Integrative functional mapping of the Escherichia coli membrane interactome

描述（由申请人提供）：膜蛋白复合物仍然是生物化学和分子生物学的最后一个主要挑战。虽然在选择膜蛋白和复合物的结晶方面取得了重大进展，但许多功能未知的膜蛋白仍有待研究。这对于膜中的复合物来说更是如此。 在这里，我们提出解决这个问题的综合分析膜蛋白复合物在E。 大肠杆菌，不仅是一个主要的微生物模型系统，但也是唯一的物种，其中所有的膜蛋白的拓扑结构已被实验确定。 在本项目中，我们将纯化大部分或全部的E.大肠杆菌中，并通过质谱分析其组成。 我们已经成功地在酵母中进行了这样的分析，从而预测获得数百种蛋白质复合物。独立地，我们将筛选E.大肠杆菌膜蛋白的相互作用，使用多个酵母双杂交系统。此外，我们将使用细菌双杂交系统绘制膜蛋白的相互作用，以确保体内测定条件。这些研究将使我们能够绘制膜蛋白复合物的拓扑结构，并将这些蛋白质与细胞质中的可溶性蛋白质联系起来。最后，我们将专注于在上述筛选中发现的未表征的膜蛋白，特别是转运蛋白，并使用突变和专门的测定方法详细分析它们的体内相互作用和功能，以使用高通量方案确定它们的活性（如转运）。生物信息学分析将协助所有三个分项目。据我们所知，以前没有尝试过这种规模的综合研究。该项目的结果将对微生物和真核生物的膜蛋白生物学产生广泛的影响，包括生物技术和医学应用。例如，膜蛋白是抗菌剂的常见靶标，该项目将鉴定新的靶标，但也阐明许多这些（迄今未表征的）跨膜蛋白的分子功能。

DESCRIPTION (provided by applicant): Membrane protein complexes remain the last major challenge in biochemistry and molecular biology. Although major advances have been made crystallizing selected membrane proteins and complexes, many membrane proteins of unknown function remain to be studied. This is even truer for complexes in the membrane. Here we propose to tackle this problem by an integrated analysis of membrane protein complexes in E. coli, not only a major microbial model system but also the only species in which the topology of all membrane proteins has been determined experimentally. In the course of this project, we will purify most or all membrane protein complexes of E. coli and analyze their composition by mass spectrometry. We have successfully carried out such an analysis in yeast and thus predict to obtain several hundred protein complexes. Independently, we will screen all cytoplasmic domains of E. coli membrane proteins for interactions using multiple yeast two-hybrid systems. In addition, we will map interactions of membrane proteins using a bacterial two-hybrid system to ensure in vivo assay conditions. These studies will allow us to map the topologies of membrane protein complexes and link those proteins to soluble proteins in the cytoplasm. Finally, we will focus on uncharacterized membrane proteins found in the aforementioned screens, especially transporters, and analyze their in vivo interactions and functions in detail using mutations and specialized assays to determine their activities (such as transport) using high throughput protocols. Bioinformatic analysis will assist all of the three subprojects. To our knowledge no such integrated study of this scale has been attempted before. The results of this project will have a broad impact on membrane protein biology in both microbes and eukaryotes, including biotechnological and medical applications. For instance, membrane proteins are common targets of antimicrobials and this project will identify new targets but also elucidate the moleculr function of many of these (hitherto uncharacterized) transmembrane proteins.