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

枯草芽孢杆菌分泌高水平的胞外酶，并在营养不良的条件下产生耐热的内孢子。为了了解枯草芽孢杆菌的蛋白分泌途径，我们分析了Ffh与SecA的相互作用，以及分泌前蛋白从Ffh向SecA的传递。含有枯草芽孢杆菌碱性蛋白酶信号肽的a-淀粉酶和b-内酰胺酶融合蛋白前体（pAprE-BlaH6）或青霉素结合蛋白5* (pPBP5*-BlaH6)在缺乏SecA或Ffh的情况下积累。这表明SRP-和sec -蛋白分泌途径在枯草芽孢杆菌中协同分泌蛋白质。枯草芽孢杆菌的SRP由scRNA、Ffh和HBsu组成。Ffh作为识别和靶向分泌前蛋白的中心蛋白。在体外，Ffh能与pre -BlaH6和pPBP5*-BlaH6结合，但不能与它们的成熟形态结合。SecA还能结合pape -BlaH6和pPBP5*-BlaH6。前体与Ffh孵育后加入SecA可使SecA-前体复合物的形成增加15 ~ 30倍，反之则无显著差异。此外，结合Ffh的前体转移到SecA。通过配体亲和印迹、6xHis标记纯化和免疫电镜观察，证实了Ffh和SecA的直接相互作用。这些结果表明，SecA和Ffh通过包括其他蛋白在内的单一蛋白分泌途径相互作用。利用双向电泳技术对胞外蛋白进行蛋白质组学分析，发现大多数分泌蛋白通过蛋白分泌途径转运到培养基、母细胞间隙和孢子前。

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.