Molecular Basis of Tail-Anchored Membrane Protein Targeting

尾锚定膜蛋白靶向的分子基础

基本信息

批准号：
9901536
负责人：
Robert J Keenan
金额：
$ 42.93万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-04-05 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9901536
关键词：
Address Binding Biochemical Biogenesis Biological Assay Biophysics C-terminal Catalysis Cell membrane Cell physiology Cell-Free System Chemicals Complex Cryoelectron Microscopy Cytosol Defect Development Diabetes Mellitus Disease Endoplasmic Reticulum Enzymes Eukaryotic Cell Event Foundations Functional disorder Goals Grant Growth Heart Diseases Human Pathology Hybrids Hydrolysis Lead Link Logic Malignant Neoplasms Mammals Mediating Membrane Membrane Proteins Modeling Molecular Molecular Conformation Molecular Target N-terminal Nature Negative Staining Neurodegenerative Disorders Pathway interactions Play Positioning Attribute Privatization Process Protein Biosynthesis Protein translocation Proteins Quality Control Reagent Recombinants Resolution Role Structure System Tail Time Transmembrane Domain Work X-Ray Crystallography Yeasts aqueous fight against human disease interdisciplinary approach novel therapeutic intervention novel therapeutics particle protein complex reconstitution reconstruction three-dimensional modeling tool trafficking virtual

项目摘要

PROJECT SUMMARY 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.

项目概要该项目的目标是建立对尾锚定（TA）如何进行详细的分子理解膜蛋白翻译后插入内质网 (ER) 膜。蛋白质占所有真核细胞膜蛋白的近 5%，几乎存在于所有细胞中它们在不同的细胞过程中发挥重要作用，包括细胞内运输， TA 蛋白生物发生中的蛋白质易位、酶催化和蛋白质质量控制缺陷是相关的。许多人类病理学，因此更好地理解这些系统的功能和功能障碍可能为多种疾病状态带来新的治疗策略。 TA 蛋白的翻译后靶向和插入 ER 膜是一个多步骤的过程由“尾锚定蛋白引导进入”(GET) 途径介导，该途径于 2007 年初首次发现。然后，我的实验室为理解 TA 蛋白的分子基础做出了基础性贡献我们在上一个授权期间进行了酵母和哺乳动物的生物发生。使用完全纯化的成分概括了该途径的早期“预靶向”步骤，确定必需的跨膜“插入酶”（称为 Get1/2）作为异二聚体复合物发挥作用。此外，我们首次确定了功能性膜蛋白靶向的高分辨率结构这项工作解决了有关 Get3-TA 蛋白复合物性质的持续争议并定义了跨膜域（TMD）在转运过程中如何被屏蔽的新范例水性细胞质。在这个项目的过程中，我们组装了一套有价值的试剂，高分辨率事实上，我们现在已经重建了利用酵母和无细胞系统的结构和功能测定。该途径中的每一步（从 TA 蛋白合成到 TA 蛋白插入）均使用一组纯化的、该系统的力量在于我们操纵重组可溶性和膜成分的能力。途径中的每个组成部分和步骤，都使用重组和化学工具。因此，我们处于独特的位置。定义该途径每一步的结构、生化和生物物理原理。在这里，我们在此技术和概念基础上解决仍然存在的两个核心问题在该领域中知之甚少。在目标 1 中，我们将定义 Get1/2 跨膜复合物如何协调 TA。在目标 2 中，我们将定义预靶向机制如何捕获 TA。蛋白质并将其转移到 Get3 靶向因子上我们将使用多学科方法来完成此任务。将功能分析与可溶性物质的混合计算和实验结构分析相结合和膜蛋白复合物。