Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V-1: NtpA(3)-B-3-D-G) and an integral membrane domain (V-0; NtpI-K-10) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V-1 portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 mu M for ADP or 3.1 mu M for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA(3)-B-3 heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA3-B3 forming V, (NtpA(3)-B-3-D-G) complex independent of nucleotides. The V-1 formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V-1 complex was as high as that of native V-1-ATPase purified from the V0V1 complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V-1 complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V-1-ATPase complex. (C) 2009 Elsevier Inc. All rights reserved.