INTER-SUBUNIT INTERACTION AND ARRANGEMENT OF THE CENTRAL STALK SUBUNITS OF ENTEROCOCCUS HIRAE V-ATPASE

V-ATPases function as ATP-dependent ion pumps in various membrane systems of living organisms. ATP hydrolysis causes rotation of the central rotor complex, which is composed of NtpC, NtpD,and NtpG subunits. We successfully cloned, expressed, and purified the central stalk subunits of Na+- translocating V-type ATPase from E. hirae. Full length DNAs for genes of NtpC, NtpD, and NtpG subunits were amplified by conventional PCR method and cloned into the plasmid vector pET23d. Recombinant DNA transformed E. coli (JM109) cells were grown in LB agar plate containing ampicillin. NtpC and NtpG subunit proteins were expressed as separate His-tagged soluble proteins in E. coli BL21(DE3) cells at 30 °C and NtpD subunit protein was stably expressed in E. coli BL21(DE3) cells at 16 °C. Expressed central stalk subunit proteins were purified by affinity chromatography followed by gel-filtration method. The amount of purified NtpC, NtpD, and NtpG subunit proteins were 13.8 mg/1 liter culture, 16.6 mg/1 liter culture, and 15.2 mg/1 liter culture, respectively. Tobacco etch virus (TEV)-protease was also expressed in E. coli BL21(DE3) cells and purified by affinity chromatography. The amount of purified TEV-protease was 8.50 mg/1 liter culture. Here, we report the stable interaction and the complex formation mechanisms of the central stalk subunits of E. hirae V-ATPase. The purified NtpD could interact directly with NtpG and NtpC could bind directly to NtpD-G complex and these three subunits could form NtpD-G-C complex. NtpD could bind to NtpG and formed NtpD-G complex. NtpD or NtpD-G complex could bind to NtpA3-B3 and formed NtpA3-B3-D or NtpA3-B3-D-G complex, respectively. NtpC could bind to NtpA3-B3-D-G and formed NtpA3-B3-D-G-C complex but could not bind to NtpA3-B3, NtpA3-B3-D, NtpD, or NtpG