Reconstitution in vitro of the catalytic portion (NtpA3-B3-D-G complex) of Enterococcus hirae V-type Na+-ATPase

Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V1; NtpA3-B3-D-G) and an integral membrane domain (V0; NtpI-K10) 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 V1 portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6 μM for ADP or 3.1 μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA3-B3 heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA3-B3 forming V1 (NtpA3-B3-D-G) complex independent of nucleotides. The V1 formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V1 complex was as high as that of native V1-ATPase purified from the V0V1 complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V1 complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V1-ATPase complex.