Characterization of yeast plasma membrane H(+)-ATPase mutant pma1-A135V and its revertants.
An A135V substitution in the first transmembrane segment of the yeast plasma membrane H(+)-ATPase (PMA1) confers cellular resistance to hygromycin B, exhibits growth sensitivity to low external pH, and results in a defective enzyme that hydrolyzes ATP at 33% of wild type level. The importance of the A135 residue was probed genetically by analysis involving both site-directed mutagenesis and randomly generated second-site intragenic suppressor mutations. No other amino acid at position 135 gave either the wild type phenotype or the normal enzyme activity of A135. Substitutions with the bulkier amino acid residues A135L, A135I, and A135F produced more severe cellular phenotypes than the original A135V mutation. The substitution of the smaller side chain residue Gly was also a mutant, although not as severe as the A135V mutant. The introduction of a bulky Trp or a polar Ser residue produced dominant lethality, while charged amino acids produced recessive lethality. Reduced rates of proton transport measured by acidification of the medium by whole cells correlate closely with the severity of cellular phenotype. Some of the mutant enzymes exhibit an apparent instability in vitro. Thus, the localized structure around A135 is highly constrained. The cellular sensitivity to low external pH of the A135V mutant was used to select intragenic revertants. Most full revertants (low pHR, HygS) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two partial revertants (low pHR, HygR) have secondary mutations at S660C or a double change at F611L-S660F in the putative ATP binding domain. These results provide additional evidence for functional coupling between the cytoplasmic domain catalyzing ATP hydrolysis and transmembrane helices 1 and 2.
Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts 02254.
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D000483:Alleles; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005947:Glucose / Q000494:pharmacology; D007700:Kinetics; D016296:Mutagenesis; D010957:Plasmids; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
D006180:Proton-Translocating ATPases; D005947:Glucose