Reconstitution in Vitro of V sub(1) Complex of Thermus thermophilus V-ATPase Revealed That ATP Binding to the A Subunit Is Crucial for V sub(1) Formation

Vacuolar-type H super(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V sub(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V sub(o) complex, responsible for proton translocation. The V sub(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A sub(3)B sub(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V sub(1) complex by reconstituting the V sub(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V sub(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A sub(1)B sub(1) and A sub(3)B sub(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V sub(1) complex. Kinetic analysis of assembly of the A and B monomers into the A sub(1)B sub(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.