MUTATIONS OF G158 AND THEIR 2ND-SITE REVERTANTS IN THE PLASMA-MEMBRANE H-ATPASE GENE (PMA1) IN SACCHAROMYCES-CEREVISIAE()

A G158D mutation residing near the cytoplasmic end of transmembrane se gment 2 of the H+-ATPase from Saccharomyces cerevisiae appears to alte r electrogenic proton transport by the proton pump (Perlin et al. (198 8) J. Biol. Chem. 263, 18118-18122.) The mutation confers upon whole c ells a pronounced growth sensitivity to low pH and a resistance to the antibiotic hygromycin B. The isolated enzyme retains high activity (7 0% of wild type) but is inefficient at pumping protons in a reconstitu ted vesicle system, suggesting that this enzyme may be partially uncou pled (Perlin et al. (1989) J. Biol. Chem. 264, 21857-21864.) In this s tudy, the acid-sensitive growth phenotype of the pma1-D158 mutant was utilized to isolate second site suppressor mutations in an attempt to probe structural interactions involving amino acid 158. Site-directed mutagenesis of the G158 locus was also performed to explore its local environment. Nineteen independent revertants of pma1-G158D were select ed as low pH-resistant colonies. Four were full phenotypic revertants showing both low pH resistance and hygromycin B sensitivity. Of three full revertants analyzed further, one restored the original glycine re sidue at position 158 while the other two carried compensatory mutatio ns V336A or F830S, in transmembrane segments 4 and 7, respectively. Pa rtial revertants, which could grow on low pH medium but still retained hygromycin B resistance, were identified in transmembrane segments 1 (V127A) and 2 (C148T, G156C), as well as in the cytoplasmic N-terminal domain (E110K) and in the cytoplasmic loop between transmembrane segm ents 2 and 3 (D170N, L275S). Relative to the G158D mutant, all reverta nts showed enhanced net proton transport in whole-cell medium acidific ation assays and/or improved ATP hydrolysis activity. Small polar amin o acids (Asp and Ser) could be substituted for glycine at the 158 posi tion to produce active, albeit somewhat defective, enzymes; larger hyd rophobic residues (Leu and Val) produced more severe phenotypes. These results suggest that G158 is likely to reside in a tightly packed pol ar environment which interacts, either directly or indirectly, with tr ansmembrane segments 1, 4 and 7. The revertant data are consistent wit h transmembrane segments 1 and 2 forming a conformationally sensitive helical hairpin structure.