STV1 GENE ENCODES FUNCTIONAL HOMOLOG OF 95-KDA YEAST VACUOLAR H-ATPASE SUBUNIT VPH1P()

The Saccharomyces cerevisiae gene, VPH1 (Vacuolar pH 1), encodes a 95- kDa integral membrane subunit of the vacuolar-type H+-ATPase (V-ATPase ) that is required for enzyme assembly; disruption of the VPH1 gene im pairs vacuolar acidification (Manolson, M. F., Proteau, D., Preston, R . A, Stenbit, A, Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992) J. Biol. Chem. 267, 14294-14 303). Here we show that STV1 (Similar To VPH1) encodes an integral mem brane polypeptide of 102 kDa with 54% identity with the peptide sequen ce of Vph1p. High copy expression of STV1 partially restores vacuolar acidification in a Delta vph1 mutant strain; solubilization and fracti onation of membrane proteins from these vacuoles show that Stv1p co-pu rifies with bafilomycin A(1)-sensitive ATPase activity and with the 60 - and 69-kDa V-ATPase subunits. Immunofluorescence microscopy of strai ns bearing a single copy of epitope tagged STV1 reveals punctate stain ing of the cytoplasm; overexpression of epitopetagged Stv1p reveals bo th punctate cytoplasmic staining and vacuolar membrane staining. North ern analysis shows that disruption of STV1 does not affect the level o f transcription of VPH1 and that disruption of VPH1 does not affect th e level of transcription of STV1. Strains bearing disruption of genes encoding other V-ATPase subunits (VMA1, VMA2, VMA3, and VMA4) fail to grow on media supplemented with 100 mM CaCl2 or 4 mM ZnCl2, media buff ered to pH 7.5, or media with a glycerol carbon source. On the same ty pes of media only a Delta vph1 Delta stv1 double disruption mutant has growth phenotypes equivalent to strains bearing a single disruption o f the VMA1, VMA2, VMA3, and VMA4 genes; a Delta vph1 strain has only m oderate growth inhibition while a Delta stv1 strain has wild type grow th on the conditions listed above. We conclude that Stv1p is a functio nal homologue of Vph1p and suggest that Stv1p and Vph1p may be equival ent subunits for V-ATPases located on different organelles. The functi on of these 100-kDa homologues may be to target or regulate other comm on V-ATPase subunits for two distinct cellular locations.