MECHANISM OF ACTION E COLI PROTEIN EXPORT FACTORS

这项拟议中的研究重点是分子伴侣在 蛋白质靶向和易位。这些细胞过程是 根本性的重要性。新合成的蛋白质的靶向是 对于新细胞的组装是必不可少的，并且有许多例子 由靶向缺陷引起的疾病。许多具有重要生理意义的 分子，如荷尔蒙，经历跨膜转移。 长期目标是了解分子伴侣在人类免疫系统中的作用 蛋白质在大肠杆菌内膜上的转运。这个 以下模型作为拟议研究的工作假设： 新生前体蛋白首先被伴侣蛋白识别，这些蛋白 都致力于蛋白质输出，如E.ColiSecB蛋白质。 陪伴者识别初生婴儿的未知结构特征 多肽。伴侣结合阻止前体折叠，防止丢失 并确保信号序列可用于 与移位设备的下一个组件相互作用， 很可能是外周膜蛋白SecA。当前体结合时 SECA，它成为膜结合的，并能够启动转位。 为了测试该模型的步骤，提出了以下具体目标： (L)鉴定对结合蛋白具有重要作用的SecB残基 新生多肽和结合的特异性。这些研究将 包括交联性和诱变性。(2)身份和身份的分析 出口蛋白质中氨基酸序列/结构的定位 促进SecB结合的前体。需要的多肽区域 将分析用于出口的SecB对SecB的绑定能力。(3) 改变SecA蛋白与SECA相互作用的SECA突变分析 SecB/前体复合体。突变体的生化特性 将在有缺陷的SecB存在的情况下改进出口。(4) 鉴定一种刺激非植物生长因子出口的细胞质因子 依赖于SecB的蛋白质。另一个出口专用的监护人将是 基因和/或生物化学鉴定的。

The proposed research focuses on the role of molecular chaperones in protein targeting and translocation. These cellular processes are of fundamental importance. Targeting of newly synthesized proteins is essential for assembly of new cells and there are numerous examples of diseases arising from defects in targeting. Many physiologically important molecules, such as hormones, undergo translocation across membranes. The long term goal is to understand the role of molecular chaperones in protein translocation across the Escherichia coli inner membrane. The following model serves as a working hypothesis for the proposed research: Nascent precursor proteins are first recognized by chaperone proteins that are dedicated to protein export, such as the E. coli SecB protein. Chaperones recognize an unknown structural feature of the nascent polypeptide. Chaperone binding blocks precursor folding, prevents the loss of export competence and ensures that the signal sequence is available for interaction with the next component of the translocation apparatus, probably the peripheral membrane protein SecA. When the precursor binds SecA, it becomes membrane bound and is able to initiate translocation. To test the steps of this model, the following specific aims are proposed: (l) Identification of residues of SecB that are important for binding of nascent polypeptides and for specificity of binding. These studies will involve crosslinking and mutagenesis. (2) Analysis of the identity and location of amino acid sequences/structures within exported protein precursors that promote SecB binding. Regions of polypeptides that require SecB for export will be analyzed for their ability to bind SecB. (3) Analysis of secA mutations that alter the interaction of SecA protein with the SecB/precursor complex. Biochemical characterization of mutants that improve export in the presence of defective SecB will be conducted. (4) Identification of a cytoplasmic factor that stimulates the export of non- SecB-dependent proteins. An alternative export-dedicated chaperone will be identified genetically and/or biochemically.