Alterations in the primary structure of the active transport carrier and changes in the function

细胞膜中营养物质的几种转运系统是Na^+偶联共转运系统。大肠杆菌中的蜜二糖转运系统是Na^+偶联共转运系统中最具特征的系统之一，我们分离出了许多突变体，它们在蜜二糖载体中表现出改变的阳离子特异性。变种人分为两种。当以蜜二糖为底物时，其中一种突变体的蜜二糖载体失去了与H^+偶联的能力，而获得了与Li^>偶联的能力。在这些突变体中，与Na^+的偶联是正常的。另一类突变体的蜜二糖载体失去了与H^<>偶联的能力，只能利用Na^+作为偶联交换。本课题对第二类突变体进行了分析，克隆了突变体的突变基因melB，并测定了其核苷酸序列。核苷酸替换导致蜜二糖载体中氨基酸残基的以下取代：Pro-142被Ser取代，Leu-232被Phe取代，或Ala-236被Thr或瓦尔取代。这些氨基酸残基位于蜜二糖载体的轻微疏水区域。这些结果有力地支持了这样一种观点，即含有这些氨基酸残基的区域或其附近区域在蜜二糖载体的H^+识别或H^+转运中起着重要作用。

Several transport systems for nutrients in cell membranes are Na^+-coupled cotransport systems. One of the best characterized Na^+-coupled cotransport systems is the melibiose transport system in Esherichia coli.We isolated many mutants which showed altered cation specificity in the melibiose carrier. There were two types of mutant. The melibiose carrier of one type of the mutants lost the ability to couple to H^+ and acquired the ability to couple to Li^<> when melibiose was the substrate. The coupling to Na^+ in those mutants was normal. The melibiose carrier of another type of the mutants lost the ability to couple to H^<> and could utilize only Na^+ as the coupling csation. We analysed the second type mutants in this project.Mutated gene, melB, of the mutants was cloned, and the nucleotide sequence was determined. The nucleotide replacements caused the following substitutions of amino acid residues in the melibiose carrier; Pro-142 with Ser, Leu-232 with Phe, or Ala-236 with Thr or Val. These amino acid residues are located in slightly hydrophobic regions of the melibiose carier. The results provide a strong support for the idea that such regions or their vicinities which contain those amino acid residues play an important role in H^+ recognition or H^+ transport by the melibiose carrier.