Reconstitution of the E.coli protein translocation machinery

几个Sec因子参与了蛋白质在大肠杆菌细胞质膜上的易位。为了阐明蛋白质易位的机制，我们建立了用纯化的Sec因子重建易位机制的条件。利用这一重构系统，我们揭示了以下几点：发现一个新因素，SecG。发现SecG能刺激蛋白脂质体的重组活性。此外，还发现SecG是一种分子质量为12kDa.2的膜蛋白。对编码SecG的SecGA基因进行了克隆和测序。构建大肠杆菌DELTASecG菌株，在体内检测其蛋白分泌情况。DELTASecG菌株在20 C时分泌缺陷，而在37 C时没有分泌缺陷，说明SecG功能对低温下蛋白转运有重要作用。SecG的作用利用重构系统对SecG的作用进行了详细的考察。我们发现SecA受体的活性需要SecY和SecE。另一方面，SesG可以刺激SecA易位偶联的atp酶，表明SecA靶向细胞膜后SecG才起作用。

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.