MOLECULAR STUDIES OF EUKARYOTIC CELL SURFACE GROWTH

多肽从胞浆转位到管腔内 内质网是分泌途径的第一步， 唯一需要亲水性蛋白质通过的事件 一种脂双层。近几年来， 这些观察结果表明，从根本上讲， 原核生物和真核生物中的蛋白质转位。在以下方面进行的工作 自上次续签以来，这笔赠款导致了一组ER的发现 构成必需元素的膜蛋白和胞质hsc7O 移位装置的。其中一种分子Sec6lp提供了 大肠杆菌核心成分与哺乳动物多肽之间的联系 转位酶。尽管许多膜、胞浆和管腔 多肽进入内质网所需的蛋白质现在已经 一旦确定，渗透的确切机制仍不清楚。 在之前的资助期间，我们开发了一种溶解酵母的方法 并在蛋白脂质体中重建易位。一层膜 含有Sec63p、Bip和两个新的SEC蛋白的蛋白质复合体 与世隔绝。所需的其他膜蛋白 易位现在被分离出来，目标是完全纯化 用来调查假定通道的性质的系统。 亚基化学计量学的关键问题，SEC之间的相互作用 蛋白质、通道组装的调节和转位能力 小分子将使用从野生动物中分离出来的SEC蛋白进行处理 类型细胞和突变细胞。 利用一组纯化的易位SEC蛋白，我们将研究 负责信号肽结合的组件，以及 参与完成蛋白质进口。我们已经建议成员 与SEC63P复合体的信号肽部分相互作用 并将它们传递到Sec6lp通道。一个 测量分离信号肽与其结合的子反应 本发明描述了蛋白质脂质体或可溶性膜蛋白。一部份 测量依赖Bip的多肽转运完成情况的反应 通过Sec6lp通道将在蛋白脂质体和 洗涤剂溶液。 Hsc7os的选择性作用将在蛋白脂质体中进行检测。 使用胞浆hsc70和Bip组成的嵌合分子进行反应。我们 我希望定义这些分子的结构域来提供拓扑 专一性。

Polypeptide translocation from the cytosol into the lumen of the endoplasmic reticulum represents the first step in the secretory pathway, and the only event that requires passage of a hydrophilic protein through a lipid bilayer. Recent years have seen a dramatic convergence of observations that suggest a fundamental conservation of the mechanism of protein translocation in prokaryotes and eukaryotes. Work conducted on this grant since the last renewal has led to the discovery of a set of ER membrane proteins and cytosolic hsc7Os that constitute essential elements of the translocation apparatus. One of these molecules, Sec6lp, provides a link between core elements of the E. coli and mammalian polypeptide translocase. Although many of the membrane, cytosolic, and lumenal proteins required for polypeptide import into the ER have now been defined, the exact mechanism of penetration remains obscure. In the previous grant period we developed a method to solubilize yeast membranes and reconstitute translocation in proteoliposomes. One membrane protein complex that contains Sec63p, BiP, and two new Sec proteins was isolated. Additional membrane proteins that are required for translocation are now being isolated with the goal being a fully purified system with which to investigate the nature of the putative channel. Crucial questions of subunit stoichiometry, interactions among the Sec proteins, regulation of channel assembly, and capacity to translocate small molecules will be addressed using Sec proteins isolated from wild type and mutant cells. With a purified set of translocation Sec proteins, we will examine the components that are responsible for signal peptide binding and that are involved in completion of protein import. We have proposed that members of the Sec63p complex interact with the signal peptide portion of presecretory proteins and deliver them to the Sec6lp channel. A subreaction that measures binding of isolated signal peptides to proteoliposomes or soluble membrane proteins is described. A partial reaction that measures BiP-dependent completion of polypeptide transport through the Sec6lp channel will be reproduced in proteoliposomes and in detergent solution. The selective-role of hsc7Os will be examined in the proteoliposome reaction using chimeric molecules made of cytosolic hsc70 and BiP. We hope to define domains of these molecules that confer topological specificity.