ROLE OF RAB PROTEINS AND CA2+ IN VESICULAR TRANSPORT

RAB 蛋白和 CA2 在囊泡运输中的作用

• 批准号: 3300803
• 负责人: William Edward Balch
• 金额: $ 28.94万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3300803
• 关键词: Golgi apparatus; adenosine triphosphate; antiidiotype antibody; calcium; calcium binding protein; cell membrane; cold temperature; electron microscopy; endoplasmic reticulum; enzyme linked immunosorbent assay; guanine nucleotide binding protein; guanosine triphosphate; guanosinetriphosphatases; immunoelectron microscopy; intracellular transport; protein engineering; protein purification; protein transport; secretion; site directed mutagenesis; transport proteins; vesicle /vacuole; virus protein

We have developed a new model system to access directly intracellular organelles of the eucaryotic cell. The plasma membrane of the eucaryotic cell can be selectively perforated to produce semi-intact cells, a cell population in which organelles of the secretory pathway (nucleus, endoplasmic reticulum (ER), Golgi, and plasma membrane) are retained in an intact, functional form and directly accessible to a wide range reagents and macromolecules. It is the broad objective of the proposal to use this model system to study in vitro the transport of protein between the ER and the cis Golgi compartment. Transport of protein from the ER to the Golgi occurs by the budding and fusion of carrier vesicles. Vesicle formation requires ATP and as yet unspecified soluble and membrane-associated components. Fusion of carrier vesicles to the Golgi compartment occurs through a series of intermediate transport steps which require GTP hydrolysis and Ca2+. We propose to specifically examine the role of the rab gene family of synthesis of peptide reagents encoding functional domains of the rab gene family. Peptides will be used as chemical analogs to inhibit transport in order to elucidate rab protein function and to prepare domain specific polyclonal and monoclonal antibodies to inhibit ER to Golgi transport in vitro. These antibodies will be used as reagents to study the role of low-molecular weight GTP binding proteins in ER to Golgi transport and to identify potentially new members of this family. In addition, recombinant rab protein will be prepared using both procaryotic and eucaryotic expression vectors for analysis of the role of known rab gene products in transport in vivo and in vitro. Site-directed binding, hydrolysis and membrane-association in transport. We will also explore the role of a newly discovered Ca2+-dependent step(s) in vesicular transport which is likely to require novel Ca2+-binding protein(s) for vesicle fusion to the Golgi compartment. These studies will include purification and characterization of 3 sequential vesicular intermediates which accumulate transported protein under 3 conditions: (1) reduced temperature (15degreesC), (2) in the absence of GTP hydrolysis, and (3) in the absence of free Ca2+. Many medically important diseases result from defects in transport between intracellular organelles and the cell surface. These include lysosomal storage diseases, familial hypercholesterolemia, cystic fibrosis, and cancer. Establishing a model in vitro system for the study of transport events occurring along the secretory pathway will have a major impact on our understanding of the biochemistry of these diseases as a result of understanding the basic mechanisms fundamental to trafficking of protein between organelles of the eucaryotic cell.

我们开发了一种新的模型系统， 真核细胞的细胞器。 真核细胞的质膜 细胞可以被选择性地穿孔以产生半完整的细胞， 其中分泌途径的细胞器（细胞核， 内质网（ER），高尔基体和质膜）被保留在一个 完整、功能性形式，可直接接触各种试剂 和大分子。 该提案的总体目标是利用这一点， 模型系统，以在体外研究蛋白质在ER和 顺式高尔基体区室。 蛋白质从内质网向高尔基体的转运 通过载体囊泡的出芽和融合发生。 囊泡形成 需要ATP和尚未明确的可溶性和膜相关的 件. 载体囊泡融合到高尔基体 通过一系列需要GTP的中间运输步骤 水解和Ca2+。 我们建议具体研究 rab基因家族合成肽试剂的功能编码 RAB基因家族的结构域。 肽将被用作化学类似物 抑制转运以阐明RAB蛋白的功能， 制备结构域特异性的多克隆和单克隆抗体以抑制ER 高尔基体的运输。 这些抗体将被用作试剂， 研究ER中低分子量GTP结合蛋白对高尔基体的作用 运输和识别这个家庭的潜在新成员。 在 此外，重组rab蛋白将使用两种原核细胞制备。 和真核表达载体用于分析已知的rab 基因产物在体内和体外的运输。 定点结合， 运输中水解和膜结合。 我们亦会探讨 一个新发现的钙离子依赖步骤在囊泡转运中的作用 其可能需要新的Ca2+结合蛋白用于囊泡融合 到高尔基体 这些研究将包括纯化和 表征3个连续的囊泡中间体， 在3种条件下转运蛋白：（1）降低温度 （2）在没有GTP水解的情况下，和（3）在没有 游离Ca2+ 许多医学上重要的疾病是由于 细胞内细胞器和细胞表面之间的运输。 这些 包括溶酶体贮积病、家族性高胆固醇血症、囊性 纤维化和癌症。 建立研究的体外系统模型 发生在分泌途径上的运输事件的沿着将有一个主要的 影响了我们对这些疾病的生物化学的理解， 通过了解贩运人口的基本机制， 真核细胞的细胞器之间的蛋白质。