Molecular Basis of Tail-Anchored Membrane Protein Targeting - Equip Suppl

帕萨特奖项目总结 该项目的目标是建立一个详细的分子理解如何尾锚定（TA） 膜蛋白质是在内质网（ER）膜上后插入的。TA 在所有真核生物膜蛋白中，几乎占5%的蛋白质存在于几乎所有的细胞中， 在细胞膜上，它们在包括细胞内运输在内的各种细胞过程中发挥重要作用， 蛋白质转运、酶催化和蛋白质质量控制。TA蛋白生物发生的缺陷与 许多人类病理，因此更好地了解这些系统的功能和功能障碍， 从而为无数疾病状态带来新的治疗策略。 翻译后靶向和TA蛋白插入ER膜是一个多步骤的过程 由“引导进入尾锚定蛋白”（GET）途径介导，首次发现于2007年初。以来 然后，我的实验室为理解TA蛋白的分子基础做出了基础性的贡献 酵母和哺乳动物的生物发生。我们在上一个资助期进行了严格的研究 使用完全纯化的艾德组分概括了该途径的早期“预靶向”步骤， 确定了必需的跨膜“插入酶”（称为Get 1/2）作为异二聚体复合物起作用。 此外，我们确定了第一个高分辨率结构的功能性膜蛋白靶向 这项工作解决了关于Get 3-TA蛋白复合物性质的持续争议 并定义了一种新的范式，用于跨膜结构域（TMD）如何在转运过程中被屏蔽。 含水胞质溶胶 在这个项目的过程中，我们已经组装了一套有价值的试剂，高分辨率 结构和利用酵母和无细胞系统的功能测定。事实上，我们现在已经重建了 从TA蛋白合成到TA蛋白插入的途径中的每一步都使用一组纯化的艾德， 重组可溶性和膜组分。这个系统的力量在于我们能够操纵 每一个组成部分和步骤的途径，使用重组和化学工具。因此，我们处于一个独特的 位置来定义结构，生物化学和生物物理学的原则，基础上的每一步的途径。 在这里，我们建立在这一技术和概念基础上，以解决两个核心问题，仍然存在 在外地了解不多。在目标1中，我们将定义Get 1/2跨膜复合物如何协调TA 蛋白质插入ER膜。在目标2中，我们将定义预定位机制如何捕获TA 蛋白质并将其转移到Get 3靶向因子上。我们将使用多学科方法来实现这一目标 该方法将功能分析与可溶性聚合物的混合计算和实验结构分析相结合， 和膜蛋白复合物。

PROJECT SUMMARY FROM PARENT AWARD The goal of this project is to establish a detailed molecular understanding for how tail-anchored (TA) membrane proteins are post-translationally inserted into the endoplasmic reticulum (ER) membrane. TA proteins, which account for nearly 5% of all eukaryotic membrane proteins, are found in virtually all cell membranes where they play essential roles in diverse cellular processes including intracellular trafficking, protein translocation, enzyme catalysis and protein quality control. Defects in TA protein biogenesis are linked to many human pathologies, and thus a better understanding of function and dysfunction in these systems may lead to new therapeutic strategies for myriad disease states. Post-translational targeting and insertion of TA proteins into the ER membrane is a multi-step process mediated by the `Guided Entry of Tail-anchored proteins' (GET) pathway, first discovered in early 2007. Since then, my lab has made fundamental contributions towards understanding the molecular basis of TA protein biogenesis in yeast and in mammals. Our rigorous studies performed during the previous granting period recapitulated the early, `pre-targeting' steps of the pathway using completely purified components and established that the essential transmembrane `insertase' (called Get1/2) functions as a heterodimeric complex. In addition, we determined the first high-resolution structures of a functional membrane protein targeting complex; this work resolved what was an ongoing controversy about the nature of the Get3-TA protein complex and defined a new paradigm for how transmembrane domains (TMDs) are shielded during transit through the aqueous cytosol. During the course of this project we have assembled a valuable suite of reagents, high-resolution structures, and functional assays that exploit yeast and cell-free systems. Indeed, we have now reconstituted every step in the pathway—from TA protein synthesis to TA protein insertion—using a set of purified, recombinant soluble and membrane components. The power of this system lies in our ability to manipulate each component and step in the pathway, using recombinant and chemical tools. Thus, we are in a unique position to define the structural, biochemical and biophysical principles that underlie every step in the pathway. Here we build on this technical and conceptual foundation to address two central questions that remain poorly understood in the field. In Aim 1, we will define how the Get1/2 transmembrane complex coordinates TA protein insertion into the ER membrane. In Aim 2 we will define how the pre-targeting machinery captures TA proteins and transfers them onto the Get3 targeting factor. We will do this using a multi-disciplinary approach that combines functional analysis with a hybrid computational and experimental structural analysis of soluble and membrane protein complexes.