Synthesis and assembly of the F0 proton channel from F0 genes cloned into bacteriophage lambda and integrated into the Escherichia coli chromosome.
The promoter region and the first four genes of the Escherichia coli proton-translocating ATPase (unc) operon, uncIBEF, were cloned into bacteriophage lambda, enabling this region to be recombined into an unc-deleted E. coli chromosome at the lambda att site. The resultant E. coli strain, carrying single-copy F0 genes, was tested for synthesis and assembly of functional F0 proton channels. Membranes isolated from this strain contained all three F0 subunits and were capable of binding purified F1 and reconstituting F1F0-dependent energy coupling activities. The presence of these F0 sectors did not affect cell growth or membrane proton permeability assayed by fluorescence quenching. When compared with wild type membranes, membranes from the single-copy F0 strain contained less a and b subunits. When the single-copy lambda F0 strain was transformed with an F1 plasmid, the cells became phenotypically and biochemically Unc+, with membrane-bound ATPase and ATP synthase activities that were 50-60% of wild type. The results demonstrate that F0 produced from single-copy genes in the absence of F1 is membrane-bound and functional (i.e. reconstitutable) but not freely permeable to protons. The presence of F1 genes and/or subunits during F0 synthesis and assembly both increases the relative amounts of membrane-bound a and b subunits and produces an F0 sector more like that found in wild type cells than is produced from the single-copy F0 genes alone.
Brusilow
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WS
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Monticello
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RA
D000255:Adenosine Triphosphate / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics*; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D009876:Operon; D010957:Plasmids; D011401:Promoter Regions, Genetic; D006180:Proton-Translocating ATPases / Q000235:genetics*
D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases