The eight subunits of the H+-ATPase of Escherichia coli are coded by the genes of the unc operon, which maps between bglB and asnA. A collection of unc mutations were transferred via P1 transduction into a strain in which A c1857 S7 was inserted into bglB. The lambda phage was induced, and asnA+ transducing phage that carried unc were selected. Transducing phage carrying mutations in the uncA, B, D, E, and F genes were used for complementation analysis with a collection of unc mutants, including mutants which had been reported previously but not genetically characterized. Some mutations gave a simple complementation pattern, indicating a single defective gene, whereas other mutations gave more complex patterns. Two mutants (uncEO05 and uncE107) altered in the proteolipid (omega) subunit of Fo were not complemented by any of the X unc phage, even though both mutants had a fully functional F, ATPase and therefore normal A and D genes. Hence, only limited conclusions can be drawn from genetic complementation alone, since it cannot distinguish normal from abnormal genes in certain classes of unc mutants. The X unc phage proved to be essential in characterizing several mutants defective in Fo-mediated H' translocation. The unc gene products were overproduced by heat induction of the lysogenized A unc phage to determine whether all the Fo subunits were in the membrane. Two mutants that gave a simple complementation pattern, indicative of one defective gene, did not assemble a three-subunit Fo. The uncB108 mutant was shown to lack the chi subunit of Fo but to retain psi and omega. Trace amounts of an altered omega subunit and normal amounts of chi and psi were found in the uncEI06 mutant. A substitution of aspartate for glycine at residue 58 of the protein was determined by DNA sequence analysis of the uncE gene cloned from the A uncE106 phage DNA. One of the omega-defective, noncomplementing mutants (uncE107) was shown to retain all three Fo subunits. The uncE gene from this mutant was also sequenced to confirm an asparagine-for-aspartate substitution at position 61 (the dicyclohexylcarbodiimide-binding site) of the omega subunit.The mechanism by which ATP is synthesized during oxidative phosphorylation is not understood at the molecular level. ATP synthesis occurs on a membrane-associated enzyme that is a reversible, H+-translocating ATPase. An electrochemical potential for H+, generated by an H+-translocating electron transport system, is postulated to be the driving force for ATP synthesis. ATP formation is thought to occur as protons are translocated through the reversible ATPase (2, 15). The ATP synthetases of bacteria, mitochondria, and chloroplasts are known to have common structural features (16,23). In each case the enzyme is composed of two structurally and functionally distinct sectors, termed F1 and Fo. F1 is the ATPase moiety of the complex and is bound to the surface of the membrane. The Fo sector extends through the membrane and functions as the H+ translocator of the complex. Each sector of the complex ...

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Mutations in the H+-translocating ATPase complex (F1F0) of Escherichia coli have been described in which aspartyl-61 of the omega subunit (uncE protein) is substituted by either glycine (uncE105) or asparagine (uncE107). Either substitution blocks the H+-translocation activity of the Fo sector of the complex. Here we report a difference in the effects of the two substitutions on the coupled ATPase activity of F1 bound to Fo. Wild-type F1 was bound to the Fo of either mutant with affinities comparable to wild-type. The ATPase activity of F1 bound to uncE107 Fo was inhibited by 50%, whereas that bound to uncE105 Fo was not inhibited. Complementation studies with a pBR322-derived plasmid that carried the E gene of the unc operon only indicated that a single mutation in the host strain was responsible for the respective phenotypes. In mutants complemented by the uncE+ plasmid, restoration of wild-type biochemical properties was only partial and may be attributed to a mixing of wild-type and mutant omega subunits in a hybrid Fo complex. The activity of membrane-bound F1 was less inhibited in the uncE+luncE107 hybrid.

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