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INTERACTION OF SRP/SRP-RECEPTOR SYSTEM AND SEC PROTEIN TRANSLOCATION PATHWAY IN Bacillus subtilis.

枯草芽孢杆菌中 SRP/SRP 受体系统与 SEC 蛋白易位途径的相互作用。

基本信息

批准号：
09460043
负责人：
YAMANE Kunio
金额：
$ 8.64万
依托单位：
University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09460043/
关键词：
Bacillus subtilis Protein secretion SRP Signal peptide Ffh SRP homologous SecA 枯草菌Bacillus subtilis SPR 相同因子 SRP 分泌蛋白質 SRP相同因子蛋白質分泌機構枯草菌蛋白質分泌 SRP受容体 Sec蛋白質蛋白質・蛋白質相互作用

项目摘要

Bacillus subtilis secretes high levels of extracellular enzymes and generates a heat-resistant endospore under poor nutrient conditions.To understand the protein secretion pathway of Bacillus subtilis, we analyzed the interaction of Ffh and SecA, as well as the passage of presecretory proteins from Ffh to SecA. Precursors of a-amylase and b-lactamase fusion proteins having signal peptide of B. subtilis alkaline protease (pAprE-BlaH6) or penicillin binding protein 5* (pPBP5*-BlaH6) accumulated in the absence of SecA or Ffh. This suggests that SRP- and Sec-protein secretion pathway co-operate to secret proteins in B. subtilis. The SRP of B. subtilis consists of scRNA, Ffh and HBsu. Ffh functions as a central protein for the recognition and targeting of presecretory proteins. Ffh binds to pAprE-BlaH6 and pPBP5*-BlaH6 in vitro, but not to their mature form. SecA also binds to both pAprE-BlaH6 and pPBP5*-BlaH6. SecA-precursor complex formation was enhanced 15 to 30 fold when the precursors and Ffh were initially incubated followed by SecA addition, but not vice versa. Moreover precursors that bind to Ffh transferred to SecA. Direct interaction of Ffh and SecA was shown by the ligand affinity blotting, 6xHis tag purification and immuno-electron microscopy. These results indicate that SecA and Ffh interact to from a single protein secretion pathway including other proteins. Proteome analysis of extracellular proteins using two-dimensional electrophoresis revealed that most secretory proteins are translocated into culture media and the interspace of the mother cell and prespore by the protein secretion pathway.

枯草芽孢杆菌在低营养条件下分泌高水平的胞外酶，并产生耐热的内孢子，为了了解枯草芽孢杆菌的蛋白质分泌途径，我们分析了FFH和SecA的相互作用，以及贮藏前蛋白从FFH到SecA的传递。含有枯草杆菌碱性蛋白酶信号肽(pAprE-BlaH6)或青霉素结合蛋白5*(pPBP5*-BlaH6)的a-淀粉酶和b-内酰胺酶融合蛋白的前体在没有SecA或FFH的情况下积累。这表明枯草杆菌中SRP-和SEC-蛋白分泌途径共同作用于分泌蛋白。枯草杆菌SRP由单链RNA、FFH和HBsu组成。FFH作为识别和靶向存储前蛋白的中心蛋白发挥作用。FFH在体外可与pAprE-BlaH6和pPBP5*-BlaH6结合，但不与其成熟形式结合。SecA还可与pAprE-BlaH6和pPBP5*-BlaH6结合。当前体和FFH最初孵育时，SecA-前体复合体的形成增加了15~30倍，然后添加SecA，但反之亦然。此外，与FFH结合的前体转移到SecA。配基亲和印迹、6xHis标记纯化和免疫电子显微镜显示FFH与SecA直接相互作用。这些结果表明，SecA和FFH相互作用形成包括其他蛋白质在内的单一蛋白质分泌途径。利用双向电泳法对胞外蛋白质进行蛋白质组分析，发现大多数分泌蛋白质通过蛋白质分泌途径转运到培养基内以及母细胞和前孢子的间隙中。