Characterization of Escherichia coli lactose carrier mutants that transport protons without a cosubstrate. Probes for the energy barrier to uncoupled transport.
The Escherichia coli lactose carrier is an energy-transducing H+/galactoside cotransport protein which strictly couples sugar and proton transport in 1:1 stoichiometry. Here we describe five lactose carrier mutants which catalyze "uncoupled" sugar-independent H+ transport. Symptoms similar to uncoupling by a proton ionophore have been observed in cells expressing these mutant carriers. The mutations occur at two separate loci, encoding substitutions either for alanine 177 (valine) or tyrosine 236 (histidine, asparagine, phenylalanine, or serine). Compared to the parent, cells expressing the valine 177 carrier grew slowly on minimal media with glucose as carbon source. When washed cells were incubated in the absence of added sugars the mutant showed a reduced protonmotive force compared with the parent. Addition of either thiodigalactoside or alpha-p-nitrophenylgalactoside reduced the defect in protonmotive force. Sugar-independent H+ entry rate into cells expressing either the normal carrier or the Val-177 mutant were measured directly using the pH electrode. Following sudden acidification of the external medium (by either oxygen-pulse or acid-pulse) protons entered more rapidly into cells expressing the Val-177 carrier. This novel sugar-independent mode of H+ transport probably depends on an acquired capacity of the Val-177 carrier to bind the transported proton with higher than normal affinity in a transition state involving the binary carrier/H+ complex.
King
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Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115.
Wilson
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TH
D001692:Biological Transport; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D008756:Methylgalactosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism; D014633:Valine
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D011522:Protons; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; C021499:lactose permease; D014633:Valine