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.