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

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.