that co-purifies with V-ATPase activity. Vphlp 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 Y.PH1, identified as an open reading frame next to the BUB2 gene).
