PROTEINS RESIDENT IN THE ER

描述(改编自申请人的摘要)：内质 网是分泌途径中的第一个细胞器，它执行一种 多种功能，包括折叠和糖基化 新合成的多肽，多聚体蛋白的组装，储存 Ca++离子和将分泌蛋白包装到囊泡中 定位于分泌链中的下一个细胞器。长期目标 调查人员的工作是了解每个人是如何 功能已完成，以及它们如何以允许 它们必须连贯一致地进行。这项拟议的工作具有以下优点 两个实验真核系统：哺乳动物细胞(或体外系统 从它们中提取)用于生化分析，以及酵母 用于遗传分析的鹿角菌。调查人员的具体目标是： 分析了BiP的结构和功能之间的关系，以及BIP的结构和功能 ER的主要伴侣蛋白。使用最近阐明的 一种密切相关的N-末端结构域的三维结构 以蛋白质(Hsc70)为指导，研究人员将分析生化 和一系列定点突变体的生理特性(I) 改变参与ATP结合和水解的氨基酸(II)防止 分子铰链区的移动(III)消除了钙离子结合 位点(Iv)修饰底物识别结构域内的序列。 为了详细分析细菌的生理生化特性， 新形式的肽基脯氨酰异构酶(sig-PPI)，似乎是 位于酵母的内质网中。探讨钙离子在维持性疾病中的作用 急诊室的完整性和功能。特别是，桑布鲁克博士将研究 酵母细胞携带的生化和生理行为 编码三种蛋白质(Ca++ATPase，钙网织蛋白， 和INS(1，4，5)P3的受体，被认为参与了 Ca++在内质网中的储存和跨ER膜的Ca++通量。

DESCRIPTION (Adapted from the applicant's abstract): The endoplasmic reticulum, the first organelle in the secretory pathway, carries out a multiplicity of functions including folding and glycosylation of newly-synthesized polypeptides, assembly of multimeric proteins, storage of Ca++ ions and packaging of secretory proteins into vesicles that are targeted to the next organelle in the secretory chain. The long-term goal of the investigators work is to understand how each of these individual functions is accomplished and how they are coordinated in a way that allows them to be carried coherently. The proposed work couples the advantages of two experimental eukaryotic systems: mammalian cells (or in vitro systems derived from them) for biochemical analysis, and the yeast Saccharomyces cerevisae for genetic analysis. The investigators specific aims are: to analyze the relationship between the structure and function of BiP, the chief chaperone protein of the ER. Using the recently-elucidated three-dimensional structure of N-terminal domain of a closely-related protein (hsc70) as a guide, the investigators will analyze the biochemical and physiological properties of a series of site-directed mutants that (i) alter the amino acids involved in ATP binding and hydrolysis (ii) prevent movement of the hinge region of the molecule (iii) eliminate Ca++ binding sites (iv) modify the sequences within the substrate recognition domain. To analyze in detail the physiological and biochemical properties of a novel form of peptidyl prolyl isomerase (sig-PPI) that appears to be located in the ER of yeast. To explore the role of Ca++ in the maintenance of ER integrity and function. In particular, Dr. Sambrook will investigate the biochemical and physiological behavior of yeast cells carrying mutations in genes coding for three proteins (Ca++ ATPase, calreticulin, and the receptor for Ins (1,4,5)P3 that are thought to be involved in the storage of Ca++ in the ER and in the flux of Ca++ across the ER membrane.