The cytoplasmic domain linking transmembrane segments 2 and 3 (LOOP1) of the yeast H+-ATPase was probed by the introduction of unique factor Xa recognition sites. Three sites, I(170)EGR, I(254)EGR and I(275)EGR, representing different structural regions of the LOOP1 domain, were engineered by site-specific mutagenesis of the PMA1 gene. In each case, multiple amino acid substitutions were required to form the factor Xa sites, which enabled an analysis of clustered mutations. Both I(170)EGR and I(275)EGR-containing mutants grew at normal rates, but showed prominent growth resistance to hygromycin B and sensitivity to low external pH. The engineered I(254)EGR site within the predicted beta-strand region produced a recessive lethal phenotype, indicating that mutations G(254)I and F(257)R were not tolerated. Mutant I(170)EGR- and I(275)EGR-containing enzymes showed relatively normal K-m and V-max values, but they displayed a strong insensitivity to inhibition by vanadate. An I(170)EGR/I(275)EGR double mutant was more significantly perturbed showing a reduced V-max and pronounced vanadate insensitivity. The I(170)EGR site within the putative alpha-helical stalk region was cleaved to a maximum of 10% by factor Xa under non-denaturing conditions resulting in a characteristic 81 kDa fragment, whereas the I(275)EGR site, near the end of the beta-strand region, showed about 30-35% cleavage with the appearance of a 70 kDa fragment. A I(170)EGR/I(275)EGR double mutant enzyme showed about 55-60% cleavage. The cleavage profile for the mutant enzymes was enhanced under denaturing conditions, but was unaffected by MgATP or MgATP plus vanadate. Cleavage at the I(275)EGR position had no adverse effects on ATP hydrolysis or proton transport by the H+-ATPase making it unlikely that this localized region of LOOP1 influences coupling. Overall, these results suggest that the local region encompassing I(275)EGR is accessible to factor Xa, while the region around I(170)EGR appears buried. Although there is no evidence for gross molecular motion at either site, the effects of multiple amino acid substitutions in these regions suggest that the LOOP1 domain is conformationally active, and that perturbations in this domain affect the distribution of conformational intermediates during steady-state catalysis.
