Protein translocation across Escherichia coli membranes

描述（由申请人提供）：蛋白质定位于不同的 细胞和细胞外区室执行各种功能， 对所有活细胞都至关重要。该项目将集中在 一般分泌途径（Sec途径）大肠杆菌，涉及 SecA、SecB、SecY、SecE、SecG、SeeD、SecF和YajC。我们将继续联合收割机 分子操作和生物化学研究，我们建立了良好的体外 系统，E. coli倒置膜囊泡。此外，我们将扩大 互补的电生理和物理方法， 为进一步研究而开发。 基于最近的意外发现，虽然有点反对 目前的教条，一些SecA整合到膜上， 在易位过程中，我们将测试这一假设， 除了催化ATP水解外，SecA的某些结构域还起作用， 重要的结构作用，在易位机制，形成的一部分， 蛋白质传导通道这一假设得到了进一步的支持， 最近的研究发现，蛋白质传导的离子电流活性 在没有SecYEG复合物的情况下可以观察到通道， 在与阴离子相互作用时形成环状和哑铃状结构 磷脂在SecA功能的背景下，这些令人兴奋的新发现 以及SecYEG在蛋白质转运中的作用， 在这个项目中进一步探索。具体目标是：（1）。进一步 表征膜SecA在Sec分泌途径中的功能：（a） 确定SecA的脂质特异性结构域在膜中的功能; (b)确定SecA在蛋白质电流活性中的作用 传导通道;（c）确定SecA相互作用后的结构 与磷脂;和2）。确定SecYEG和其他蛋白质的作用 在蛋白质易位中：（a）确定SecYEG的功能;（B） 确定其他蛋白质的作用;和（c）测试一个简化的工作 模型 表征SecYEG和SecA在本文提出的膜中的作用 对了解细菌的作用机制具有重要意义。 蛋白质分泌，具有重要的医学和工业应用。

DESCRIPTION (provided by applicant): The localization of proteins to different cellular and extracellular compartments to carry out various functions is of fundamental importance to all living cells. This project will center on the general secretion pathway (the Sec pathway) Escherichia coli which involves SecA, SecB, SecY, SecE, SecG, SeeD, SecF and YajC. We will continue to combine molecular manipulation and biochemical studies in our well established in vitro systems with E. coli inverted membrane vesicles. In addition, we will extend the complementary electrophysiological and physical approaches that are newly developed for further studies. Building on the recent unexpected findings, though if somewhat against the current dogma, that some SecA integrates into membranes and does not cycle on and off membranes during translocation, we will test the hypothesis that in addition to catalyzing ATP hydrolysis, certain domains of SecA play an important structural role in the translocation machinery, forming part of the protein-conducting channels. This hypothesis is gaining further support with the recent findings that ionic current activity of the protein-conducting channels can be observed in the absence of SecYEG complex, and that SecA alone forms ring-like and dumbbell structures upon interaction with anionic phospholipids. These new exciting findings in the context of how SecA functions in the membranes, as well as the roles of SecYEG in protein translocation will be further explored in this project. The specific aims are: 1.) To further characterize the functions of membrane SecA in the Sec secretion pathway: (a) to determine the function of lipid-specific domains of SecA in the membranes; (b) to determine the role of SecA in electro-current activity of the protein conducting channel; (c) to determine the structures of SecA upon interaction with phospholipids; and 2). To determine the roles of SecYEG and other proteins in protein translocation: (a) to determine the functions of SecYEG; (b) to determine the roles of other proteins; and (c) to test a simplified working model. Characterizing the roles of SecYEG and SecA in the membranes as proposed here is of fundamental significance to understanding the mechanisms of bacterial protein secretion, which has important medical and industrial applications.