STUDIES OF PROTEINS RESIDENT IN THE ER

描述（改编自申请人摘要）：内质网 网织细胞是分泌途径中的第一个细胞器， 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， Ca++离子和分泌蛋白包装成囊泡， 定位于分泌链中的下一个细胞器 远景目标 研究人员的工作就是了解这些个体 功能的实现以及它们如何以允许 要连贯地进行。 拟议的工作夫妇的优点， 两个实验性真核系统：哺乳动物细胞（或体外系统 来源于它们）用于生化分析，以及酵母菌属（Saccharomyces cerevisae进行遗传分析。 调查人员的具体目标是： 分析BiP的结构与功能的关系， ER的主要伴侣蛋白。 使用最近阐明的 一个密切相关的N-末端结构域的三维结构 蛋白质（hsc 70）作为指导，研究人员将分析生化 和生理特性的一系列定点突变体，（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Ca++结合 位点（iv）修饰底物识别结构域内的序列。 详细分析了一种新的抗氧化剂的生理生化特性， 一种新形式的肽基脯氨酰异构酶（sig-PPI）， 位于酵母的内质网中。 探讨Ca ~（++）在维持心肌细胞功能中的作用 的完整性和功能。 特别是，萨姆布鲁克博士将调查 酵母细胞的生理生化行为 编码三种蛋白质（Ca++ ATP酶，钙网蛋白， 和Ins（1，4，5）P3的受体，它们被认为参与了 Ca++在ER中的储存和Ca++穿过ER膜的通量。

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.