STRUCTURE AND FUNCTION OF THE NUCLEAR PORE COMPLEX

核孔复合体的结构和功能

• 批准号: 3304271
• 负责人: RONALD A MILLIGAN
• 金额: $ 20.49万
• 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/3304271
• 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）是一个大型组装体 1200-1400 直径 埃，厚度 600-800 埃，跨越 核膜。 NPC 是各部门之间的沟通渠道 细胞核和细胞质，允许小分子被动扩散 分子和离子并主动运输大分子和 这两个区室之间存在核糖核蛋白颗粒。 阿德诺 疱疹病毒在将 DNA 释放到 NPC 之前与 NPC 结合 核。 完整的逆转录病毒衣壳通过 全国人大。 尽管 NPC 在牢房中的显着位置及其 控制（例如）激素的核质交换的重要性 受体复合物、调节蛋白、mRNA、核糖体和病毒 组件，对此组件知之甚少： 无法分离足够数量的 NPC 进行生化分析 通过 X 射线方法表征或分析，仅非常低 分辨率 3-D 信息可用于其结构变化很少 (<10%) NPC蛋白已被鉴定及其分子机制 的运输过程是未知的。 这项工作的长期目标是了解 NPC 高分辨率并阐明分子机制 主动核质运输。 的成就 本文所述的工作将代表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将 用于确定投影图和 3 维地图 改进的制备方法使分辨率达到约 50 埃 洗涤剂释放的 NPC。 随后，一个数字的 3D 位置 重要的 NPC 蛋白质——包括核所需的蛋白质 运输——将通过免疫电子显微镜确定 图像分析。 冷冻干燥、金属阴影的检查 手动展开核膜的准备工作将揭示 核侧和细胞质侧的结构差异 全国人大。 这些发现将与 3D 地图中的细节相关。 使用合成核定位信号肽的实验已 旨在调查中央插头的身份 NPC 并可能提供有关运输的结构信息 设备。