FUNCTION OF THE COOH-TERMINAL DOMAIN OF VPH1P IN ACTIVITY AND ASSEMBLY OF THE YEAST V-ATPASE

We have previously shown that mutations in buried charged residues in the last two transmembrane helices of Vph1p (the 100-kDa subunit of th e yeast V-ATPase) inhibit proton transport and ATPase activity (Leng, X. H,, Manolson, M,, Liu, Q., and Forgac, M, (1996) J, Biol, Chem. 271 , 22487-22493), In this report we have further explored the function o f this region of Vph1p (residues 721-840) using a combination of site- directed and random mutagenesis, Effects of mutations on stability of Vph1p, assembly of the V-ATPase complex, 9-amino-6-chloro-2-methoxyacr idine quenching (as a measure of proton transport), and ATPase activit y were assessed, Additional mutations were analyzed to test the import ance of Glu-789 in TM7 and His-743 in TM6. Although substitution of As p for Glu at position 789 led to a 50% decrease in 9-amino-6-chloro-2- methoxyacridine quenching, substitution of Ala at this position gave a mutant with 40% quenching relative to wild type, suggesting that a ne gative charge at this position is not absolutely essential for proton transport, Similarly a positive charge is not essential at position Hi s-743, since the H743Y and H743A mutants retain 20 and 60% of wild-typ e quenching, respectively, Interestingly, H743A approaches wild-type A TPase activity at elevated pH while the E789D mutant shows a slightly lower pH optimum than wild type, suggesting that these residues are in a location to influence V-ATPase activity, The low pumping activity o f the double mutant (E789H/H743E) suggests that these residues do not form a simple ion pair, Random mutagenesis identified a number of addi tional mutations both inside the membrane (L739S and L746S) as well as external to the membrane (H729R and V803D) which also significantly i nhibited proton pumping and ATPase activity, By contrast, a cluster of five mutations were identified between residues 800 and 814 in the so luble segment just COOH-terminal to TM7 which affected either assembly or stability of the V-ATPase complex, Two mutations (F809L and G814D) may also affect targeting of the 100-kDa subunit, These results sugge st that this segment of Vph1p plays a crucial role in organization of the V-ATPase complex.