Evidence that the NH2 terminus of Vph1p, an integral subunit of the V-0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V-1 sector

The vacuolar-type H+-ATPase (V-ATPase) is composed of a peripherally bound (V-1) and a membrane-associated (V-0) complex. V-1 ATP hydrolysis is though t to rotate a central stalk, which in turn, is hypothesized to drive V-0 pr oton translocation. Transduction of torque exerted by the rotating stalk on V-0 requires a fixed structural link (stator) between the complexes to pre vent energy loss through futile rotation of V-1 relative to V-0; this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, h as a cytosolic NH2 terminus (Nt-Vph1p) and a membrane-associated COOH termi nus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the cataly tic V-1 subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confi rmed the interaction. Expression of Nt-Vph1p in a Delta vph1 mutant was nec essary to recruit Vma13p to V-1. Vma13p bound to Nt-Vph1p in vitro demonstr ating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocati on while not reducing bafilomycin A(1)-sensitive ATP hydrolysis, Trypsin cl eaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by sub stitution of arginine 53 to serine partially protected vacuolar acidificati on from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V-1 ATP hydrol ysis to V-0 proton translocation.