Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p).
The B subunit of the vacuolar (H+)-ATPase (V-ATPase) has previously been shown to participate in nucleotide binding and to possess significant sequence homology with the alpha subunit of the mitochondrial F-ATPase, which forms the major portion of the noncatalytic nucleotide binding sites and contributes several residues to the catalytic sites of this complex. Based upon the recent x-ray structure of the mitochondrial F1 ATPase (Abrahams, J.P., Leslie, A.G., Lutter, R., and Walker, J.E. (1994) Nature 370,621-628), site-directed mutagenesis of the yeast VMA2 gene has been carried out in a strain containing a deletion of this gene. VMA2 encodes the yeast V-ATPase B subunit (Vma2p). Mutations at two residues postulated to be contributed by Vma2p to the catalytic site (R381S and Y352S) resulted in a complete loss of ATPase activity and proton transport, with the former having a partial effect on V-ATPase assembly. Interestingly, substitution of Phe for Tyr-352 had only minor effects on activity (15-30% inhibition), suggesting the requirement for an aromatic ring at this position. Alteration of Tyr-370, which is postulated to be near the adenine binding pocket at the noncatalytic sites, to Arg, Phe, or Ser caused a 30-50% inhibition of proton transport and ATPase activity, suggesting that an aromatic ring is not essential at this position. Finally, mutagenesis of residues in the region corresponding to the P-loop of the alpha subunit (H180K, H180G, H180D, N181V) also inhibited proton transport and ATPase activity by approximately 30-50%. None of the mutations in either the putative adenine binding pocket nor the P-loop region had any effect on the ability of Vma2p to correctly fold nor on the V-ATPase to correctly assemble. The significance of these results for the structure and function of the nucleotide binding sites on the B subunit is discussed.
Liu
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Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Kane
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PM
Newman
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Forgac
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D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D001693:Biological Transport, Active; D017931:DNA Primers / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018942:Macrolides; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000201:enzymology
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004791:Enzyme Inhibitors; D018942:Macrolides; D046911:Macromolecular Substances; C040929:bafilomycin A1; D006180:Proton-Translocating ATPases