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

枯草芽孢杆菌（Bacillussubtilis）分泌大量胞外酶，在营养不良的条件下产生耐热的内生孢子，为了了解枯草芽孢杆菌的蛋白分泌途径，我们分析了Ffh和SecA的相互作用，以及分泌前蛋白从Ffh到SecA的传递。具有B的信号肽的α-淀粉酶和β-内酰胺酶融合蛋白的前体。枯草杆菌碱性蛋白酶（pAprE-BlaH 6）或青霉素结合蛋白5*（pPBP 5 *-BlaH 6）在不存在SecA或Ffh的情况下积累。这表明，SRP-和Sec-蛋白分泌途径协同作用以分泌B中的蛋白。枯草杆菌。B.枯草芽孢杆菌中的RNA由scRNA、Ffh和HBsu组成。Ffh作为一种识别和靶向分泌前蛋白的中心蛋白。Ffh在体外与pAprE-BlaH 6和pPBP 5 *-BlaH 6结合，但不与它们的成熟形式结合。SecA还结合pAprE-BlaH 6和pPBP 5 *-BlaH 6。SecA-前体复合物的形成增强15至30倍时，前体和Ffh最初孵育，然后SecA添加，但反之亦然。此外，与Ffh结合的前体转移到SecA。通过配体亲和印迹、6xHis标签纯化和免疫电镜显示Ffh和SecA的直接相互作用。这些结果表明SecA和Ffh相互作用形成包括其他蛋白的单一蛋白分泌途径。利用双向电泳对胞外蛋白质进行蛋白质组学分析，发现大多数分泌蛋白质通过蛋白质分泌途径被转运到培养基中以及母细胞和前孢子的间隙中。

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.