MOLECULAR STUDIES OF EUKARYOTIC CELL SURFACE GROWTH

真核细胞表面生长的分子研究

• 批准号: 2174792
• 负责人: RANDY W. SCHEKMAN
• 金额: $ 12.35万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174792
• 关键词: MOLECULAR; STUDIES; EUKARYOTIC; CELL; SURFACE

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.

多肽从细胞质移位到细胞腔 内质网代表分泌途径的第一步， 而唯一需要亲水性蛋白质通过 脂质双层。 近年来， 观察表明，一个基本的保护机制， 原核生物和真核生物中的蛋白质易位。 开展的工作 自从上次更新以来，这项资助已经导致了一组ER的发现。 膜蛋白和细胞溶质hsc70构成的基本要素 转移器官的一部分 其中一种分子Sec6lp， E的核心元素之间的联系。大肠杆菌和哺乳动物多肽 转位酶 尽管许多细胞膜、细胞质和内腔 多肽输入ER所需的蛋白质现在已经被 定义，渗透的确切机制仍然不清楚。 在前一个赠款期间，我们开发了一种方法， 膜和蛋白脂质体中的重组易位。 一个膜 一种含有Sec63p、BiP和两种新的Sec蛋白的蛋白复合物， 与世隔绝 另外的膜蛋白是必需的 现在正在分离易位，目标是完全纯化 该系统用于调查假定信道的性质。 亚基化学计量学的关键问题， 蛋白质、通道组装的调节和转运能力 小分子将使用从野生型中分离的Sec蛋白来解决。 类型和突变细胞。 用一组纯化的Sec易位蛋白，我们将研究 负责信号肽结合的组分， 参与完成蛋白质进口。 我们建议， Sec63p复合物的信号肽部分相互作用， 分泌前蛋白质并将其递送至Sec6lp通道。一 亚反应，其测量分离的信号肽与 描述了脂蛋白体或可溶性膜蛋白。 部分 测量多肽转运的BiP依赖性完成的反应 通过Sec6lp通道的蛋白质将在蛋白脂质体和蛋白质中复制 洗涤剂溶液。 将在蛋白脂质体中检查hsc70的选择性作用。 使用由胞质hsc70和BiP制成的嵌合分子进行反应。 我们 希望能定义这些分子的结构域， 的特异性