Defective H⁺-ATPase of hygromycin B-resistant pma1 mutants from Saccharomyces cerevisiae

Mutations in the plasma membrane H⁺-ATPase gene (PMA1) of Saccharomyces cerevisiae that confer growth resistance to hygromycin B have been shown recently to cause a marked depolarization of whole cell membrane potential (Perlin, D.S., Brown, C.L., and Haber, J.E. (1988) J. Biol. Chem. 263, 18118-18122). In this report, the biochemical and genetic properties of H⁺-ATPases from four prominent hygromycin B-resistant pma1 mutants, pma1-105, pma1-114, pma1-147, and pma1-155, are described. Single base pair changes were identified in pma1-105, pma1-114, pma1-147, and pma1-155, are described. Single base pair changes were identified in pma1-105, pma1-114, and pma1-147 that resulted in amino acid substitutions of Ser-368 → Phe, Gly-158 → Asp, Pro-640 → Leu, respectively. An A → G transition mutation at -39 in the 5'-untranslated region of the mRNA of pma1-155 was also found. This mutation creates an out-of-Frame upstream AUG initiation codon that apparently reduces normal translation of PMA1. DNA sequence analysis of PMA1 from strain Y55 identified 9 base pair substitutions that resulted in 6 amino acid changes in nonconserved regions when compared to the published sequence for strain S288C. Plasma membranes of three of the four pma1 mutants contained normal amounts of H⁺-ATPase; membranes from pma1-155 contained enzyme at 62% of the wild-type level. The kinetics of ATP hydrolysis were most strongly altered for enzymes from pma1-105 and pma1-147 which showed changes in both K(m) and V(max). A striking pH dependence for these parameters was found for enzyme from pma1-105 which resulted in a precipitous decline in K(m) and V(max) below pH 6.5 ATP hydrolysis by enzymes from pma1-105 and pma1-147 was insensitive to inhibition by vanadate. These enzymes, in contrast to wild-type and vanadate-sensitive mutant enzymes, were poorly protected from trypsin-induced inactivation by MgATP and vanadate or P(i) alone. These results are pertinent to the mechanism of vanadate-induced enzyme inhibition and suggest that Ser-368 and Pro-640 influence the affinity of the phosphate-binding site for P(i). All mutant enzymes catalyzed ATP-induced pH gradient formation following purification and reconstitution into liposomes. Finally, these results further demonstrate the usefulness of hygromycin B as a generalized screening tool for isolating diverse plasma membrane ATPase mutants.