title: Growth control strength and active site of yeast plasma membrane ATPase studied by site-directed mutagenesis.
abstract: Several amino acids which are conserved in cation-pumping ATPases with phosphorylated intermediate have been mutagenized in the yeast plasma membrane H+-ATPase. The mutant genes have been selectively expressed in a yeast strain where the wild-type ATPase is only expressed in galactose medium. A series of mutants with decreasing levels of activity demonstrates that the ATPase is rate-limiting for growth and that decreased ATPase activity correlates with decreased intracellular pH. Enzymatic and transport studies of mutant ATPases indicate that (a) Lys474 is the target for the inhibitor fluorescein 5'-isothiocyanate and this residue can be replaced by either arginine or histidine with partial retention of activity; (b) the sensitivity to inhibition by vanadate is affected by the mutations Thr231----Gly, Cys376----Leu, Lys379----Gln and Asp634----Asn; (c) the mutation Ser234----Ala causes uncoupling between ATP hydrolysis and proton transport and reduces the ATP content of the cells; (d) the mutation Asp730----Asn, which affects a polar residue conserved in hydrophobic stretches of H+-ATPases, abolishes ATPase activity and proton transport but not the formation of a phosphorylated intermediate.
authors.affiliation: European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
authors.forename: F
authors.initials: F
authors.lastname: Portillo
authors.forename: R
authors.initials: R
authors.lastname: Serrano
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D005800:Genes, Fungal; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014638:Vanadates / Q000494:pharmacology
chemical_list: D014638:Vanadates; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases