Molecular mechanisms of post-translational targeting of tail-anchored proteins.

尾锚定蛋白翻译后靶向的分子机制。

基本信息

批准号：
8762368
负责人：
Shu-ou Shan
金额：
$ 35.58万
依托单位：
CALIFORNIA INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8762368
关键词：
ATP phosphohydrolase Address Amber Binding Biochemical Biogenesis Biological Biological Assay Cell physiology Cells Cellular Membrane Complex Coupled Dimerization Dyes Endoplasmic Reticulum Ensure Enzymatic Biochemistry Evolution Family Fluorescence Fluorescence Resonance Energy Transfer Fluorescence Spectroscopy Fluorescent Dyes Goals Guanosine Triphosphate Phosphohydrolases Hydrolase Hydrolysis Individual Kinetics Knowledge Laboratories Life Logic Mediating Membrane Membrane Proteins Mind Molecular Molecular Chaperones Monitor Nucleotides Pathology Pathway interactions Physiology Property Proteins Proteome Reaction Reagent Regulation Series Signal Recognition Particle Site Specificity Stress Structure System Tail Testing Thermodynamics Time Transmembrane Domain Work base dimer driving force insight member novel public health relevance receptor signal recognition particle receptor single-molecule FRET time use tool

项目摘要

DESCRIPTION (provided by applicant): Membrane proteins comprise ~30% of a cell's proteome, and their efficient and accurate localization is essential for the structure and proper functioning of all cells. Compared to the well-studied co-translational protein targeting pathway, post-translational membrane protein targeting poses additional challenges due to the presence of highly hydrophobic transmembrane domains on the substrate protein. Deciphering the molecular strategies to escort such aggregation-prone substrates to the correct target site is a fundamental mechanistic challenge. In the Guided Entry of Tail-anchor (GET) pathway, a complex cascade of protein interactions mediates the post-translational delivery of TA proteins to the endoplasmic reticulum membrane, providing an excellent opportunity to address these questions. Our general goal is to decipher, at the biochemical and biophysical level, the molecular mechanisms underlying the targeting of TA proteins by this novel pathway. Our specific goal is to understand how Get3, the central ATPase in this pathway, uses its ATPase cycle to drive and coordinate the complex cascade of protein interactions during the GET pathway. To this end, we will establish a precise framework for the Get3 ATPase cycle and identify conformational changes that occur during this cycle. We will define when, where and how the upstream and downstream interaction partners of Get3 regulate its ATPase cycle and reciprocally, how this ATPase cycle drives an ordered cascade of interactions of Get3 with its effector proteins. We will develop novel assays to dissect individual steps of the targeting reaction in real time and use this to decipher how highly specific substrate selection is achieved by the pathway. These studies will significantly advance our understanding of the molecular mechanisms that underlie the post- translational targeting of membrane proteins. Further, Get3 represents the first eukaryotic ATPase that belongs to a novel class of 'dimerization-activated' nucleotide hydrolases; studies of this ATPase dimer will be instrumental to test, expand, and generalize the regulatory principles for this growing class of novel cellular regulators.

描述（由申请人提供）：膜蛋白约占细胞蛋白质组的 30%，其有效且准确的定位对于所有细胞的结构和正常功能至关重要。与经过充分研究的共翻译蛋白靶向途径相比，由于底物蛋白上存在高度疏水性跨膜结构域，翻译后膜蛋白靶向带来了额外的挑战。破译将这种易于聚集的底物护送至正确靶位点的分子策略是一个基本的机械挑战。在尾锚引导 (GET) 通路中，复杂的蛋白质相互作用级联介导 TA 蛋白翻译后递送至内质网膜，为解决这些问题提供了绝佳的机会。我们的总体目标是在生物化学和生物物理水平上破译这种新途径靶向 TA 蛋白的分子机制。我们的具体目标是了解该途径中的中心 ATP 酶 Get3 如何利用其 ATP 酶循环来驱动和协调 GET 途径中蛋白质相互作用的复杂级联。为此，我们将为 Get3 ATPase 循环建立精确的框架，并识别该循环期间发生的构象变化。我们将定义 Get3 的上游和下游相互作用伙伴何时、何地以及如何调节其 ATP 酶循环，以及该 ATP 酶循环如何驱动 Get3 与其效应蛋白的有序级联相互作用。我们将开发新的检测方法来实时剖析靶向反应的各个步骤，并用它来破译该途径如何实现高度特异性的底物选择。这些研究将显着增进我们对膜蛋白翻译后靶向分子机制的理解。此外，Get3 代表了第一个真核 ATP 酶，属于一类新型“二聚化激活”核苷酸水解酶；对这种 ATP 酶二聚体的研究将有助于测试、扩展和推广这一不断增长的新型细胞调节剂的调节原理。