MECHANISM OF PROTEIN LOCALIZATION IN ESCHERICHIA COLI

我们的总体目标是使用大肠杆菌作为模型系统来阐明 蛋白质定位的基本机制，发生在所有 活细胞 我们的长远目标是（i）界定及 表征大肠杆菌蛋白质输出机制，（ii）分配 （三）了解其各个组成部分的功能; 这些功能是相互关联的， 出口途径，以及（iv）确定该途径是如何调节的 并与其他重要的细胞过程相协调。 在 目前，9个推定的出口机械部件已被 鉴定，但没有指定特定的生物化学功能 这些组件中的任何一个。 这些具体目标 研究是为了进一步定义和阐明secA的作用， 在大肠杆菌中催化和调节蛋白质输出的基因产物。 使用突变分析和SecA依赖性体外蛋白 易位系统，我们将定义易位 SecA蛋白催化的活性，确定哪些部分 SecA多肽编码这种活性，并鉴定 与SecA蛋白相互作用的出口相关组分 履行这一职能。 我们将剖析secA的协同调节 与蛋白质出口熟练程度进一步使用的组合， 突变分析以及体内和体外测定，以表征 SecA多肽的哪些部分编码其自身的 翻译阻遏物活性，确定位置和结构 输出响应翻译操作符，并阐明 负责观察到的协同调节的生化机制。 由于蛋白质定位的机制是中心的， 生物学的重要性，从各个层面的运作， 单个蛋白质分子的生物合成和维持 正常和异常的细胞状态，并且由于这些机制具有 在整个进化过程中都是保守的，这些对大肠杆菌的研究 应该具有广泛的意义。

Our overall goal is to use E.coli as a model system to elucidate the basic mechanisms of protein localization that occur in all living cells. Our long-term objective is (i) to define and characterize the E.coli protein export machinery, (ii) to assign functions to its individual components, (iii) to understand how these functions are interelated to produce an efficient protein export pathway, and (iv) to determine how this pathway is regulated and coordinated with other essential cellular processes. At present, 9 putative export machinery components have been identified, but no specific biochemical function has been assigned to any of these components. The specific objective of these studies is to further define and elucidate the role of the secA gene product in catalyzing and regulating protein export in E.coli. Using a mutational analysis and a SecA-dependent in vitro protein translocation system, we will define the translocation activity(ies) catalyzed by SecA protein, determine which portions of the SecA polypeptide encodes this activity, and identify the export-related components that SecA protein interacts with in fulfilling this function. We will dissect the coregulation of secA with protein export proficiency further using a combination of mutational analysis and in vivo and in vitro assays to characterize which portions of the SecA polypeptide encodes its autogenous translational repressor activity, define the location and structure of the export-responsive translational operator, and elucidate the biochemical mechanism responsible for the coregulation observed. Since the mechanisms of protein localization are of central biological importance at all levels, from the functioning of individual protein molecules to the biogenesis and maintenance of normal and abnormal cell states, and since such mechanisms have been conserved throughout evolution, these studies with E.coli should be of broad significance.