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

糖、氨基酸和离子的跨膜转运对于细胞吸收营养物质、维持细胞内稳态和能量传递非常重要。本课题从生物化学和遗传学的角度分析了糖和氨基酸的Na^+偶联或H^+偶联主动转运、ATP合成和Na^+/H^+反向转运蛋白。我们分离到许多突变体，它们表现出转运载体特异性的阳离子改变。我们克隆了突变基因（melB），并测定了核苷酸序列。我们发现蜜二糖载体中某些氨基酸残基的取代引起阳离子特异性的改变。我们还利用phoA-melB融合技术对蜜二糖载体的膜拓扑结构进行了分析，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调节很重要。我们在铜绿假单胞菌中发现了支链氨基酸的Na^+偶联转运系统，并对其进行了表征;在鼠伤寒沙门氏菌中也发现了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。因此，揭示了H^+-柠檬酸盐载体的结构特征。对H^+偶联和Na^+偶联ATP合成系统的结构和功能进行了分析。

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.