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
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References
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 press
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.
Preston RA, Reinagel PS, Jones EW, (1992) Genes required for vacuolar acidity in Saccharomyces cerevisiae. Genetics, 131: 551–558.
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© 1993 Springer-Verlag Berlin Heidelberg
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Manolson, M.F. et al. (1993). The Membrane-Bound 95 KDA Subunit of the Yeast Vacuolar Proton-Pumping ATPase is Required for Enzyme Assembly and Activity. In: Morré, D.J., Howell, K.E., Bergeron, J.J.M. (eds) Molecular Mechanisms of Membrane Traffic. NATO ASI Series, vol 74. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-662-02928-2_22
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DOI: https://doi.org/10.1007/978-3-662-02928-2_22
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