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 在翻译后转录中穿过内质网（ER）膜， 方式这个过程必须在基本的机械方面不同于 更好理解的共翻译模式。我们最近 建立了一个重组系统， 蛋白质转运途径与蛋白脂质体含有纯化的 来自S.啤酒。这种SEC复合体 含有异源三聚体Sec61p-复合物，类似于哺乳动物，但 在基因筛选中发现的所有其他膜蛋白， 易位成分有效的翻译后转运还 需要管腔Kar2p（BiP）和ATP。我们现在建议使用这个系统， 确定翻译后转运的分子机制， 比较它与共翻译的一个，并确定如何两者 系统相互作用。具体而言，我们将讨论以下问题： 1.不同的蛋白质复合物参与共翻译和翻译后 路径？我们将检验Sec61p复合物作为 一个核心组成部分，与其他Sec-蛋白质以及 在翻译后途径中与Kar2p结合， 共翻译途径中的SRP受体。 为此我们将 表征和改善重建的翻译后系统，和 建立复制共翻译途径的重组系统 在酵母中。 2.翻译后蛋白质转运的机制是什么？使用 重组系统和采用各种其他方法，以及 突变的Sec-蛋白质，我们将研究如何信号序列是 在翻译后易位途径中识别，该过程如何 是能量驱动的，是否存在蛋白质传导 通道，以及该通道是否由Kar2p或其他蛋白质门控。 3.还有哪些因素参与易位？我们将分析 最近发现的，推定的，第二个Sec61p-复合物的功能， 酵母，鉴定参与翻译后 易位途径，寻找哺乳动物TRAM蛋白的同源物 在酵母，并确定一级结构和功能的四个 信号肽酶复合物的亚基，这些亚基尚未被 表征了 这些研究将使我们对人类的认识有重大进展。 分泌蛋白生物合成的第一步。虽然 项目将局限于酵母S.很可能是因为， 结果对于共翻译和翻译后蛋白质具有重要意义 所有生物体的运输途径。

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.