Reconstitution of the recombinant 70-kDa subunit of the clathrin-coated vesicle H⁺ ATPase

Vacuolar-type proton pumps are complex hetero-oligomers. When dissociated into subcomplexes and subunits, the partial reactions of ATP hydrolysis and trans-membranous proton flow can be assigned to isolated domains. Data suggest that the molecular site of ATP hydrolysis resides within the 70-kDa subunit but that ATPase activity likely requires at least three additional subunits of 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now cloned and sequenced the 70-kDa subunit from bovine brain and have expressed the protein in insect Sf9 (Spodoptera frugiperda) cells with a recombinant baculovirus. When purified, the protein has no significant ATPase activity but can be photoaffinity labeled with [α³²P]ATP and UV irradiation with an apparent K(d) of 35 μM. When reconstituted with biochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombinant 70-kDa subunit restores Ca²⁺-activated ATP hydrolysis to a specific activity of 0.6 μmol P(i) · mg protein^-1 · min^-1, thus demonstrating that ATP hydrolysis in vacuolar-type proton pumps is dependent upon both the 70-kDa subunit as well as multi-subunit interactions.