Energy Transduction in Sodium Driven-flagagellar Motor

在弧菌Na^+型鞭毛中发现了MotX、MotY、PomA和PomB四种马达蛋白。它们是溶藻弧菌膜蛋白中不可或缺的一部分，也是Na^+驱动的极性鞭毛马达旋转的必要组成部分。为了研究PomA的功能，我们用羟胺对pomA基因进行了随机突变。结果表明，H^+马达的PomA和MotA具有非常相似的结构和功能。所以产生扭矩的基本机制在质子和钠马达中是相似的。完整的motX基因从溶藻弧菌中克隆，并显示出互补三个mot突变，motX 94，motX 115和motX 119，以及副溶血性弧菌的motX突变体。鞭毛马达的研究主要集中在H^+-马达中，有五种核心蛋白（MotA、MotB、FliG、FliM和FliN）参与鞭毛马达的功能。在马达的转子部分中，三种可溶性蛋白质FliG、FliM和FliN是力产生、鞭毛组装和控制马达旋转方向的组分。我们克隆了Na^+驱动的极鞭毛马达的fliG基因，并利用His标签进行纯化。为了验证马达蛋白是否形成复合物，进行免疫沉淀。PomB与PomA通过抗PomA抗体共沉淀，并且抗PomB抗体也与PomA共沉淀。这些直接证明了PomA和PomB在功能上相互作用。

Four motor proteins, MotX, MotY, PomA and PomB have been identified in the Na^+-type flagella of Vibrio spp. They are integral membrane proteins and essential components for the rotation of the Na^+-driven polar flagellar motor of Vibrio alginolyticus. To investigate PomA function, we carried out random mutagenesis of the pomA gene by using hydroxylamine. As the results, it is suggested that PomA and MotA of H^+ motor have very similar structure and roles. So the basic mechanism for torque generation will be similar in the proton and sodium motor. The complete motX gene was cloned from Vibrio alginolyticus and was shown to complement three mot mutations, motX94, motX115 and motX119, as well as a motX mutant of V.parahaemolyticus. The study of the flagellar motor has been intensively done in the H^+-motor and there are five core proteins (MotA, MotB, FliG, FliM and FliN) involved in the motor function. In the rotor part of the motor, three soluble proteins, FliG, FliM and FliN, are the components for force-generation, flagellar assembly and controlling the direction of motor rotation. Among them, FliG has a direct role in force generation with the cytoplaslmic region of MotA.We have clnoed the fliG gene of Na^+-driven polar flagellar motor and the product could be purified using the His tag. To verify whether the motor proteins form a complex, immunoprecipitation was carried out. PomB was coprecipitated with PomA by anti-PomA antibody, and anti-PomB antibody coprecipitated with PomA as well. Those directly demonstrated that PomA and PomB functionally interact with each other.