Brooker R J
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108.
J Biol Chem. 1991 Mar 5;266(7):4131-8.
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.
对含有Val177或Val177/Asn319的乳糖通透酶的糖特异性突变体进行了H⁺和糖共转运偶联能力的分析。两个突变体都能在很大程度上使乳糖顺浓度梯度运输。Val177突变体在半乳糖苷的主动积累方面存在部分缺陷，而Val177/Asn319突变体在糖的逆浓度梯度积累方面完全缺陷。关于偶联，已表明Val177突变体在很大程度上催化H⁺的非偶联运输。这导致在有氧条件下H⁺电化学梯度降低，并且当在厌氧条件下产生瞬时H⁺电化学梯度时也导致更快的H⁺摄取。有趣的是，已表明半乳糖苷会降低Val177菌株中非偶联H⁺运输的速率。Val177/Asn319菌株也催化非偶联H⁺运输，但程度低于单一的Val177突变体。此外，已表明Val177/Asn319突变体在有或没有H⁺的情况下都能运输半乳糖苷。在双突变体中观察到的H⁺/乳糖化学计量比为0.30，而在野生型菌株中为0.98。当跨膜产生H⁺电化学梯度时，如果不可代谢的半乳糖苷已在膜上达到平衡，Val177/Asn319突变体通透酶被证明会促进极其快速的净H⁺泄漏。这种泄漏的机制与涉及H⁺/半乳糖苷内流和非偶联半乳糖苷外流的循环途径一致。在双突变体中，在存在不可代谢半乳糖苷的情况下H⁺泄漏的程度非常大，以至于低浓度的某些半乳糖苷（即0.5 mM硫代半乳糖苷）会导致生长完全受到抑制。针对阳离子和糖与乳糖通透酶结合可能在共同识别位点涉及直接物理偶联的可能性对这些结果进行了讨论。
