Structure and mechanism in cation-coupled transport systems

阳离子/底物同调是细胞膜内溶质主动转运的主要机制。在许多阳离子/衬底共模体系中，Na^+或H^+被用作耦合阳离子。也就是说，对称的驱动力是Na^+或H^+的电化学电位。Na^+的电化学电位由Na^+/H^+反转运体确定。在本研究中，我们研究了转运蛋白在正转运蛋白和反转运蛋白中的结构和转运机制。大肠杆菌和鼠伤寒沙门氏菌的糖二糖转运系统利用Na^+、H^+或Li^+作为同调的偶联阳离子，这取决于所转运的底物。我们分离出许多类型的突变体，它们表现出阳离子特异性改变，底物特异性改变，活性改变或温度敏感性改变。我们克隆了突变型melB基因，鉴定了甲基二糖转运蛋白中核苷酸的替换和氨基酸的替换残基。我们发现了许多Pro到Ser的替代品。然后，我们通过位点定向诱变构建了多种突变型的糖二糖转运蛋白。分析了突变的糖二糖转运蛋白的特性。因此，我们揭示了氨基酸残基在糖二糖转运蛋白中的结构-功能关系和作用。二糖转运系统的功能受磷酸转移酶系统的调控。我们鉴定了二糖转运体中的氨基酸残基和结构域，这些氨基酸残基和结构域参与了与调节蛋白的相互作用。我们还鉴定了Na^+/H^+反转运蛋白，并克隆了编码该反转运蛋白的nhaA基因并对其进行了测序。由此，我们获得了Na^+/H^+反转运子的结构-功能关系信息。

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.