Abstract
Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.
Highlights
From the $Department of Biological Science, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, the IlDepartment of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, the **DepartmentBoiof chemistry, Beckmn Center, Stanford University, Stanford,California 94305, and the $$Department of Genetics, Stanford University Medical Center, Stanford, California 94305
Vacuoles purified from yeast bearing the v p h l - 1 mutation had no detectable The yeast vacuole, like the mammalian lysosome, requires bafilomycin-sensitiveATPase activity or ATP-de- an acidic lumen to perform certain of its cellular functions
The V P H l gene was cloned by complementation of the vphl-1 mutation andindependently cloned by screening a Xgt 11 expression library with antibodies directed againsta 95-kDa vacuolar integral membrane protein.Deletion disruption of the V P H l gene revealed that theV P H l gene is not essential for viability butis required for vacuolarH’ATPase assembly andvacuolar acidification
Summary
Plaque-forming units were screened (approximately 8 yeast genomes Cloning VPHl-The VPHl gene was cloned twice in our worth), resulting in four identical positives. To further confirm that the isopropyl @-D-thiogalactopyranoside-inducedprotein was in-frame and contiguous to @-galactosidaseand encoded by a genuine yeast gene, about 300 bp were sequenced starting just before and going through the putativfuesion protein's EcoRI junction siteusing the Xgtll forward primer (New England Biolabs, Catalog No 1218).Sequencing revealed a continuous open reading frame, in-frame and contiguous to lacZ, with a codon bias in agreepVIP1-78 DNA.pVIP1-78, upontransformationintothe uphl-1 mutant strain BJ5035 and the uphl deletion-disrupted strain BJ6718, complementedbothuphlmutations,asassayed by fluorescence. Similarities were noticed between the restriction maps for average GRAVY score for sequenced soluble proteins is -0.4 DNA encoding VPHl and the Xgtll-selected DNA contained with hydrophobic values lying above and hydrophilic scores within plasmids pVIP1-78 andpVIP1-79.
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