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.

在大肠杆菌中蛋白质易位分析的领域中，它是在体外分析各种SEC突变体的最紧急主题，尤其是以纯化的形式比较其生化性质。我在这个项目中的目的是纯化野生型和突变体Secy蛋白，包括单体和SECE和SECG的复合物。使用his_6 tagging方法，我成功地净化了野生型和6种突变形式的secy 39，包括secy39，包括secy的secyeg secy，在secyeg secyeg sepy and secyeg copplectes和sece sece sece单体中。通过此过程，纯化的野生型Secyeg复合物保留了通过重建Secyeg蛋白质体测量的蛋白质易位活性。现在，我正在尝试从以下观点分析和比较纯化的复合物； 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我发现，如果将His_6标记插入成熟OMPA域的N末端区域，则发现其易位获得了Ni^<2+>灵敏度。 Ni^<2+>的抑制作用被组氨酸和使用从PRLA3制备的膜囊泡中和，这是SECY的等位基因，被认为扩大了转运酶的信号识别特异性。 Ni^<2+>仅在蛋白质易位的靶向后阶段有效，并且还抑制了Pro-oMPA和SECY之间的化学交联。这些结果表明，SEC YEG复合物可能在易位的早期阶段直接识别分泌的前体。