Structure and mechanism in active transport systems for sugars, amino acids and ions.

糖、氨基酸和离子的跨细胞膜运输对于营养物质的吸收、维持细胞内的动态平衡和细胞内的能量传递是非常重要的。本课题试图从生化和遗传学的角度分析Na~+偶联和H~(++)偶联的糖和氨基酸的主动转运、三磷酸腺苷的合成以及Na~+/H~(++)逆向转运蛋白。我们分离到了许多突变体，这些突变体表现出运输载体的特异性阳离子改变。我们克隆了突变基因(Melb)，并测定了其核苷酸序列。我们发现，蜂蜜二糖载体中某些氨基酸残基的取代导致了阳离子的特异性改变。我们还利用PhoA-Melb融合技术分析了蜜二糖载体的膜拓扑结构，并确定了膜中载体的拓扑结构。我们还分析了Na~+/H~+逆向转运蛋白，发现它对细胞内pH的调节很重要。我们在铜绿假单胞菌中发现了支链氨基酸的钠离子偶联转运系统，并对其进行了表征。我们还在鼠伤寒沙门氏菌中发现了一个H^+偶联的柠檬酸转运系统。克隆了该基因(CITA)并测定了其核苷酸序列。从而揭示了H~(2+)-柠檬酸载体的结构特征。分析了H^+偶联和安娜^+偶联的三磷酸腺苷合成系统的结构和功能。

Transport of sugars, amino acids and ions across cell membranes is very important for absorption of nutrients, maintenance of intracellular homeostasis and energy transduction in cells. In this project, we tried to analyze Na^+-coupled or H^+-coupled active transport of sugars and amino acids, ATP synthesis, and Na^+/H^+ antiporter from biochemical and genetic aspects.The melibiose transport system of Escherichia coli is an Na^+-coupled system. We isolated many mutants which showed altered cation specificly of the transport carrier. We cloned the mutated gene (melB), and determined the nucleotide sequences. We found that substitutions of some amino acid residues in the melibiose carrier caused alteration in cation specificly. We also analyzed the membrane topology of the melibiose carrier by using phoA-melB fusion, and determined the topology of the carrier in membrane. We also analyzed the Na^+/H^+ antiporter, and found that it was important for intracellular pH regulation. We found Na^+-coupled transport system for branched-chain amino acids in Pseudomonas aeruginosa, and characterized it. We also found an H^+-coupled citrate transport system in Salmonella typhimurium. The gene (citA) was cloned and the nucleotide sequences were determined. Thus, structural characteristics of the H^+-citrate carrier were revealed. Structure and function of an H^+-coupled and an Na^+- coupled ATP synthesizing systems were also analyzed.