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.

一些 Sec 因子参与跨大肠杆菌细胞质膜的蛋白质易位 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，我们揭示了.1.新因子SecG的发现。SecG被发现可以刺激蛋白脂质体的重构活性。此外，SecG被发现是一种具有一定分子量的膜蛋白12kDa.2.编码SecG的SecGA基因的基因分析被克隆并测序。此外，构建了大肠杆菌DELTASecG菌株并检查了体内蛋白质分泌。DELTASecG菌株在20°C下有分泌缺陷，但在37°C下没有分泌缺陷。 C,表明SecG功能对于蛋白质低温易位很重要。3.SecG的作用。使用重构系统对SecG进行了详细检查，发现SecY和SecE都是SecA受体活性所必需的，另一方面，SesG被发现可以刺激SecA的易位偶联ATP酶，表明SecA靶向后SecG起作用。到膜。

项目成果

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。”

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。”

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。”