ROLE OF GTP-BINDING PROTEINS IN INTRACELLULAR TRANSPORT

GTP 结合蛋白在细胞内运输中的作用

• 批准号: 3302462
• 负责人: PAUL R MELANCON
• 金额: $ 14.04万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1992-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302462
• 关键词: Golgi apparatus; Lagomorpha; cell free system; eukaryote; genetic library; guanine nucleotide binding protein; immunoelectron microscopy; immunofluorescence technique; laboratory mouse; membrane proteins; monoclonal antibody; protein sequence; transport proteins

The long term objective of this project is to elucidate the mechanisms of intracellular transport of proteins in eukaryotic cells. Biochemical and genetic evidence demonstrates that small, ras-like, GTP-binding proteins are involved in this process. The objective of this proposal is to identify and functionally characterize (a) GTP-binding protein(s) involved in the transport of proteins through the Golgi. The major experimental system will be a cell-free transport assay that measures the transfer of proteins between cis and medial Golgi compartments. The non-hydrolysable analog GTPgammaS inhibits transport in the cell-free system; current evidence suggests that this effect is mediated by the binding of a cytosolic GTP-binding protein to an effector on the membrane. The specific aims of the project are to (1) Purify the GTP-binding protein using its inhibitory effect on the cell-free transport assay, (3) Use the pure protein to obtain a cDNA and specific antibodies in order to study the function of the GTP-binding protein in the transport reaction. (5) Study the regulation of nucleotide exchange on the GTP-binding protein that is responsible for its activation. (6) Identify the effector to which GGBF binds tightly on the membranes in order to study its role in transport. The cell-free transport assay provides a system with which to functionally characterize specific GTP-binding proteins. The work described in the proposal paves the way for one of the first detailed description of the mechanism of action of a small GTP-binding protein of the ras super-family.

该项目的长期目标是阐明 真核细胞中蛋白质的细胞内转运。 生化和 遗传证据表明，小的、ras 样的 GTP 结合蛋白 都参与到这个过程中。 该提案的目的是 鉴定并功能表征所涉及的 (a) GTP 结合蛋白 通过高尔基体运输蛋白质。主要实验 系统将是一种无细胞转运测定，可测量 顺式和内侧高尔基体之间的蛋白质。 不可水解的类似物 GTPgammaS 抑制无细胞转运 系统;目前的证据表明，这种效应是由 胞质 GTP 结合蛋白与膜上效应子的结合。 该项目的具体目标是（1）纯化GTP结合蛋白 利用其对无细胞转运测定的抑制作用，（3）使用 纯蛋白质以获得 cDNA 和特异性抗体，以便研究 GTP 结合蛋白在转运反应中的功能。 （五）学习 GTP 结合蛋白上核苷酸交换的调节 负责其激活。 (6) 识别GGBF所针对的效应器 紧密结合在膜上以研究其在运输中的作用。 无细胞转运测定提供了一个系统，可以在功能上 表征特定的 GTP 结合蛋白。 中描述的工作 提案为第一个详细描述铺平了道路 ras 超家族的小 GTP 结合蛋白的作用机制。