STRUCTURE-FUNCTION-RELATIONSHIPS IN MEMBRANE SEGMENT-5 OF THE YEAST PMA1 H-ATPASE()

Membrane segment 5 (M5) is thought to play a direct role in cation tra nsport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+,K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plas ma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secret ory vesicles as described previously (Nakamoto, R, K,, Rao, R,, and Sl ayman, C, W, (1991) J, Biol, Chem, 266, 7940-7949), Three substitution s (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evid enced by extreme sensitivity to trypsin; the altered proteins were arr ested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in suff icient 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 E-1-to-E-2 s tep of the reaction cycle. Alanine substitution at an adjacent positio n (V692A) had substantial ATPase activity (54%), but was likewise affe cted in the E-1-to-E-2 step, as evidenced by shifts in its apparent af finity for ATP, H+, and orthovanadate, Among the mutants that were suf ficiently active to be assayed for ATP-dependent H+ transport by acrid ine orange fluorescence quenching, none showed an appreciable defect i n the coupling of transport to ATP hydrolysis, The only residue for wh ich the data pointed to a possible role in cation liganding was Ser-69 9, where removal of the hydroxyl group (S699A and S699C) led to a mode st acid shift in the pH dependence of the ATPase, This change was subs tantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+,K+-ATPase (Arguello, J, M,, and Lingr el, 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 ye ast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany th e reaction cycle. It is therefore worth noting that the mutationally s ensitive residues lie along one face of a putative alpha-helix.