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）：膜蛋白占细胞蛋白质组的30％，其有效而准确的定位对于所有细胞的结构和正常功能至关重要。与研究经过良好的共同翻译蛋白靶向途径相比，翻译后膜蛋白靶向的靶向构成了额外的挑战，因为底物蛋白上存在高度疏水性跨膜结构域。将这种易于聚集的底物护送到正确的目标位点的分子策略是一个基本的机械挑战。在尾锚径（GET）途径的指导条件下，一系列复杂的蛋白质相互作用介导了TA蛋白向内质网膜的翻译后递送，提供了解决这些问题的绝佳机会。我们的总体目标是在生化和生物物理水平上解密该新型途径对TA蛋白靶向的分子机制。我们的具体目标是了解该途径中的中央ATPase如何使用其ATPase循环驱动和协调GET途径期间蛋白质相互作用的复杂级联。为此，我们将为GET3 ATPase周期建立一个精确的框架，并确定在此周期中发生的构象变化。我们将定义GET3的上游和下游相互作用伙伴的何时，何地和下游相互作用伙伴如何调节其ATPase周期和相互调节，该ATPase周期如何驱动GET3相互作用的有序级联与其效应子蛋白的相互作用。我们将开发新的测定方法，以实时剖析靶向反应的各个步骤，并使用它来破译该途径如何实现高度特定的底物选择。这些研究将显着提高我们对膜蛋白转化后靶向的分子机制的理解。此外，GET3代表了属于新型“二聚化激活”核苷酸水解酶的第一个真核ATPase。对该ATPase二聚体的研究将有助于测试，扩展和推广这种不断增长的新型细胞调节剂的调节原理。