Mutational analysis of the catalytic subunit of the yeast vacuolar proton-translocating ATPase.
In order to generate a set of tools for probing structure-function relationships in the catalytic subunit of the yeast vacuolar H(+)-ATPase, the gene encoding this subunit (VMA1) was randomly mutagenized. Mutant plasmids unable to complement the growth defects of yeast cells lacking an intact VMA1 gene were isolated and sequenced. Eight different mutant alleles of VMA1 were examined for levels of the catalytic subunit and other subunits of the enzyme, assembly of the ATPase complex, targeting to the vacuolar membrane, and concanamycin A-sensitive ATPase activity. The mutations S811P and E740D resulted in mutant enzymes that assembled fully but were incapable of ATP hydrolysis, and the mutation E785G generated a similar but somewhat less severe phenotype (17% of the ATPase activity of wild-type vacuoles). When MgATP-dependent stripping of the peripheral subunits by 100 mM KNO3 was examined in these three mutants, only the E785G mutant exhibited significant stripping, suggesting that ATP hydrolysis, even at relatively low levels, generates a conformation susceptible to dissociation. Plasmids containing the mutations E751G and F752S partially complemented the growth defects and resulted in partial defects in ATPase activity that appear to reflect reduced catalytic efficiency. Partial defects in growth and ATPase activity were also seen in the Y797H mutant, but this mutation caused an assembly defect manifested as a preferential loss of two of the peripheral subunits of the enzyme. The phenotypes of these mutants are interpreted in the context of homologies with other V-type and F-type ATPases.
Liu
J
J
Department of Biochemistry and Molecular Biology, State University of New York, Syracuse 13210, USA.
Kane
P M
PM
D000483:Alleles; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship; D014617:Vacuoles / Q000201:enzymology*
D017931:DNA Primers; D011522:Protons; D006180:Proton-Translocating ATPases