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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.

在大肠杆菌蛋白质易位分析领域，对各种SEC突变体进行体外分析，尤其是对它们的生化性质进行纯化比较，是目前最迫切的课题。本课题的目的是从SecE和SecG的单体和复合体中分离纯化野生型和突变型SecY蛋白。利用His_6标记的方法，成功地从SecYEG复合体和野生型SecE单体中提纯了SecY的野生型和6种突变形式，其中包括SecY的cs等位基因SecY39。用此方法纯化的野生型SecYEG复合体通过重组SecYEG蛋白脂质体保持了蛋白转位活性。作为下一步，我试图从以下几个角度对纯化的复合体进行分析和比较：1)对分泌前体的亲和力，2)对SecA ATPase的亲和力，3)对SecA膜插入和去插入的影响，4)对蛋白质转位早期的影响，通过对Ni^&lt；2+&gt；抑制His_6标记的前OmpA转位的分析。如果将His_6标签插入到成熟OmpA结构域的N-末端区域，则具有敏感性。组氨酸和由Secy的等位基因prlA3制备的膜小泡可扩大转位酶的信号识别特异性，从而中和了Ni^&lt；2+&gt；的抑制作用。Ni^&lt；2+&gt；仅在蛋白质转位的后靶向阶段有效，并抑制原OmpA和SecY之间的化学交联。这些结果表明，SEC YEG复合体可能在转位的早期直接识别分泌前体。