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 靶向和易位通过生物膜， 简单细菌，大肠杆菌，作为一种容易遗传和生化 系统在这项事业中。 虽然在这方面取得了迅速进展， 阐明了E.大肠杆菌蛋白分泌，大多数 这一途径中的步骤没有被足够详细地理解， 构建一个连贯的生化系统 为了实现这一目标，我们将重点 在这个系统的中心组成部分，SecA ATP酶，它与 前蛋白，SecB分子伴侣，阴离子磷脂，完整膜 蛋白质SecY/SecE，以及它自己的mRNA，是易位 前蛋白插入和穿过内膜所需的ATP酶 膜的 其中许多相互作用将进一步定义， 分子基础确定，其调节核苷酸结合， 水解研究，和他们的真实的目的，在催化 蛋白质分泌被破译。 为了理解的功能（S） 这种复杂的ATP酶，催化的独特的生物化学活动， 将确定SecA的两个单独的ATP结合结构域。 基础 SecA识别前蛋白的研究将采用 结合遗传和生化策略来阐明结构 前蛋白的信号肽和成熟区的特征， 允许SecA认可，SecA地区在促进这些方面很重要 将确定相互作用。 SecA的外围设备和 将研究与内膜的整体关联 进一步通过鉴定SecA受体和确定 促进和调节SecA与膜的整体结合。 一 采用半胱氨酸特异性切割的新作图技术将被使用 以高精度定位ATP结合，前蛋白结合，脂质结合， SecA一级结构上的mRNA结合位点。 一个精致 SecA蛋白结构知识将通过合作获得 研究结晶SecA蛋白或重要的SecA亚结构， 以原子分辨率确定其三维结构。 最后， SecA调节的基础及其与蛋白质的协调 细胞的分泌状态将使用遗传和 生物化学方法来定位分泌反应元件， geneX-secA mRNA，评估geneX翻译终止的重要性 和SecA蛋白结合在此元件中的功能，并鉴定 secA法规中的任何其他重要因素。 这些研究 对理解这些基本问题， 正常和异常细胞状态中的其他蛋白质分泌系统， 以及在试图修改输出时的实际重要性 途径，以适应新的基板。

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.