IN-VIVO SYNTHESIS OF ATPASE COMPLEXES OF PROPIONIGENIUM-MODESTUM AND ESCHERICHIA-COLI AND ANALYSIS OF THEIR FUNCTION

Expression studies of Propionigenium modestum ATPase genes in various combinations with Escherichia coli ATPase genes were performed in the unc deletion mutant strain E. coli DK8. Plasmids containing the whole une operon from P. modestum were unable to complement the E. coli unc deletion mutant. Although all ATPase subunits were expressed from the plasmids, there was no detectable ATP hydrolysing activity, indicating that the F-1 part was not functional. Transformants expressing an E. coli F-1-P. modestum F-0 hybrid exhibited considerable ATPase activiti es. Binding of the F-1 part to the membrane was very weak, however, an d the coupling between ATP hydrolysis and Nai transport was impaired. After combining the genes for E. coli ATPase subunits alpha, beta, gam ma, delta and epsilon and the hydrophilic part of subunit b with P. mo destum ATPase subunits a and c and the hydrophobic part of subunit b o n a plasmid, a non-functional hybrid ATPase was expressed in E. coli. The ATPase was only loosely bound to the membrane, from which it was s olubilized with Triton X-100 and purified. Subunit b and a proteolytic degradation product were the only F-0 subunits detectable in the puri fied enzyme. A stable F-0 complex is thus not formed with the hybrid b subunit. The absence of a functional F-0 complex was in accord with p roton-conduction measurements with bacterial vesicles. The only functi onal Na+-translocating ATPase expressed in E. coli thus far consists o f E. coli subunits alpha, beta, gamma and epsilon, and P. modestum sub units delta, a, b and c [Kaim, G. & Dimroth, P. (1993) Eur. J. Biochem . 218, 937-944]. During the cloning conducted in our present study, er rors in the sequence entry into the EMBL data bank (accession no. X584 61) for the P. modestum ATPase alpha and beta subunits became evident, which are corrected in this paper.