Abstract
Energy transduction in the anaerobic, thermophilic bacterium Clostridium fervidus relies exclusively on Na+ as the coupling ion. The Na+ ion gradient across the membrane is generated by a membrane-bound ATPase (G. Speelmans, B. Poolman, T. Abee, and W. N. Konings, J. Bacteriol. 176:5160-5162, 1994). The Na+-ATPase complex was purified to homogeneity. It migrates as a single band in native polyacrylamide gel electrophoresis and catalyzes Na+-stimulated ATPase activity. Denaturing gel electrophoresis showed that the complex consists of at least six different polypeptides with apparent molecular sizes of 66, 61, 51, 37, 26, and 17 kDa. The N-terminal sequences of the 66- and 51-kDa subunits were found to be significantly homologous to subunits A and B, respectively, of the Na+-translocating V-type ATPase of Enterococcus hirae. The purified V1V0 protein complex was reconstituted in a mixture of Escherichia coli phosphatidylethanolamine and egg yolk phosphatidylcholine and shown to catalyze the uptake of Na+ ions upon hydrolysis of ATP. Na+ transport was completely abolished by monensin, whereas valinomycin stimulated the uptake rate. This is indicative of electrogenic sodium transport. The presence of the protonophore SF6847 had no significant effect on the uptake, indicating that Na+ translocation is a primary event and in the cell is not accomplished by an H+-translocating pump in combination with an Na+-H+ antiporter.