Reconstitution of the E.coli protein translocation machinery

大肠杆菌蛋白质易位机制的重建

• 批准号: 05454620
• 负责人: TOKUDA Hajime
• 金额: $ 3.71万
• 依托单位: University of Tokyo, Institute of Molecular and Cellular Biosciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05454620/
• 关键词: Protein translocation machinery; Reconstitution system; Proteoliposome; SecG; Membrane protein; 蛋白質膜透過; 再構成系; 遺伝子破壊; p12

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.

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。

项目成果

Nishiyama, K.: "Disruption of the gene encoding p12 (SecG) reveals the direct involvement and important function of SecG in the protein translocation of Escherichia coli at low temperature." EMBO J.13. 3272-3277 (1994)

Nishiyama, K.：“p12 编码基因 (SecG) 的破坏揭示了 SecG 在大肠杆菌低温下蛋白质易位中的直接参与和重要功能。”

Hanada, M.: "Reconstitution of an efficient protein translocation machinery comprising SecA and the three membrane proteins, SecY,SecE,and SecG (p12)." J.Biol.Chem.269. 23625-23631 (1994)

Hanada, M.：“重建包含 SecA 和三种膜蛋白 SecY、SecE 和 SecG (p12) 的有效蛋白质易位机制。”

Kawasaki, S.: "Membrane vesicles containing overproduced SecY and SecE exhibit high translocation ATPase activity and counter movement of protons in a SecA-and presecretory protein-dependent manner." J.Biol.Chem.268. 8193-8198 (1993)

Kawasaki, S.：“含有过量产生的 SecY 和 SecE 的膜囊泡表现出高易位 ATP 酶活性，并以 SecA 和分泌前蛋白依赖性方式实现质子的反向运动。”

Tokuda, H.: "Biochemical characterization of the presecretory protein translocation machinery of Escherichia cali." FEBS Lett.346. 65-68 (1994)

Tokuda, H.：“卡利埃希氏菌分泌前蛋白易位机制的生化特征。”

Tokuda,H.: "Histidine residues are involred in translocation-coupled ATP hydrolysis of SecA protein." Biochem.Biophys.Res.Commun.195. 1415-1421 (1993)

Tokuda, H.：“组氨酸残基参与 SecA 蛋白的易位偶联 ATP 水解。”