Biogenesis of multi-pass membrane proteins at the ER

项目总结 近30%的真核生物基因组编码完整的膜蛋白，这些蛋白服务于许多必需的 作为受体、酶、锚和转运体发挥作用。细胞表面的膜蛋白和大多数 细胞内的隔室首先在内质网(ER)组装。(fi)这些蛋白质是 通过信号识别颗粒共翻译到内质网并通过Sec61插入到双层中 很复杂。在最简单的观点中，核心的Sec61复合体通过引导新生疏水性来介导插入 跨膜结构域(TMD)进入中央水孔，该孔横向打开，允许TMD进入 双层的。 虽然这个模型对于理解TMD插入的基本机制很有价值，但它的 在生理底物的生物发生中的应用--特别是那些具有多个TMD的底物--已经被 很有挑战性。这一挑战源于真核膜蛋白的极端多样性，这些蛋白具有 插入、折叠、修饰fi阳离子和组装的截然不同的拓扑和生物物理要求 变成了功能实体。这些不同的步骤是由“易位”协调的，这是一个糟糕的fiNed和动态的 由Sec61复合体和各种附属亚基组成的系综体。这些建筑， 大多数这种机械的化学计量和功能，以及它们在膜中的作用，人们知之甚少 蛋白质的生物发生在很大程度上还没有被探索。了解不同的转位蛋白复合体如何介导 膜生物发生是细胞生物学中的一个基本问题 我们最近克隆了一种保守但知之甚少的人类蛋白质，称为TMCO1，作为fi的成员 一个先前未知的蛋白质超家族，参与膜蛋白的生物发生。与一致 这项任务，我们的初步数据表明，TMCO1是多组分组件的一部分，该组件 包括Sec61复合体和核糖体，并将其直接连接到共翻译插入、折叠中的一个角色 和/或组装一大群膜蛋白。 在这里，我们建立在这个概念和技术基础上，以了解fiNe TMCO1在 膜蛋白生物发生。在目标1中，我们将在全球范围内确定一组新生的TMCO1底物并使用 这些都进一步阐明了TMCO_1的作用机制。在目标2中，我们将严格分析交互伙伴 对于TMCO1和DefiNe，含有TMCO1的核糖体-Sec61复合物的结构。我们将使用一个 结合生化、遗传和结构分析的多学科方法。

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.