Mechanisms of Polytopic Protein Biogenesis in the ER

项目描述（申请人提供）：本项目的长期目标是确定水通道蛋白水通道折叠、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家族，在肾、肺、脑和其他组织的正常和病理水稳态中起着重要作用。水通道蛋白功能的分子基础是通过精确排列六个跨膜段和两个短螺旋，以双重倒立对称围绕单体水选择孔。最近的研究表明，这种显著结构的形成是通过新生多肽和核糖体-转座子复合物（RTC） ER之间精确有序的相互作用而精心安排的。然而，RTC促进水通道蛋白折叠并反过来受水通道蛋白结构调节的潜在机制才刚刚开始被理解。因此，需要新的方法来检查新生膜蛋白在其天然折叠环境。本建议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。具体目标将：1。表征通过转座子控制膜整合和进展的新生多肽的结构和功能特性。2. 确定核糖体易位复合体控制新生多肽进入不同细胞区室的机制。3. 确定共翻译AQP折叠的时间和分子环境。拟议的实验将使用翻译结合探针直接进入RTC内新生多肽的分子环境。与截断的功能性易位中间体的光交联将定义新生AQP多肽的结构特征如何控制TM片段进入、进展和从Sec61 a易位孔退出时膜整合的顺序阶段。合并荧光团的碰撞猝灭将决定合成的精确阶段，在该阶段管状和细胞质肽环可以进入相应的细胞区室。最后，将使用Forester共振能量转移来确定合成阶段和RTC内a-螺旋形成和早期三级结构形成的位置。这种结合生化和生物物理的方法将定义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。这些研究结果将为我们理解多构体蛋白折叠的正常和病理机制提供重要的进展。

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.