Effects of inducing expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase.
To evaluate whether expression of cloned genes for the F0 proton channel of the Escherichia coli F1F0 ATPase is sufficient to cause membrane proton permeability, plasmids carrying different combinations of the uncB, E, and F genes, encoding the a, c, and b subunits of the F0 sector, cloned behind the inducible lac promoter in pUC9 or pUC18, were constructed. The effects of inducing F0 synthesis in an unc deletion strain were monitored by measuring cell growth rate, quantitating F0 subunits by immunoblotting, and measuring the ability of membranes to maintain a respiration-induced proton gradient and to bind F1 and carry out energy-coupling reactions. The levels of functional reconstitutable F0 in membranes could be increased four- to sixfold with no change in cellular growth rate or membrane proton permeability (assayed by fluorescence quenching). These results were obtained in uninduced cultures, so the F0 genes were presumably being transcribed from some promoter besides lac. Induction of transcription of all three F0 genes produced increased amounts of F0 subunits in membranes as determined by immunoblot and F1-binding assays, but, when reconstituted with F1, the F0 in membranes isolated from induced cultures was significantly less functional than the F0 in membranes isolated from uninduced cultures. Such induction did result in growth inhibition, but there was no correlation between growth inhibition and either increased membrane proton permeability or the presence of functional, reconstitutable F0.
Monticello
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Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.
Angov
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Brusilow
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D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics; D025243:Bacterial Proton-Translocating ATPases; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000502:physiology*; D004252:DNA Mutational Analysis; D004790:Enzyme Induction; D004926:Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins; D007473:Ion Channels / Q000502:physiology*; D007763:Lac Operon; D009243:NAD / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000502:physiology; D011522:Protons; D011994:Recombinant Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007473:Ion Channels; D011522:Protons; D011994:Recombinant Proteins; C074429:atpE protein, E coli; C074430:uncF protein, E coli; D009243:NAD; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C074428:atpB protein, E coli