Structure and mechanism in cation-coupled transport systems

阳离子/底物共转运蛋白是溶质在细胞膜中主动转运的主要机制。在许多阳离子/底物共载体体系中，使用Na~(++)或H~(2+)作为偶联阳离子。也就是说，符号的驱动力是Na~(++)或H~(++)的电化学势。Na~(++)/H~(++)反向转运蛋白建立了Na~(++)的电化学势。在本研究中，我们研究了转运蛋白的结构及其在转运蛋白和对转运蛋白中的转运机制。大肠杆菌和鼠伤寒沙门氏菌的转运蛋白系统利用Na~+、H~(++)或Li~(++)作为转运蛋白的偶联阳离子，依赖于底物的转运。我们分离到了多种类型的突变体，它们表现出阳离子专一性、底物专一性、活性和温度敏感性的变化。我们克隆了突变类型的Melb基因，鉴定了核苷酸的替代，并鉴定了Melibiose转运蛋白中取代的氨基酸残基。我们发现了许多从Pro到Ser的替代品。然后，我们通过定点突变构建了多种突变类型的蜜糖转运蛋白。对突变的蜜糖转运蛋白的性质进行了分析。因此，我们揭示了甘露糖运输蛋白中氨基酸残基的结构-功能关系和作用。甘露糖运输系统的功能受磷酸转移酶系统的调节。我们鉴定了蜜二糖转运蛋白中与调节蛋白相互作用的氨基酸残基和结构域，我们还鉴定了Na+/H+逆向转运蛋白，并克隆和测序了编码该反向转运蛋白的nhaA基因。因此，我们获得了Na~(++)/H~(++)逆向转运蛋白结构-功能关系的信息。

Cation/substrate symport is a major mechanism of active solute transport in cell membranes. In many cation/substrate symport systems, Na^+ or H^+ is utilized as the coupling cation. Namely, the driving force for the symport is an electrochemical potential of Na^+ or H^+. An electrochemical potential of Na^+ is established by Na^+/H^+ antiporter. In this study, we investigated structure of the transport proteins and mechanism of transport in the symporter and the antiporter.The melibiose transport system of Escherichia coli and Salmonella Typhimurius utilizes either Na^+, H^+ or Li^+ as the coupling cation for symport depending on substrate transported. We isolated many types of mutant which showed altered cation specificity, altered substrate specificity, altered activity or altered temperature sensitivity. We cloned the mutant types melB gene, identified substitution of nucleotide and identified substituted amino acid residues in the melibiose transport protein. We found many Pro to Ser replacements. Then we constructed many mutant types of the melibiose transport protein by site-directed mutagenesis. Properties of the mutated melibiose transport proteins were analyzed. Thus, we revealed structure-function relationship and role of amino acid residues in the melibiose transport protein.Function of the melibiose transport system is regulated by phospho-transferase system. We identified amino acid residues and domain in the melibiose transporter, which are involved in the interaction with a regulatory protein.We also characterized Na^+/H^+ antiporter, and cloned and sequenced nhaA gene encoding the antiporter. Thus, we obtained information about structure-function relationship in the Na^+/H^+ antiporter.