Protein translocation across the endoplasmic reticulum

该提案中描述的研究旨在阐明 新生的多肽被转运并整合到粗面内质网（PER）中 膜的将特别强调（a）分析信号识别颗粒（SRP）， 在易位反应中的SRP受体依赖性步骤，最终与选择性连接的a （B）分析核糖体-新生链复合物（RNC）的功能， 在新生多肽转运和膜蛋白整合过程中的转运通道。一个目标 第一个具体目的是通过以下方式表征SRP受体（SR）和核糖体之间的相互作用： 鉴定与核糖体相互作用的最小SR结构域。大分子上SR的结合位点 核糖体亚基将使用生物化学方法和冷冻电子技术的组合来确定。 显微镜第二个目的是了解Sec 61 p的细胞质环在SRP中的作用。 依赖的共翻译易位途径。第二个具体目标的目的是调查 使用一系列的生物化学方法， 泛素易位测定（UTA）报告基因。我们将测试的假设，横向运动的一个 蛋白质易位通道内的新生跨膜跨度相对于蛋白质易位通道是缓慢的事件。 合成延伸率这些实验应该提供对细胞的体内动力学的洞察。 核糖体通道连接关于蛋白质转运机制的几个关键问题是 提出了最近解决的X射线晶体结构的M。jannaschii SecYEB复合体。酵母分子 遗传学方法将用于测试目前的蛋白质易位模型，通过进行结构- 辅助Sshlp易位通道的功能分析。上述三个研究目标 解决蛋白质易位反应的知之甚少的方面。 完整膜蛋白、分泌蛋白和溶酶体蛋白的准确高效生物合成 蛋白质是人类细胞的基本功能，也是芽殖酵母等简单模式生物的基本功能。 粗面内质网中蛋白质的修饰或折叠缺陷是导致 越来越多的人类疾病被称为“ER质量控制”疾病。

The research described in this proposal is directed towards elucidating the molecular mechanism by which nascent polypeptides are translocated across on integrated into the rough endoplasmic reticulum (PER) membrane. Particular emphasis will be placed upon (a) analyzing the signal recognition particle (SRP) and SRP receptor-dependent steps in the translocation reaction that culminate with the selective attachment of a ribosome-nascent chain complex (RNC) to the translocation channel and (b) analyzing the function of the translocation channel during nascent polypeptide transport and membrane protein integration. One objective of the first specific aim is to characterize the interaction between the SRP receptor (SR) and the ribosome by indentifying the minimal SR domain that interacts with the ribosome. The binding site for the SR on the large ribosomal subunit will be determined using a combination of biochemical methods and cryoelectron microscopy. A second objective is to understand the role of cytoplasmic loops of Sec61p in the SRP- dependent cotranslational translocation pathway. The objective of the second specific aim is to investigate the in vivo kinetics of membrane protein integration in the yeast Saccharomyces cerevisiae using a series of ubiquitin-translocation assay (UTA) reporters. We will test the hypothesis that the lateral movement of a nascent transmembrane span within the protein translocation channel is a slow event relative to the protein synthesis elongation rate. These experiments should provide insight into the in vivo dynamics of the ribosome-channel junction. Several crucial questions about the mechanism of protein translocation were raised by the recently solved X-ray crystal structure of the M. jannaschii SecYEb complex. Yeast molecular genetics methods will be used to test current models of protein translocation by conducting a structure- function analysis of the auxiliary Sshlp translocation channel. The three research objectives outlined above address poorly understood aspects of the protein translocation reaction. The accurate and efficient biosynthesis of integral membrane proteins, secreted proteins and lysosomal proteins is an essential function in human cells, as well as in simple model organisms like budding yeast. Defects in the modification or folding of proteins in the rough endoplasmic reticulum are responsible for a growing list of human diseases that are termed "ER-quality control" diseases.