Nucleotide labeling and reconstitution of the recombinant 58-kDa subunit of the vacuolar proton-translocating ATPase.

Evidence suggests that ATP hydrolysis catalyzed by the clathrin-coated vesicle proton-translocating ATPase requires at least four polypeptides of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D.K. (1988) J. Biol. Chem. 263, 9859-9867). To further investigate the subunit requirements for ATP hydrolysis, histidine-tagged, 58-kDa polypeptide was expressed in insect Sf9 (Spodoptera frugiperda) cells. After purification by Ni(2+)-nitrolotriacetic acid chromatography, the 58-kDa protein was found to lack significant ATPase activity. However, the subunit was photoaffinity labeled with [alpha-32P]ATP, [14C]ADP, or 35S-labeled ADP and UV irradiation in a divalent cation-dependent manner. The labeling was saturable with an apparent Kd of 4 microM for both ATP and ADP. ATP and ADP competition labeling experiments indicate that the two nucleotides share the same binding site. When reconstituted with recombinant 70-kDa subunit and a biochemically prepared catalytic sector (Vc) depleted of the 70- and 58-kDa subunits, the 58-kDa component restores Ca(2+)-activated ATP hydrolysis to a specific activity of 0.19 mumol Pi x mg protein-1 x min-1, thus demonstrating that ATP hydrolysis in vacuolar type proton pumps is dependent upon the 58-kDa subunit as well as multi-subunit interactions.