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The Membrane-Bound 95 KDA Subunit of the Yeast Vacuolar Proton-Pumping ATPase is Required for Enzyme Assembly and Activity

  • M. F. Manolson
  • D. Proteau
  • R. A. Preston
  • M. E. Colosimo
  • B. T. Roberts
  • M. A. Hoyt
  • E. W. Jones
Conference paper
Part of the NATO ASI Series book series (volume 74)

Abstract

Yeast vacuoles, like mammalian lysosomes, are maintained at an acidic pH by a vacuolar-type proton-pumping ATPase (V-ATPase). A genetic screen using the pH sensitive fluorescence of 6-carboxyfluorescein diacetate was developed to identity yeast defective in vacuolar acidification (Preston et al., 1992). Yeast bearing the vphl-1 (Vacuolar pH1-1) mutation have neutral vacuoles in vivo (Preston et al., 1989) and have no detectable bafilomycin-sensitive ATPase activity or ATP dependent proton-pumping associated with purified vacuoles. The peripherally bound nucleotide-binding subunits (Vmaip and Vma2p) are present in wild type levels in yeast whole cell extract yet are not associated with the vacuolar membrane. The VPH1 gene was cloned by screening a λgt11 expression library with antibodies directed against a 95 kDa vacuolar integral membrane protein and independently cloned by complementation of the vphl-1 mutation. Disruption of the gene revealed that the VPH1 gene product is required for V-ATPase assembly and vacuolar acidification but is not essential for cell viability or for targeting and maturation of vacuolar proteases. Through cell fractionation and immunobloting, the Vphlp antigen was shown to co-purify with alkaline phosphatase activity, a specific marker for vacuolar membranes. Furthermore, the Vphlp antigen was enriched and co-purified with vacuolar ATPase activity. H+-ATPase activity. The inability of alkaline Na2CO3

Keywords

Vacuolar Membrane Entire Open Reading Frame Putative Polypeptide Vacuolar Protease Yeast Vacuole 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. Manolson MF, Proteau D, Preston RA, Stenbit A, Roberts BT, Hoyt MA, Preuss D, Mulholland J, Botstein D, Jones EW (1992) The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H+ -ATPase. J. Biol. Chem. in pressGoogle Scholar
  2. Preston RA, Murphy RF, Jones EW, (1989) Assay of vacuolar pH in yeast and identification of acidification-defective mutants. Proc. Natu. Acad. Sci. U.S.A. 86: 7027–7031.Google Scholar
  3. Preston RA, Reinagel PS, Jones EW, (1992) Genes required for vacuolar acidity in Saccharomyces cerevisiae. Genetics, 131: 551–558.PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1993

Authors and Affiliations

  • M. F. Manolson
    • 1
  • D. Proteau
    • 1
  • R. A. Preston
    • 1
  • M. E. Colosimo
    • 1
  • B. T. Roberts
    • 2
  • M. A. Hoyt
    • 2
  • E. W. Jones
    • 1
  1. 1.Dept. of Biol. Sci.Carnegie Mellon Univ.PittsburghUSA
  2. 2.Dept. of Biol.The Johns Hopkins Univ.BaltimoreUSA

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