Molecular Basis of Tail-Anchored Membrane Protein Targeting

尾锚定膜蛋白靶向的分子基础

• 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
• 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

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.

项目总结 这个项目的目标是建立对尾部锚定(TA)的详细分子理解。 膜蛋白在翻译后插入内质网(ER)膜。标记 蛋白质几乎存在于所有细胞中，占所有真核膜蛋白的近5%。 它们在不同的细胞过程中发挥重要作用的膜，包括细胞内TRAFfi转录， 蛋白质易位、酶催化和蛋白质质量控制。TA蛋白生物发生中的缺陷与 从而更好地了解这些系统中的功能和功能障碍 为无数疾病状态带来新的治疗策略。 TA蛋白的翻译后靶向和插入内质网是一个多步骤的过程 fi在2007年初发现，在“尾部锚定蛋白的引导进入”(GET)途径的介导下，该基因被发现。自.以来 然后，我的实验室为理解TA蛋白的分子基础做出了根本性的贡献 酵母和哺乳动物的生物发生。我们在之前的授权期内进行了严格的研究 用完全提纯的成分概括了该通路的早期的“前靶向”步骤，并 确定了必要的跨膜‘插入酶’(称为Get1/2)的功能是异源二聚体复合体。 此外，我们还确定了一个功能性膜蛋白靶向的fi第一高分辨结构。 复合体；这项工作解决了关于Get3-TA蛋白复合体性质的持续争议 和DefiNed提出了跨膜结构域(TMD)在转运过程中如何被屏蔽的新范例 含水的胞浆。 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 结构，以及利用酵母和无细胞系统的功能分析。事实上，我们现在已经重组了 途径中的每一步--从TA蛋白合成到TA蛋白插入--使用一组纯化的fi， 重组可溶性和膜组分。这个系统的力量在于我们操纵的能力 使用重组和化学工具，在途径中的每个组成部分和步骤。因此，我们处于一个独特的 定位于define的结构、生化和生物物理原理，这些原理是该途径中每一步的基础。 在这里，我们建立在这个技术和概念的基础上，以解决仍然存在的两个核心问题 在fi领域，人们对此知之甚少。在目标1中，我们将了解Get1/2跨膜复合体是如何与TA配位的 蛋白质插入内质网细胞膜。在目标2中，我们将介绍fiNe预定位机制如何捕获TA 蛋白质并将它们转移到Get3靶向因子上。我们将使用多学科的方法来实现这一点 它将泛函分析与混合计算和实验结构分析相结合 和膜蛋白复合体。