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Study of molecular mechanism of protein translocation through biochemical analysis of mutant secY in Escherichia coli

通过大肠杆菌突变体secY的生化分析研究蛋白质易位的分子机制

基本信息

批准号：
06680582
负责人：
YOSHIHISA Tohru
金额：
$ 1.41万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06680582/
关键词：
Translocation SecY Escherichia coli Secretion シグナル配列 His_6タグ

项目摘要

In the field of protein translocation analyzes in Escherichia coli, it is the most urgent theme to analyze a variety of sec mutants in vitro, especially, to compare their biochemical nature in a purified form. My aim in this project is purification of wild type and mutant SecY proteins both in a monomer and a complex with SecE and SecG.Using His_6-tagging method, I succeeded to purify wild type and 6 mutant forms of SecY including SecY39, a cs allele of secY,in SecYEG complexes and the wild type SecE monomer. The purified wild type SecYEG complex by this procedure retained protein translocation activity measured by reconstitution of SecYEG proteoliposome. Now I am trying, as the next step, to analyze and compare the purified complexes from the point of the following views ; 1) affinity to secretory precursors, 2) affinity to SecA ATPase, 3) effects on the membrane insertion and de-insertion of SecA,and 4) effects on an early stage of protein translocation through the analysis of Ni^<2+> inhibition of His_6-tagged pro-OmpA translocation.During the purification and protein translocation analysis of His_6-tagged pro-OmpA,a model protein of secretory precursor I used, I found that its translocation acquired Ni^<2+> sensitivity if a His_6 tag is inserted into the N-terminal region of the mature OmpA domain. The inhibitory effect by Ni^<2+> is neutralized by histidine and by using membrane vesicles prepared from prlA3, an allele of secY that is thought to broaden signal recognition specificity of translocase. Ni^<2+> is only effective in a post-targeting stage of the protein translocation, and also inhibited chemical cross-linking between pro-OmpA and SecY.These results demonstrated a possibility that Sec YEG complex directly recognizes secretory precursors in an early stage of the translocation.

在大肠杆菌蛋白易位分析领域，对多种sec突变体进行体外分析，特别是对其纯化后的生化性质进行比较是目前最为迫切的课题。在这个项目中，我的目标是纯化野生型和突变型SecY蛋白，无论是在单体还是与SecE和SecG复合物中。利用his_6标记法，我成功地纯化了SecYEG复合物和野生型SecE单体中的SecY39 （SecY的一个cs等位基因）和6个突变型SecY。通过该方法纯化的野生型SecYEG复合体保留了通过重组SecYEG蛋白脂质体测量的蛋白质易位活性。现在，作为下一步，我试图从以下观点来分析和比较纯化的配合物；1)与分泌前体的亲和力，2)与SecA ATPase的亲和力，3)对SecA膜插入和去插入的影响，4)通过分析Ni^<2+>对his_6标记的pro-OmpA易位的抑制作用，对早期蛋白质易位的影响。在我使用的分泌前体模型蛋白His_6-tagged pro-OmpA的纯化和蛋白质易位分析中，我发现如果在成熟的OmpA结构域n端插入His_6标签，其易位获得Ni^<2+>敏感性。Ni^<2+>的抑制作用被组氨酸和prlA3制备的膜囊所中和，prlA3是一种被认为可以扩大转座酶信号识别特异性的secY等位基因。Ni^<2+>仅在蛋白易位的后靶向阶段有效，并且也抑制了pro-OmpA和SecY之间的化学交联。这些结果表明，Sec YEG复合物在易位的早期阶段直接识别分泌前体的可能性。