RECONSTITUTION OF THE RECOMBINANT 70-KDA SUBUNIT OF THE CLATHRIN-COATED VESICLE H-ATPASE()

Vacuolar-type proton pumps are complex heterooligomers. When dissociat ed into subcomplexes and subunits, the partial reactions of ATP hydrol ysis and transmembranous proton flow can be assigned to isolated domai ns. Data suggest that the molecular site of ATP hydrolysis resides wit hin the 70-kDa subunit but that ATPase activity likely requires at lea st three additional subunits of 58, 40, and 33 kDa (Xie, X.-S., and St one, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have now cloned a nd sequenced the 70-kDa subunit from bovine brain and have expressed t he protein in insect Sf9 (Spodoptera frugiperda) cells with a recombin ant baculovirus. When purified, the protein has no significant ATPase activity but can be photoaffinity labeled with [alpha(32)P]ATP and UV irradiation with an apparent K-d of 35 mu M. When reconstituted with b iochemically prepared 58-, 40-, and 33-kDa polypeptides, the recombina nt 70-kDa)a subunit restores Ca2+-activated ATP hydrolysis to a specif ic activity of 0.6 mu mol P-i.mg protein(-1).min(-1), thus demonstrati ng that ATP hydrolysis in vacuolar-type proton pumps is dependent upon both the 70-kDa subunit as well as multi-subunit interactions.