MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

我们的总体目标是阐明前蛋白的分子细节。 靶向生物膜并通过生物膜转运 作为一种易于遗传和生化的简单细菌--大肠杆菌 在这项事业中的制度。尽管在以下方面取得了快速进展 阐明大肠杆菌蛋白质分泌的一些基本特征，MOST 此途径中的步骤未得到足够详细的理解 构建一个连贯的生化方案。为了实现这一目标，我们将集中精力 在这个系统的中心组件SecA ATPase上，它与 前蛋白、SecB伴侣、阴离子磷脂、整体膜 蛋白SecY/SecE，以及其自身的mRNA，是易位的 前蛋白插入和通过内部所需的ATPase 薄膜。其中许多相互作用将被进一步定义，他们的 确定的分子基础，它们通过核苷酸结合和 研究了水解性，以及它们在催化中的真正目的 蛋白质分泌被破译。为了理解(S)的作用 这种复杂的ATPase，其催化的不同生化活性 将确定SecA的两个单独的ATP结合结构域。其基础是 前蛋白的SecA识别将使用 结合遗传和生化策略来阐明结构 前蛋白信号肽和成熟区的特征 允许塞卡得到承认，塞卡的区域在促进这些方面很重要 相互作用将被确定。塞卡的外周基础和 将研究与内膜的整体关联。 此外，通过鉴定SECA受体并定义 促进和调节SECA与膜的整体结合。一个 将使用使用半胱氨酸特异性切割的新的图谱技术 高精度定位ATP结合、前蛋白结合、脂质- 和SECA一级结构上的mRNA结合位点。一个精致的 通过协作获得SecA蛋白的结构知识 研究结晶SecA蛋白或一个重要的SecA亚结构和 在原子分辨率下确定它的三维结构。最后， SecA的调控基础及其与蛋白质的协同作用 细胞的分泌状态将通过遗传和 定位分泌反应元件的生化方法 Genex-SecA mRNA，评估Genex翻译终止的重要性 和SecA蛋白结合在一起的这个元件的功能，并鉴定 任何其他在SECA法规中重要的因素。这些研究 应该对理解这些基本问题具有广泛的意义 正常和异常细胞中的其他蛋白质分泌系统包括 并且在尝试修改导出时具有实际重要性 适应新底物的途径。

Our overall goal is to elucidate the molecular details of preprotein targeting to and translocation through biological membranes using the simple bacterium, Escherichia coli, as a facile genetic and biochemical system in this undertaking. Although rapid progress has been made in elucidating some of the basic features of E. coli protein secretion, most steps in this pathway are not understood in sufficient detail to construct a coherent biochemical scheme. Towards this goal we will focus on a central component of this system, SecA ATPase, which interacts with preproteins, the SecB chaperone, anionic phospholipids, integral membrane proteins SecY/SecE, as well as its own mRNA, and is the translocation ATPase required for preprotein insertion and passage across the inner membrane. Many of these interactions will be defined further, their molecular basis determined, their regulation by nucleotide binding and hydrolysis investigated, and their real purpose in the catalysis of protein secretion deciphered. In order to understand the function(s) of this complex ATPase, the distinct biochemical activities catalyzed by the two separate ATP-binding domains of SecA will be determine. The basis of SecA recognition of preproteins will be investigated employing a combined genetic and biochemical strategy to elucidate the structural features of the signal peptide and mature region of the preprotein that allow SecA recognition, and regions of SecA important in promoting these interactions will be determined. The basis of SecA's peripheral and integral associations with the inner membrane will be investigated further by identifying the SecA receptor and defining components that promote and regulate SecA's integral association with the membrane. A novel mapping technique employing cysteine-specific cleavage will be used to locate with high precision the ATP-binding, preprotein-binding, lipid- binding, and mRNA-binding sites on SecA's primary structure. A refined structural knowledge of SecA protein will be gained through collaborative studies to crystallize SecA protein or an important SecA substructure and determine its three dimensional structure at atomic resolution. Finally, the basis for SecA regulation and its coordination with the protein secretion status of the cell will be determine using genetic and biochemical approaches to locate the secretion-responsive element on geneX-secA mRNA, assess the importance of geneX translation termination and SecA protein binding in the function of this element, and identify any additional factors important in secA regulation. These studies should be of broad significance to understanding these basic problems in other protein secretion systems in normal and abnormal cells states as well as of practical importance in attempting to modify the export pathway to accommodate novel substrates.