Protein translocation across Escherichia coli membranes

描述(申请人提供)：不同蛋白质的定位 执行各种功能的细胞和胞外隔间是 对所有活细胞的根本重要性。这个项目将集中在 一般分泌途径(SEC途径)大肠杆菌，涉及 Seca、SecB、Secy、Sece、SecG、Seed、SecF和YajC。我们将继续结合 我们已建立的体外分子操作和生化研究 具有大肠杆菌倒置膜小泡的系统。此外，我们还将延长 相互补充的电生理和物理方法是新的 为进一步研究而开发的。 建立在最近意想不到的发现基础上，尽管有点反对 目前的教条是，一些SecA整合到膜上，不再循环 并在转位过程中脱膜，我们将测试假设在 除了催化ATP水解外，SecA的某些结构域还发挥着 在转位机制中的重要结构作用，构成了 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复合体的情况下，可以观察到通道，并且仅有SecA 与阴离子相互作用形成环状和哑铃状结构 磷脂。在SECA如何运作的背景下，这些令人兴奋的新发现 以及SecYEG在蛋白质转位中的作用 将在这个项目中进一步探索。具体目标是：1.为了进一步 表征膜SecA在SEC分泌途径中的功能：(A) 确定膜中SECA的脂质专一性结构域的功能； (B)确定SecA在蛋白质电流活动中的作用 传导通道；(C)根据相互作用确定SecA的结构 含磷脂；和2)。确定SecYEG和其他蛋白质的作用 在蛋白质转位中：(A)确定SecYEG的功能；(B) 确定其他蛋白质的作用；以及(C)测试简化的工作 模特。 表征SecYEG和SecA在膜中的作用 对了解细菌的致病机制具有重要意义 蛋白质分泌，具有重要的医疗和工业应用。

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.