Reconstitution of the E.coli protein translocation machinery

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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.