Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation

Abstract

Vacuolar-type H+-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V1 complex, responsible for ATP hydrolysis, and a membrane-embedded Vo complex, responsible for proton translocation. The V1 complex of Thermus thermophilus V-ATPase has the subunit composition of A3B 3DF, 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 V1 complex by reconstituting the V1 complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V1 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 A1B1 and A3B3 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 V1 complex. Kinetic analysis of assembly of the A and B monomers into the A1B1 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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.