Mechanisms of Polytopic Protein Biogenesis in the ER

内质网中多位蛋白生物合成的机制

• 批准号: 7118633
• 负责人: WILLIAM R SKACH
• 金额: $ 29.41万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118633
• 关键词: crosslink; dogs; endoplasmic reticulum; fluorescence resonance energy transfer; fluorescent dye /probe; laboratory rabbit; membrane activity; membrane biogenesis; membrane transport proteins; molecular assembly /self assembly; molecular chaperones; photochemistry; protein biosynthesis; protein folding; protein isoforms; protein localization; protein structure function; protein transport; ribosomes; tissue /cell culture; water channel

DESCRIPTION (provided by applicant): The long-term goal of this project is to define the general principles and molecular mechanisms by which aquaporin water channels fold, integrate and assemble into the endoplasmic reticulum (ER) membrane. Aquaporins comprise a highly conserved protein family that plays a major role in normal and pathological water homeostasis in the kidney, lung, brain and other tissues. The molecular basis of aquaporin function is achieved by a precise arrangement of six transmembrane segments and two short helices in a two-fold inverted symmetry surrounding a monomeric water-selective pore. Recent studies have demonstrated that formation of this remarkable structure is orchestrated via precise and ordered interactions between the nascent polypeptide and the ribosome-translocon complex (RTC) ER. However, the underlying mechanisms by which the RTC facilitates aquaporin folding and is in turn regulated by aquaporin structure is only beginning to be understood. New approaches are therefore needed to examine nascent membrane proteins in their native folding environment. The studies outlined in this proposal will address two fundamental aspects of this process. First, they will directly define how the RTC facilitates aquaporin folding and membrane integration of transmembrane segments. Second they will determine how structural properties of aquaporins act in a reciprocal fashion to control RTC structure and function. The Specific Aims will: 1. Characterize structural and functional properties of the nascent polypeptide that control membrane integration and progression through the translocon. 2. Define the mechanism by which the ribosome translocon complex controls accessibility of the nascent polypeptide to different cellular compartments. 3. Define the timing and molecular environment of cotranslational AQP folding. Proposed experiments will use translationally incorporated probes to directly access the molecular environment of the nascent polypeptide within the RTC. Photocrosslinking to truncated functional translocation intermediates will define how structural features within the nascent AQP polypeptide control sequential stages of membrane integration during TM segment entry, progression, and exit from the Sec61 a translocon pore. Collisional quenching of incorporated fluorophores will determine the precise stage of synthesis at which lumenal and cytosolic peptide loops gain access to their appropriate cellular compartments. Finally, Forester Resonance Energy Transfer will be used to determine the stage of synthesis and location within the RTC at which a-helix formation takes place and early tertiary structure is formed. Together this combined biochemical and biophysical approach will define how the RTC facilitates early aquaporin folding, how 2x and 3x structure formation impacts interactions between the nascent polypeptide and RTC, and how these interactions regulate RTC structure to direct protein topology and membrane insertion. Results of these studies will provide a major advance in our understanding of normal and pathological mechanisms of polytopic protein folding.

描述(由申请人提供)：该项目的长期目标是确定水通道折叠、整合和组装到内质网(ER)膜的一般原理和分子机制。水通道蛋白是一个高度保守的蛋白质家族，在肾、肺、脑和其他组织的正常和病理性水平衡中发挥重要作用。水通道蛋白功能的分子基础是通过六个跨膜片段和两个短螺旋的精确排列实现的，这些螺旋围绕着单体水选择孔形成两个倒置的对称性。最近的研究表明，这种特殊结构的形成是通过新生多肽和核糖体-转位蛋白复合体(RTC)ER之间精确而有序的相互作用来调控的。然而，RTC促进水通道蛋白折叠并进而受水通道蛋白结构调控的潜在机制才刚刚开始被理解。因此，需要新的方法来研究新生的膜蛋白在其天然折叠环境中的情况。本提案中概述的研究将涉及这一进程的两个基本方面。首先，他们将直接定义RTC如何促进水通道蛋白折叠和跨膜片段的膜整合。其次，他们将确定水通道蛋白的结构特性如何以相互作用的方式控制RTC的结构和功能。具体目标如下：1.确定控制转运子的膜整合和进展的新生多肽的结构和功能特性。2.确定核糖体转位复合体控制新生多肽与不同细胞隔间的可及性的机制。3.确定共翻译AQP折叠的时机和分子环境。拟议的实验将使用翻译结合的探针来直接访问RTC内新生多肽的分子环境。截短的功能转位中间体的光交联将定义新生AQP多肽中的结构特征如何控制TM片段进入、进展和退出Sec61a转位孔时膜整合的连续阶段。被掺入的荧光团的碰撞猝灭将决定合成的精确阶段，在这个阶段，管腔和胞质多肽环可以进入它们适当的细胞室。最后，Forester的共振能量转移将被用来确定合成的阶段和在RTC中发生a-螺旋形成和早期三级结构形成的位置。这一结合的生化和生物物理方法将定义RTC如何促进早期水通道蛋白折叠，2x和3x结构形成如何影响新生多肽和RTC之间的相互作用，以及这些相互作用如何调节RTC结构以指导蛋白质拓扑和膜插入。这些研究的结果将为我们理解多聚体蛋白折叠的正常和病理机制提供重要的进展。