Mutational Analysis of the Catalytic Subunit of the Yeast Vacuolar Proton-Translocating ATPase

Abstract

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.

Photoaffinity Labeling of Wild-type and Mutant Forms of the Yeast V-ATPase A Subunit by 2-Azido-[32P]ADP

“…The plasmid pPK17-7, containing VMA1 lacking the entire VMA1-derived endonuclease (VDE) subcloned into the yeast shuttle vector pSEYC68 at BamHI and SalI sites (24,35) was from Dr. Kane. Yeast cells were grown in yeast extract-peptone-dextrose (YEPD) medium or in supplemented synthetic dextrose medium (SD).…”

“…Deletion of genes encoding subunits of the V-ATPase results in a conditional lethal phenotype (35,47,48), which is also observed for vma mutants possessing less than approximately 20% of wild-type V-ATPase activity (24). Strains bearing these mutations are able to grow on medium buffered to acidic pH (5.0 -5.5) but are unable to grow at neutral pH (7.5) or in neutral medium containing 50 mM CaCl 2 .…”

“…The F-ATPases function in ATP synthesis in mitochondria, chloroplasts, and bacteria (18 -20). The x-ray crystal structures of F 1 indicate that the nucleotide binding sites are located at the interface of the ␤ and ␣ subunits, with the catalytic sites residing primarily on the ␤ subunits and the noncatalytic sites residing primarily on the ␣ subunits (21,22).Although mutagenesis studies of the yeast V-ATPase have begun to identify residues important for catalytic activity (23)(24)(25)(26), the structure of the nucleotide binding sites on the VATPase has yet to be determined. One approach to characterizing the structure of nucleotide binding sites is the use of photoaffinity analogs.…”

“…Although mutagenesis studies of the yeast V-ATPase have begun to identify residues important for catalytic activity (23)(24)(25)(26), the structure of the nucleotide binding sites on the VATPase has yet to be determined. One approach to characterizing the structure of nucleotide binding sites is the use of photoaffinity analogs.…”

Structural Analysis of the N-terminal Domain of Subunit a of the Yeast Vacuolar ATPase (V-ATPase) Using Accessibility of Single Cysteine Substitutions to Chemical Modification

“…All mutant strains were tested for growth on YEPD buffered to pH 5.5 or 7.5. Yeast that contain Ͻ20% wild-type V-ATPase activity are unable to grow at neutral pH (35). This conditional lethality is referred to as the vma Ϫ phenotype (36).…”