An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. I. Val177 and Val177/Asn319 permeases facilitate proton uniport and sugar uniport.
The sugar specificity mutants of the lactose permease containing Val177 or Val177/Asn319 were analyzed with regard to their ability to couple H+ and sugar co-transport. Both mutants were able to transport lactose downhill to a significant degree. The Val177 mutant was partially defective in the active accumulation of galactosides, whereas the Val177/Asn319 mutant was completely defective in the uphill accumulation of sugars. With regard to coupling, the Val177 mutant was shown to catalyze the uncoupled transport of H+ to a substantial degree. This led to a decrease in the H+ electrochemical gradient under aerobic conditions and also resulted in faster H+ uptake when a transient H+ electrochemical gradient was generated under anaerobic conditions. Interestingly, galactosides were shown to diminish the rate of uncoupled H+ transport in the Val177 strain. The Val177/Asn319 strain also catalyzed uncoupled H+ transport, but to a lesser degree than the single Val177 mutant. In addition, the Val177/Asn319 mutant was shown to transport galactosides with or without H+. The observed H+/lactose stoichiometry was 0.30 in the double mutant compared to 0.98 in the wild-type strain. When an H+ electrochemical gradient was generated across the membrane, the Val177/Asn319 mutant permease was shown to facilitate an extremely rapid net H+ leak if nonmetabolizable galactosides had been equilibrated across the membrane. The mechanism of this leak is consistent with a circular pathway involving H+/galactoside influx and uncoupled galactoside efflux. The magnitude of the H+ leak in the presence of nonmetabolizable galactosides was so great in the double mutant that low concentrations of certain galactosides (i.e. 0.5 mM thiodigalactoside) resulted in a complete inhibition of growth. These results are discussed with regard to the possibility that cation and sugar binding to the lactose permease may involve a direct physical coupling at a common recognition site.
Brooker
R J
RJ
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
D001692:Biological Transport; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D006863:Hydrogen-Ion Concentration; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008756:Methylgalactosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008756:Methylgalactosides; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; D013862:Thiogalactosides; C009468:thiomethylgalactoside; D007544:Isopropyl Thiogalactoside; C021499:lactose permease; D007785:Lactose