POSTTRANSLATIONAL PROTEIN TRANSPORT INTO YEAST ER

这个项目的目标是了解蛋白质是如何运输的。 翻译后穿过内质网(ER)膜 举止。这一过程必须在基本机制方面不同于 这是更好地理解合作翻译模式的结果。我们最近做了 建立了一个重新构建的系统，再现了翻译后的 含纯化蛋白脂质体的蛋白质转运途径 酿酒酵母ER蛋白的七聚体复合体。这个SEC-Complex 含有一个异三聚体的Sec61P-复合体，与哺乳动物中的类似，但 也包括在基因筛查中发现的所有其他膜蛋白 移位组件。高效的翻译后运输也 需要腔内K2p(Bip)和三磷酸腺苷。我们现在建议使用这一系统来 确定翻译后转运的分子机制，以 将其与协译进行比较，并确定两者如何 系统相互作用。具体来说，我们将解决以下问题： 1.不同的蛋白质复合体是否参与共翻译和翻译后 小路？我们将检验Sec61p-复合体作为 与其他SEC蛋白以及 与Kar2p在翻译后途径中的作用，并与 共翻译途径中的SRP受体。为此，我们将 描述和改进重组后的翻译后系统，以及 建立重现共翻译途径的重组系统 放在酵母里。 2.翻译后蛋白质转运的机制是什么？使用 重组系统，并采用各种其他方法以及 在突变的SEC蛋白中，我们将研究信号序列是如何 在翻译后易位途径中，识别的过程是如何 是能量驱动的，是否存在一种蛋白质传导 通道，以及通道是否被Kar2p或其他蛋白质门控。 3.易位还涉及哪些其他因素？我们将分析 最近发现的第二个Sec61P-络合物的功能 酵母，确定参与翻译后的胞浆因子 易位途径，寻找哺乳动物TRAM蛋白的同源物 在酵母中，并确定了这四个化合物的一级结构和功能 信号肽复合体的亚基还没有被 特色化的。 这些研究将使我们对 分泌蛋白质生物合成的第一步。尽管 该项目将仅限于酿酒酵母，很可能 结果将对共翻译蛋白和翻译后蛋白具有重要意义 所有生物体中的运输路径。

The goal of this project is to understand how proteins are transported across the endoplasmic reticulum (ER) membrane in a posttranslational manner. This process must differ in fundamental mechanistic aspects from that of the better understood cotranslational mode. We have recently established a reconstituted system that reproduces the posttranslational pathway of protein transport with proteoliposomes containing a purified heptameric complex of ER proteins from S. cerevisiae. This Sec- complex contains a heterotrimeric Sec61p-complex, similar to that in mammals, but also all other membrane proteins found in genetic screens for translocation components. Efficient posttranslational transport also requires lumenal Kar2p (BiP) and ATP. We now propose to use this system to determine the molecular mechanism of posttranslational transport, to compare it with the cotranslational one and to determine how the two systems interact. Specifically, we will address the following questions: 1. Are distinct protein complexes involved in co- and posttranslational pathways? We will test the hypothesis that the Sec61p- complex serves as a core component that associates with the other Sec- proteins as well as with Kar2p in the posttranslational pathway, and functions together with the SRP-receptor in the cotranslational pathway. To this end, we will characterize and improve the reconstituted posttranslational system, and establish reconstituted systems that reproduce the cotranslational pathway in yeast. 2. What is the mechanism of posttranslational protein transport? Using the reconstituted systems and employing various other methods as well as mutants in the Sec- proteins, we will study how the signal sequence is recognized in the posttranslational translocation pathway, how the process is energetically driven, whether there exists a protein- conducting channel, and whether the channel is gated by Kar2p or other proteins. 3. What other factors are involved in translocation? We will analyze the function of a recently discovered, putative, second Sec61p- complex in yeast, identify cytosolic factors involved in the posttranslational translocation pathway, search for a homolog of the mammalian TRAM protein in yeast, and determine the primary structure and functions of the four subunits of the signal peptidase complex that have not yet been characterized. These studies will lead to a significant progress in our understanding of the first steps in the biosynthesis of secretory proteins. Although the project will be confined to the yeast S. cerevisiae, it is likely that the results will be of significance for the co- and posttranslational protein transport pathways in all organisms.