MECHANISMS OF INTRACELLULAR MEMBRANE FUSION

细胞内膜融合机制

• 批准号: 2458726
• 负责人: JAMES ROTHMAN
• 金额: $ 45.62万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2458726
• 关键词: Golgi apparatus; SDS polyacrylamide gel electrophoresis; adenosinetriphosphatase; affinity chromatography; cell adhesion molecules; cell free system; cellular pathology; electron microscopy; gel filtration chromatography; immunochemistry; intracellular membranes; laboratory mouse; laboratory rabbit; membrane activity; membrane fusion; molecular cloning; polymerase chain reaction; protein structure function; receptor expression; transport proteins; western blottings

The long term goal of this project is to understand the mechanism of intracellular membrane fusion events. In the last project period, utilizing a cell-free system that reconstitutes intercisternal transport in the Golgi stack as an assay system, we discovered and purified an N- ethylmaleimide-sensitive fusion ATPase (NSF) that is required for fusion processes involving multiple compartments in yeast and animal cells. We also found and purified three NSF attachment proteins (SNAPs) that assemble with NSF and an integral membrane SNAP receptor to form a 20S particle that seems likely to be involved in fusion processes. We propose to build upon this foundation by better characterizing the structure and function of NSF, SNAPs and snap receptor, to definitively establish their roles in fusion in living cells, and to isolate additional fusion components. We will test our hypothesis that the 20S NSF-containing particle is the "core" of a general intracellular membrane fusion machinery that assembles from dispersed subunits upon demand when a fusion event is required, and disassembles in the course of catalyzing fusion.

该项目的长期目标是了解 细胞内膜融合事件。 在最后一个项目期间， 利用一个无细胞的系统，该系统在 Golgi堆栈作为测定系统，我们发现并纯化了N- 融合所需的乙基马来酰亚胺敏感融合ATPase（NSF） 涉及酵母和动物细胞中多个隔室的过程。 我们 还发现并纯化了三个组装的NSF附件蛋白（快照） 使用NSF和一个整体膜捕捉受体形成20S粒子 似乎可能参与融合过程。 我们建议以 通过更好地表征NSF的结构和功能，该基础， 快照和快照受体，以明确地在融合中建立角色 活细胞，并隔离其他融合成分。 我们将测试 我们假设20S含NSF的粒子是A的“核心” 一般细胞内膜融合机械，从 在需要融合事件时根据需要分散亚基，并且 在催化融合过程中分解。