title: An analysis of lactose permease "sugar specificity" mutations which also affect the coupling between proton and lactose transport. II. Second site revertants of the thiodigalactoside-dependent proton leak by the Val177/Asn319 permease.
abstract: The double mutant of the lactose permease containing Val177/Asn319 exhibits proton leakiness by two pathways (see Brooker, R. J. (1991) J. Biol Chem. 266, 4131-4138). One type of H+ leakiness involves the uncoupled influx of H+ (leak A pathway) while a second type involves the coupled influx of H+ and galactosides in conjunction with uncoupled galactoside efflux (leak B pathway). In the current study, 14 independent lactose permease mutants were isolated from the Val177/Asn319 parent which were resistant to thiodigalactoside growth inhibition but retained the ability to transport maltose. All of these mutants contained a third mutation (besides Val177/Asn319) at one of two sites. Eight of the mutants had Ile303 changed to Phe, while six of the mutants had Tyr236 changed to Asn or His. Each type of triple mutant was characterized with regard to sugar transport, H+ leakiness, and sugar specificity. Like the parental strain, all three types of triple mutant showed moderate rates of downhill lactose transport and were defective in the uphill accumulation of sugars. However, with regard to proton leakiness, the triple mutants fell into two distinct categories. The mutant containing Phe303 was generally less H+ leaky than the parent either via the leak A or leak B pathway. In contrast, the triple mutants containing position 236 substitutions (Asn or His) were actually more H+ leaky via the leak A pathway and exhibited similar H+ leakiness via the leak B pathway at high thiodigalactoside concentrations. The ability of the position 236 mutants to grow better than the parent in the presence of low concentrations of thiodigalactoside appears to be due to a decrease in affinity for this particular sugar rather than a generalized defect in H+ leakiness. Finally, the triple mutants showed a sugar specificity profile which was different from either the Val177/Asn319 parent, the single Val177 mutant, or the wild-type strain. These results are discussed with regard to the effects of mutations on both the sugar and H+ transport pathways.
authors.affiliation: Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
authors.forename: J A
authors.initials: JA
authors.lastname: Eelkema
authors.forename: M A
authors.initials: MA
authors.lastname: O'Donnell
authors.forename: R J
authors.initials: RJ
authors.lastname: Brooker
mesh_terms: D001483:Base Sequence; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001692:Biological Transport; D002475:Cellobiose / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005697:Galactosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007785:Lactose / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D008553:Melibiose / Q000378:metabolism; D008564:Membrane Potentials; D026901:Membrane Transport Proteins / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D027981:Symporters; D013862:Thiogalactosides / Q000378:metabolism
chemical_list: D001565:Benzoates; D029968:Escherichia coli Proteins; D005697:Galactosides; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D013862:Thiogalactosides; D002475:Cellobiose; D008320:Maltose; C031803:thiodigalactoside; D019817:Benzoic Acid; C021499:lactose permease; D008553:Melibiose; D007785:Lactose