NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts
NSF- and SNARE-mediated membrane fusion is required for nuclear envelope formation and completion of nuclear pore complex assembly in Xenopus laevis egg extracts
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DOI：
10.1242/jcs.010181
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发表时间：
2007-08-15
影响因子：
4
通讯作者：
Meyer, Hemmo H.
中科院分区：
文献类型：
--
作者：
Baur, Tina;Ramadan, Kristijan;Meyer, Hemmo H.
尽管在了解有丝分裂后核膜（NE）重组方面取得了进展，但仍不清楚是什么驱动了所需的膜融合以及这与核孔复合体（NPC）组装到底是如何协调的。在这里，我们发现，与其他细胞内融合反应一样，非洲爪蟾卵提取物中的 NE 融合是由需要 NSF 激活的 SNARE 蛋白介导的。非洲爪蟾 NSF 抗体、NSF 耗竭或显性失活 NSFE329Q 变体特异性抑制 NE 形成。分期实验进一步表明，在信封密封完成之前，需要 NSF。此外，过量的外源性 α-SNAP 会阻断 SNARE 功能，从而阻止膜融合并导致染色质表面不平坦的囊泡积聚。在这些条件下，核孔蛋白 Nup107 和 gp210 被完全募集，而含有 FxFG 重复序列的核孔蛋白的组装被阻断。我们共同将 NSF 和 SNARE 介导的膜融合事件定义为 Nup107 招募下游 NE 形成过程中以及膜扁平化和 NPC 组装完成的上游过程中的重要步骤。
Despite the progress in understanding nuclear envelope (NE) reformation after mitosis, it has remained unclear what drives the required membrane fusion and how exactly this is coordinated with nuclear pore complex (NPC) assembly. Here, we show that, like other intracellular fusion reactions, NE fusion in Xenopus laevis egg extracts is mediated by SNARE proteins that require activation by NSF. Antibodies against Xenopus NSF, depletion of NSF or the dominant-negative NSFE329Q variant specifically inhibited NE formation. Staging experiments further revealed that NSF was required until sealing of the envelope was completed. Moreover, excess exogenous alpha-SNAP that blocks SNARE function prevented membrane fusion and caused accumulation of non-flattened vesicles on the chromatin surface. Under these conditions, the nucleoporins Nup107 and gp210 were fully recruited, whereas assembly of FxFG-repeat-containing nucleoporins was blocked. Together, we define NSF- and SNARE-mediated membrane fusion events as essential steps during NE formation downstream of Nup107 recruitment, and upstream of membrane flattening and completion of NPC assembly.