STRUCTURE AND FUNCTION OF THE NUCLEAR PORE COMPLEX

核孔复合体的结构和功能

• 批准号: 3304272
• 负责人: RONALD A MILLIGAN
• 金额: $ 22.98万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-11 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3304272
• 关键词: Xenopus; Xenopus oocyte; cell nucleus; computer graphics /printing; computer simulation; cryoscopy; cytoplasm; detergents; fluorescent dye /probe; freeze etching; image processing; immunoelectron microscopy; intracellular transport; membrane proteins; membrane structure; molecular asymmetry; monoclonal antibody; nonhistone nucleoprotein; nuclear membrane; protein sequence

The nuclear pore complex (NPC) is a large assembly 1200-1400 angstroms in diameter and 600-800 angstroms thick which spans the nuclear envelope. The NPC is the communication pathway between the nucleus and the cytoplasm, allowing the passive diffusion of small molecules and ions and actively transporting large macromolecules and ribonucleoprotein particles between these two compartments. Adeno and herpes viruses bind to the NPC prior to release of their DNA into the nucleus. Intact retrovirus capsids are transported through the NPC. Despite the NPC's prominent location in the cell and its importance in controlling nucleoplasmic exchange of (e.g.) hormone receptor complexes, regulatory proteins, mRNPs, ribosomes and virus components, there is relatively little known about this assembly: the NPC cannot be isolated in sufficient quantities for biochemical characterization or analysis by x-ray methods, only very low resolution 3-D information is available on its structure vary few (<10%) NPC proteins have been identified and the molecular mechanisms of the transport processes are unknown. The long term goals of the work are to understand the structure of the NPC ar high resolution and to elucidate the molecular mechanism of active nucleocytoplasmic transport. The accomplishment of the work described herein will represent major progress towards attaining these goals. Cryo-electron microscopy coupled with computer image processing will be used to determine projection maps and a 3-dimensional map at a resolution of about 50 Angstroms from improved preparations of detergent-released NPCs. Subsequently, the 3-D location of a number of important NPC proteins-- including those necessary for nuclear transport -- will be determined by immunoelectron microscopy and image analysis. An examination of freeze-dried, metal-shadowed preparations of manually spread nuclear envelopes will reveal structural differences on the nuclear and cytoplasmic sides of the NPC. These findings will be related to details in the 3-D map. Experiments using synthetic nuclear localization signal peptides have been designed to investigate the identity of the central plug of the NPC and may provide structural information on the transport apparatus.

核孔复合体（NPC）是一个1200-1400 直径600-800埃，厚度跨越 核膜 全国人大是人民代表大会与政府之间的沟通渠道。 细胞核和细胞质，允许小的被动扩散， 分子和离子，并积极运输大分子， 核糖核蛋白颗粒之间的这两个隔间。 腺 而疱疹病毒在释放DNA进入细胞之前与NPC结合， 原子核 完整的逆转录病毒衣壳通过 届全国人大 尽管NPC在细胞中的突出位置及其 控制核质交换的重要性（例如）激素 受体复合物、调节蛋白、mRNP、核糖体和病毒 组件，对该组件的了解相对较少： NPC不能以足够的量分离用于生化 通过X射线方法进行表征或分析，只有极低 分辨率的三维信息，其结构变化不大 （<10%）NPC蛋白已被鉴定，其分子机制 运输过程是未知的。 这项工作的长期目标是了解 NPC的高分辨率和阐明分子机制 活跃的核质运输。 的完成而 本文所述的工作将是实现以下目标的重大进展： 这些目标。 冷冻电子显微镜与计算机图像处理相结合， 用于确定投影图和三维图， 从改进的制备物中分离约50埃 清洁剂释放的NPC 随后，一个数字的三维位置 重要的NPC蛋白质-包括那些必要的核 运输-将通过免疫电子显微镜测定， 图像分析 对冷冻干燥的金属阴影 人工展开的核包裹的准备工作将揭示 细胞核和细胞质侧的结构差异 届全国人大 这些发现将与3D地图中的细节相关。 使用合成的核定位信号肽的实验具有 被设计为调查中央插头的身份， NPC，并可提供运输的结构信息 设备.