Biogenesis of multi-pass membrane proteins at the ER

项目摘要 近30%的真核生物基因组编码整合膜蛋白，这些蛋白在许多重要的生物学功能中起作用。 作为受体、酶、锚和转运蛋白发挥作用。细胞表面的膜蛋白， 细胞内区室首先在内质网（ER）组装。这些蛋白质 通过信号识别颗粒协同靶向ER并通过Sec 61插入双层 复杂.在最简单的观点中，核心Sec 61复合物通过引导新生的疏水性蛋白介导插入。 跨膜结构域（TMD）进入中央，水孔横向打开，允许TMD进入 双层。 虽然该模型已被证明对理解TMD插入的基本机制很有价值， 应用于生理底物的生物发生，特别是具有多种TMD的底物， 挑战性这一挑战来自于真核生物膜蛋白的极端多样性， 插入、折叠、修饰和组装的拓扑结构和生物物理要求截然不同 功能实体。这些不同的步骤由“translocon”协调，translocon是一个定义不明确的动态 在一个实施方案中，所述复合物是包含Sec 61复合物与多种辅助亚基缔合的整体。结构， 大多数这种机制的化学计量和功能知之甚少，它们在膜 蛋白质的生物合成在很大程度上尚未探索。了解不同的易位子复合物如何介导 膜生物发生是细胞生物学中的一个基本问题 我们最近艾德一种保守但知之甚少的人类蛋白质TMCO 1归类为一个成员， 一个以前未被认识的参与膜蛋白生物合成的蛋白质超家族。符合 在这项任务中，我们的初步数据表明，TMCO 1是多组分组装的一部分， 包括Sec 61复合物和核糖体，并直接将其连接到共翻译插入、折叠 和/或一大群膜蛋白的组装。 在这里，我们建立在这一概念和技术基础上，以定义TMCO 1如何在 膜蛋白生物合成在目标1中，我们将在全球范围内识别新生TMCO 1底物集，并使用 这些来定义TMCO 1的作用机制。在目标2中，我们将严格分析互动伙伴 并定义了含TMCO 1的核糖体-Sec 61复合物的结构。我们将使用 多学科的方法，结合了生物化学，遗传和结构分析。

PROJECT SUMMARY Nearly 30% of the eukaryotic genome encodes integral membrane proteins, which serve many essential functions as receptors, enzymes, anchors and transporters. Membrane proteins of the cell surface and most intracellular compartments are first assembled at the endoplasmic reticulum (ER). These proteins are cotranslationally targeted to the ER by the signal recognition particle and inserted into the bilayer by the Sec61 complex. In the simplest view, the core Sec61 complex mediates insertion by guiding nascent hydrophobic transmembrane domains (TMDs) into a central, aqueous pore which opens laterally to allow TMD entry into the bilayer. While this model has proven valuable for understanding the basic mechanism of TMD insertion, its application to the biogenesis of physiologic substrates—especially those with multiple TMDs—has been challenging. This challenge arises from the extreme diversity of eukaryotic membrane proteins, which have drastically different topologies and biophysical requirements for insertion, folding, modification and assembly into functional entities. These different steps are coordinated by the `translocon', a poorly defined and dynamic ensemble comprising the Sec61 complex in association with a variety of accessory subunits. The structures, stoichiometry and functions of most of this machinery are poorly understood, and their roles in membrane protein biogenesis are largely unexplored. Understanding how different translocon complexes mediate membrane biogenesis is a fundamental question in cell biology We recently classified a conserved but poorly understood human protein called TMCO1 as a member of a previously unrecognized superfamily of proteins involved in membrane protein biogenesis. Consistent with this assignment, our preliminary data demonstrate that TMCO1 is part of a multi-component assembly that includes the Sec61 complex and ribosomes, and directly link it to a role in the cotranslational insertion, folding and/or assembly of a large group of membrane proteins. Here we build on this conceptual and technical foundation to define how TMCO1 functions in membrane protein biogenesis. In Aim 1, we will globally identify the set of nascent TMCO1 substrates and use these to define the mechanism of TMCO1 action. In Aim 2, we will rigorously analyze the interaction partners of TMCO1 and define the structure of TMCO1-containing ribosome-Sec61 complexes. We will do this using a multi-disciplinary approach that combines biochemical, genetic and structural analyses.