EXPORT OF PROTEINS IN ESCHERICHIA COLI

新合成的特异体的移位现象 穿过或进入生物膜的多肽是常见的 真核分泌、细胞器生物发生与原核蛋白 出口。尽管这两个国家之间存在明显的差异 系统，潜在的原则似乎是共享的，并且 分子机制可能存在相似之处；因此， 研究人员试图阐明这种疾病的分子机制 对于蛋白质定位，重要的是要记住 在每个模型系统中获得的结果。 这里提出的研究计划旨在阐明 大肠杆菌中蛋白质输出机制的研究 Coli.三个主要目标大致可概括为：1) 通过定义中存在的步骤来描述出口的途径 并确定先导肽是否在体内发挥作用 在这些步骤中的每一个步骤中的作用，2)表征胞浆因子 参与出口的职能和性质 与前体的相互作用，3)阐明 前体在出口过程中的构象和折叠 进程。体内和体外研究的平衡将是 在工作过程中进行维护。因此，在体内 指数型细胞脉冲标记鉴定中间体 将在体外分析它们的生长阶段以确定它们的功能 关于前体与其相互作用的途径和性质 这台仪器。旨在确定折叠率的研究 在完整细胞中的研究将与使用 纯化的蛋白质和荧光光谱。试图 纯化胞浆因子将涉及到交联剂的使用 试剂和标准柱层析法。 周质蛋白麦芽糖结合蛋白被选为 学习不仅是因为它有很好的特点，而且还因为我们 可以利用产生出口的突变菌株- 我们可以利用的有缺陷的蛋白质种类。这个 这里提出的研究应该是对其他 实验室，并有助于理解 蛋白质定位的基本过程。

The phenomenon of translocation of specific, newly synthesized polypeptides across or into biological membranes is common to eukaryotic secretion, organelle biogenesis and prokaryotic protein export. Although there are obvious differences among the systems, it seems likely that underlying principles are shared and similarities may exist in the molecular mechanisms; thus, as investigators attempt to elucidate the molecular mechanism of protein localization, it will be important to keep in mind the results obtained in each of the model systems. The research plan proposed here is aimed at elucidating the mechanism of export of protein in the bacterium, Escherichia coli. The three main objectives can be broadly stated as: 1) to delineate the pathway of export by defining the steps that exist in vivo and determining whether or not the leader peptide plays a role at each of these steps, 2) to characterize the cytosolic factor involved in export with respect to its function and the nature of interaction with the precursor, 3) to elucidate the role of conformation and folding of the precursors during the export process. A balance of in vivo and in vitro studies will be maintained during the course of the work. Thus, in vivo intermediates identified by pulse-labelling of cells in exponential growth phase will be analysed in vitro to determine their function in the pathway and the nature of interaction of the precursor with the apparatus. Studies designed to determine the rate of folding in intact cells will be combined with studies of folding using purified proteins and fluorescence spectroscopy. Attempts to purify the cytosolic factor will involve use of cross-linking reagents and standard column chromatography. The periplasmic protein maltose-binding protein was chosen for study not only because it is well characterized but also so that we can make use of the mutated strains which produce export- defective species of the protein that are available to us. The studies proposed here should complement those in other laboratories and contribute to the understanding of the fundamental process of protein localization.