Abstract
Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix.
Highlights
§ To whom correspondence and reprint requests should be addressed: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510
The results have identified five amino acid residues that play a significant role in the reaction cycle, along with three others that are required for proper protein folding and transit through the secretory pathway
In analyzing the data from this study, the sequence alignment of Fig. 6 can serve as a useful guide. It illustrates a modest degree of evolutionary conservation along membrane segment 5 of the P-ATPases and highlights six residues that are discussed in detail below
Summary
Yeast Strains—Two related strains of Saccharomyces cerevisiae were used in this study: SY4 (MATa, ura, leu112, his619, sec6-4ts GAL2, pma1::YIpGAL-PMA1) and NY605 (MATa, ura, leu112, GAL2). To determine the level of expressed Pma protein relative to a wild-type control, secretory vesicles (5–20 g) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted [18], followed by PhosphorImager (Molecular Dynamics) analysis; typically, the analysis was carried out at two protein concentrations within the linear range, and the expression level was calculated from the average of the two determinations. Metabolic Labeling and Immunoprecipitation—To measure the synthesis of mutant ATPases that were unable to reach the secretory vesicles, SY4 cells were shifted from galactose medium at 23 °C to glucose medium at 39 °C as described above and metabolically labeled with [35S]methionine [26]. A, secretory vesicles were isolated from yeast expressing either wild-type (WT) or mutant (R695A, H701A, and L706A) ATPase and subjected to immunoblotting. Protein Determination—Protein concentrations were determined by a modification of the method of Lowry et al [27] as described by Ambesi et al [22] using bovine serum albumin as a standard
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