RECONSTITUTION OF RECOMBINANT 40-KDA SUBUNIT OF THE CLATHRIN-COATED VESICLE H-ATPASE()

We have proposed a model of the ATP hydrolytic sector of the clathrin- coated vesicle H+-ATPase wherein significant catalysis requires four s ubunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X.-S., and Stone, D. K. (1988) J. Biol. Chem. 263, 9859-9867). We have cloned an d expressed the 40-kDa component in Escherichia coli and have purified the recombinant protein to homogeneity. This subunit lacks ATP hydrol ytic capacity, but when reconstituted to a 40 kDa-depleted hydrolytic sector, there is a greater than 20-fold increase in calcium-activated, N-ethylmaleimide-sensitive ATP hydrolysis, indicating that this subun it is required for vacuolar-type proton pump function.