HYBRID F-0 COMPLEXES OF THE ATP SYNTHASES OF SPINACH-CHLOROPLASTS AND ESCHERICHIA-COLI - IMMUNOPRECIPITATION AND MUTANT ANALYSES

Abstract

Hybrid F-0 complexes of the ATP synthases of spinach chloroplast (CF0) and Escherichia coli (EF(0)) were investigated. Immunoprecipitations with polyclonal antibodies against the different F-0 subunits clearly revealed that hybrid F-0 complexes derived from CF0 subunit III and EF(0) subunits a and b were formed in vivo. In addition, the ATPase activities of the hybrid ATP synthase, measured in everted cytoplasmic membranes of an atpE mutant strain transformed with the atpH gene coding for CF0 III, were comparable to activities obtained for the same mutant strain complemented with the atpE gene (EF(0) c). Nevertheless, CF0 III was not able to replace EF(0) c functionally, since the strain containing the hybrid ATP synthase was not able to grow on succinate. In order to investigate the reason for this lack of function, hybrid proteolipids of CF0 III and EF(0) c were constructed. Only a chimaeric protein comprising the seven N-terminal amino acid residues from CF0 III and the remaining part of EF(0) c was able to replace wild-type EF(0) c, whereas hybrid proteins with 13 and 33 N-terminal amino acids of CF0 III were not functional. The results suggested that a network of interactions between the subunits essential for proton translocation and/or coupling of the F-1 part exists, which was optimized for each species during evolution, although the overall structure of F0F1 complexes has been conserved.