Alanine-scanning mutagenesis along membrane segment 4 of the yeast plasma membrane H+-ATPase. Effects on structure and function.

Membrane segment 4 of P-type cation pumps has been suggested to play a critical role in the coupling of ATP hydrolysis to ion translocation. In this study, structure-function relationships in M4 of the yeast (Saccharomyces cerevisiae) plasma membrane H+-ATPase have been explored 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). One substitution (I329A) led to arrest of the enzyme at an early stage of biogenesis, and three others (G333A, L338A, G349A) reduced ATP hydrolysis to near-background levels. The remaining 26 mutants were expressed well enough in secretory vesicles (44-121% of wild type) and had sufficient ATPase activity (16-123% of wild type) to be characterized in detail. When acridine orange fluorescence quenching was used to measure rates of ATP-dependent proton pumping over a range of ATP concentrations, only minor changes were seen. In kinetic studies, however, seven of the mutant enzymes (I331A, I332A, V334A, V336A, V341A, V342A, and M346A) were resistant to vanadate inhibition, and three of them (I332A, V336A, and V341A) also had a decreased Km and increased pH optimum for ATP hydrolysis. Limited trypsinolysis was used to probe the structure of two different Val-336 substitutions, V336A, described above, and V336R, which displayed little or no ATPase activity. Both were cleaved at a relatively normal rate to give a pattern of fragments essentially identical to that seen with the wild-type enzyme. However, while vanadate, ADP, and ATP were able to protect the wild-type and V336A enzymes against trypsinolysis, the V336R ATPase was protected only by ADP and ATP. Taken together, the data suggest that key residues in the M4 segment may help to communicate the E1-E2 conformational change to ion-binding sites in the membrane.