Abstract
Structural features of the putative helical hairpin region comprising transmembrane segments 1 (TM1) and 2 (TM2) of the yeast plasma membrane H(+)-ATPase were probed by site-directed mutagenesis. The importance of phenylalanine residues Phe-116, Phe-119, Phe-120, Phe-126, Phe-144, Phe-159, and Phe-163 was explored by alanine replacement mutagenesis. It was found that substitutions at all positions, except Phe-120 and Phe-144, produced viable enzymes, although a range of cellular growth phenotypes were observed like hygromycin B resistance and low pH sensitivity, which are linked to in vivo action of the H(+)-ATPase. Lethal positions Phe-120 and Phe-144, could be replaced with tryptophan to produce viable enzyme, although the F144W mutant was highly perturbed. ATP hydrolysis measurements showed that Km was not significantly altered for most mutant enzymes, whereas Vmax was moderately reduced with two mutants, F144W and F163A, showing less than 50% of the normal activity. Double Phe-->Ala mutations in TM1 and TM2 were constructed to examine whether such substitutions would result in a higher degree of enzyme destabilization. Mutant F116A/F119A was viable and gave a normal phenotype, while F159A/F163A was not viable. Other double mutants, F116A/F159A and F119AF/159A, which are predicted to lie juxtaposed on TM1 and TM2, produced non-functional enzymes. However, a viable F119V/F159A mutant was isolated and showed hygromycin B resistance. These results suggest that double mutations eliminating 2 phenylalanine residues strongly destabilize the enzyme. A putative proline kink at Gly-122/Pro-123 in TM1 is not essential for enzyme action since these residues could be variously substituted (G122A or G122N; P123A, P123G, or P123F) producing viable enzymes with moderate effects on in vitro ATP hydrolysis or proton transport. However, several substitutions produced prominent growth phenotypes, suggesting that local perturbations were occurring. The location of Pro-123 is important because Gly-122 and Pro-123 could not be exchanged. In addition, a double Pro-Pro created by a G122P mutation was lethal, suggesting that maintenance of an alpha-helical structure is important. Other mutations in the hairpin, including modification of a buried charged residue, E129A, were not critical for enzyme action. These data are consistent with the view that the helical hairpin comprising TM1 and TM2 has important structural determinants that contribute to its overall stability and flexibility.