title: Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants fromSaccharomyces cerevisiae.
abstract: Mutations in the plasma membrane H(+)-ATPase gene (PMA1) of Saccharomyces cerevisiae that confer growth resistance to hygromycin B have been shown recently to cause a marked depolarization of whole cell membrane potential (Perlin, D. S., Brown, C. L., and Haber, J. E. (1988) J. Biol. Chem. 263, 18118-18122). In this report, the biochemical and genetic properties of H+-ATPases from four prominent hygromycin B-resistant pma1 mutants, pma1-105, pma1-114, pma1-147, and pma1-155, are described. Single base pair changes were identified in pma1-105, pma1-114, and pma1-147 that resulted in amino acid substitutions of Ser-368----Phe, Gly-158----Asp, Pro-640----Leu, respectively. An A----G transition mutation at -39 in the 5'-untranslated region of the mRNA of pma1-155 was also found. This mutation creates an out-of-Frame upstream AUG initiation codon that apparently reduces normal translation of PMA1. DNA sequence analysis of PMA1 from strain Y55 identified 9 base pair substitutions that resulted in 6 amino acid changes in nonconserved regions when compared to the published sequence for strain S288C. Plasma membranes of three of the four pma1 mutants contained normal amounts of H(+)-ATPase; membranes from pma1-155 contained enzyme at 62% of the wild-type level. The kinetics of ATP hydrolysis were most strongly altered for enzymes from pma1-105 and pma1-147 which showed changes in both Km and Vmax. A striking pH dependence for these parameters was found for enzyme from pma1-105 which resulted in a precipitous decline in Km and Vmax below pH 6.5. ATP hydrolysis by enzymes from pma1-105 and pma1-147 was insensitive to inhibition by vanadate. These enzymes, in contrast to wild-type and vanadate-sensitive mutant enzymes, were poorly protected from trypsin-induced inactivation by MgATP and vanadate or Pi alone. These results are pertinent to the mechanism of vanadate-induced enzyme inhibition and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for Pi. All mutant enzymes catalyzed ATP-induced pH gradient formation following purification and reconstitution into liposomes. Finally, these results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants.
authors.affiliation: Department of Biochemistry, Public Health Research Institute, New York, New York 10016.
authors.forename: D S
authors.initials: DS
authors.lastname: Perlin
authors.forename: S L
authors.initials: SL
authors.lastname: Harris
authors.forename: D
authors.initials: D
authors.lastname: Seto-Young
authors.forename: J E
authors.initials: JE
authors.lastname: Haber
mesh_terms: D000483:Alleles; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular / Q000379:methods; D004352:Drug Resistance, Microbial / Q000235:genetics; D005800:Genes, Fungal; D006921:Hygromycin B / Q000494:pharmacology*; D007700:Kinetics; D009154:Mutation; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D014638:Vanadates / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D014638:Vanadates; D006921:Hygromycin B; D006180:Proton-Translocating ATPases