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 hygrom ycin 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 inv olving both site-directed mutagenesis and randomly generated second-si te intragenic suppressor mutations. No other amino acid at position 13 5 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 Gl y was also a mutant, although not as severe as the A135V mutant. The i ntroduction of a bulky Trp or a polar Ser residue produced dominant le thality, while charged amino acids produced recessive lethality. Reduc ed rates of proton transport measured by acidification of the medium b y whole cells correlate closely with the severity of cellular phenotyp e. 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 pH(R), Hyg( S)) restored A135, but second-site mutations in putative transmembrane segments 2 (V146I and V157F) and 4 (L327V) were also observed. Two pa rtial revertants (low pH(R), Hyg(R)) 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 bet ween the cytoplasmic domain catalyzing ATP hydrolysis and transmembran e helices 1 and 2.