Mechanisms of Polytopic Protein Biogenesis in the ER

描述（由申请人提供）：该项目的长期目标是定义水通道蛋白水通道折叠、整合和组装到内质网（ER）膜中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水稳态中起重要作用。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在围绕单体水选择性孔的双重反向对称中的精确排列来实现的。最近的研究表明，这种显着的结构的形成是通过新生多肽和核糖体-转位子复合物（RTC）ER之间的精确和有序的相互作用来协调的。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的膜蛋白在其天然折叠环境。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。具体目标是：1。表征通过易位子控制膜整合和进展的新生多肽的结构和功能特性。2.定义核糖体转位子复合物控制新生多肽进入不同细胞区室的机制。3.定义AQP折叠的时间和分子环境。所提出的实验将使用附加掺入的探针直接进入RTC内新生多肽的分子环境。与截短的功能性易位中间体的光交联将定义新生AQP多肽内的结构特征如何控制TM片段进入、进展和从Sec 61 a易位子孔退出期间膜整合的顺序阶段。掺入的荧光团的碰撞猝灭将决定内腔和胞质肽环进入其适当细胞区室的精确合成阶段。最后，Forester共振能量转移将用于确定合成阶段和RTC内发生α-螺旋形成和形成早期三级结构的位置。这种生物化学和生物物理相结合的方法将共同定义RTC如何促进早期水通道蛋白折叠，2x和3x结构的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结构和膜插入。这些研究的结果将提供一个重大的进展，在我们的理解正常和病理机制的多面体蛋白质折叠。

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.