Mutagenesis of the yeast plasma membrane H(+)-ATPase. A novel expression system.
The plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae is a prototype for the mutagenic analysis of structure-function relationships in P-type cation pumps. Because a functional H+ pump is required for viability, wild-type ATPase must be maintained in the plasma membrane for normal cell growth. Our expression strategy involves a rapid switch in expression from the wild-type ATPase gene to a mutant allele followed by entrapment of the newly synthesized mutant enzyme in an internal, secretory vesicle pool. The isolated vesicles prove to be ideally suited for the study of the catalytic and transport properties of the ATPase. Work to date has focused on conserved residues in the vicinity of the aspartyl-phosphate reaction intermediate. Substitution of Asp378 with Glu, Ser, or Asn and of Lys379 with Gln prevents normal biogenesis of the mutant ATPase. The more conservative Lys379----Arg mutation was tolerated, but with a sixfold loss of activity and substantial alterations in Km for ATP and Ki for vanadate. Nonconservative replacement of Thr380, Thr382, or Thr384 with Ala led to inactive enzyme, whereas the conservative change to Ser caused a two to threefold reduction in ATP hydrolysis and H(+)-pumping. Taken together, the results are consistent with an essential role for these invariant residues in phosphate-binding and ATP hydrolysis.
Rao
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Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510.
Slayman
C W
CW
D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D002462:Cell Membrane / Q000201:enzymology; D015870:Gene Expression; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D006180:Proton-Translocating ATPases