Reconstitution of the E.coli protein translocation machinery

几种Sec因子参与蛋白质跨大肠杆菌细胞质膜的易位。为了阐明蛋白质易位的机制，我们已经建立了纯化的Sec因子重建易位机制的条件。利用该重组系统，我们发现了以下几点：1.发现了一种新的因子SecG。SecG是一种膜蛋白，分子量约为12kDa。2.对编码SecG的SecGA基因进行了克隆和测序。此外，构建了大肠杆菌DELTASecG菌株并检查了体内蛋白分泌。DELTASecG菌株在20 ℃时分泌缺陷，而在37 ℃时不分泌，表明SecG功能在低温下蛋白质转运中起重要作用。3. SecG的作用利用重组系统详细研究了SecG的作用。发现SecA受体活性需要SecY和SecE两者。另一方面，发现SesG刺激SecA的易位偶联ATP酶，表明SecG在SecA靶向膜后起作用。

Several Sec factors are involved in the protein translocation across the E.coli cytoplasmic membrane. To elucidate the mechanism of protein translocation, we have established conditions for the reconstitution of the translocation machinery with purified Sec factors. Using this reconstitution system, we have revealed followings.1.Discovery of a novel factor, SecG.SecG was found to stimulate the reconstituted activity of proteoliposomes. Furthermore, SecG was found to be a membrane protein having a molecular mass of 12kDa.2.Genetic analysis of SecGA gene encoding SecG was cloned and sequenced. Furthermore, E.coli DELTASecG strain was constructed and examined for protein secretion in vivo. The DELTASecG strain was defective in secretion at 20ﾟC but not at 37ﾟC,indicating that the SecG function is important for protein translocation at low temparature.3.Roles of SecG.Roles of SecG was examined in detail using the reconstitution system. It was found that both SecY and SecE are required for SecA receptor activity. On the the other hand, SesG was found to stimulate the translocation-coupled ATPase of SecA,indicating that SecG function after SecA targets to the membrane.