EXPORT OF PROTEINS IN ESCHERICHIA COLI

新合成的特异性 多肽穿过或进入生物膜是常见的， 真核分泌、细胞器生物发生和原核蛋白 导出. 虽然有明显的差异， 系统，似乎潜在的原则是共享的， 相似性可能存在于分子机制中;因此，作为 研究人员试图阐明 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 大肠杆菌蛋白质输出的机制 杆菌 三个主要目标可以概括为：1） 通过定义存在的步骤来描绘出口途径， 并确定前导肽是否在体内起作用。 在每个步骤中的作用，2）表征胞质因子 参与出口有关其功能和性质， 与前体的相互作用，3）阐明的作用， 前体在输出过程中的构象和折叠 过程 将平衡体内和体外研究， 在工作过程中保持。 因此，在体内 通过指数细胞脉冲标记鉴定的中间体 将在体外分析生长期以确定其功能 在前体的相互作用的途径和性质， 设备。 旨在确定折叠速率的研究 在完整的细胞中，将结合使用 纯化的蛋白质和荧光光谱。 试图 纯化胞质因子将涉及使用交联 试剂和标准柱色谱法。 选择周质蛋白麦芽糖结合蛋白， 研究不仅是因为它具有很好的特征， 可以利用突变的菌株， 有缺陷的蛋白质种类。 的 本文提出的研究应补充其他领域的研究。 实验室，并有助于了解 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.