Structure and mechanism in cation-coupled transport systems

阳离子/基质共输是细胞膜中溶质主动转运的主要机制。在许多阳离子/底物同向转运体系中，Na^+或H^+被用作偶联阳离子。也就是说，同向迁移的驱动力是Na^+或H^+的电化学电势。Na^+/H^+反向转运蛋白可以确定Na^+的电化学电位。在本研究中，我们研究了转运蛋白的结构以及同向转运体和反向转运体的转运机制，大肠杆菌和鼠伤寒沙门氏菌的蜜二糖转运系统利用Na^+、H^+或Li^+作为偶联阳离子进行同向转运，这取决于所转运的底物。我们分离出许多类型的突变体，这些突变体显示出改变的阳离子特异性、改变的底物特异性、改变的活性或改变的温度敏感性。我们克隆了突变型melB基因，鉴定了蜜二糖转运蛋白中的核苷酸替换和替换的氨基酸残基。我们发现了许多Pro到Ser的替代品。然后通过定点突变的方法构建了多种蜜二糖转运蛋白突变体。分析了突变蜜二糖转运蛋白的性质。因此，我们揭示了蜜二糖转运蛋白的结构与功能的关系以及氨基酸残基的作用，蜜二糖转运系统的功能受磷酸转移酶系统的调控。我们鉴定了蜜二糖转运蛋白中参与与调节蛋白相互作用的氨基酸残基和结构域，鉴定了Na^+/H^+反向转运蛋白，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^+反向转运蛋白的结构与功能关系的信息。

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.