Importin β negatively regulates nuclear membrane fusion and nuclear pore complex assembly
Importin β negatively regulates nuclear membrane fusion and nuclear pore complex assembly
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DOI：
10.1091/mbc.e03-05-0275
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发表时间：
2003-11-01
影响因子：
3.3
通讯作者：
Forbes, DJ
中科院分区：
文献类型：
--
作者：
Harel, A;Chan, RC;Forbes, DJ
真核细胞核的组装涉及三个不同的事件：膜募集、融合形成双核膜以及核孔复合体 (NPC) 组装。我们报告说，importin beta 负向调节其中两个事件：膜融合和 NPC 组装。当过量的 importin 0 添加到完整的非洲爪蟾核重建反应中时，囊泡被招募到染色质，但它们的融合被阻止。 Ran-GTP 膜融合的输入β下调是可逆的。事实上，过量的 RanGTP (RanQ69L) 单独刺激过度的膜融合，导致核内膜小管和细胞质环形片状结构。我们提出，需要精确平衡输入β到Ran来创建正确的双核膜，同时抑制不需要的融合事件。有趣的是，截短的 importin beta 45-462 允许膜融合，但产生缺乏任何 NPC 的细胞核。这揭示了 NPC 组装的独特输入β调节。当添加到预融合的核中间体中时，过量的全长导入蛋白 β 和 13 45-462 的作用类似，即两者都阻止 NPC 组装。 importin beta NPC 块映射 NPC 组装中 GTPgammaS 和 BAPTA 敏感步骤的下游，可通过胞质溶胶逆转。值得注意的是，它不能被 25 muM RanGTP 逆转，该浓度很容易逆转融合抑制。该报告使用完整的重构系统和天然染色质底物，显着扩展了输入 P 的功能。其作用现在包括对核组装的两个主要事件的负调控：膜融合和 NPC 组装。
Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin beta negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin 0 is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin beta down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin beta to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin beta 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin beta-regulation of NPC assembly. Excess full-length importin beta and 13 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin beta NPC block, which maps downstream of GTPgammaS and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 muM RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin P. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly.