ABSTRACT Xenopus egg extracts are a powerful in vitro tool for studying complex biological processes, including nuclear reconstitution, nuclear membrane and pore assembly, and spindle assembly. Extracts have been further used to demonstrate a moonlighting regulatory role for nuclear import receptors or importins on these cell cycle assembly events. Here we show that exportins can also play a role in these events. Addition of Crm1, Exportin-t, or Exportin-5 decreased nuclear pore assembly in vitro. RanQ69L-GTP, a constitutively active form of RanGTP, ameliorated inhibition. Both Crm1 and Exportin-t inhibited fusion of nuclear membranes, again counteracted by RanQ69L-GTP. In mitotic extracts, Crm1 and Exportin-t negatively impacted spindle assembly. Pulldowns from the extracts using Crm1- or Exportin-t-beads revealed nucleoporins known to be essential for both nuclear pore and spindle assembly, with RanQ69L-GTP decreasing a subset of these target interactions. This study suggests a model where exportins, like importins, can regulate major mitotic assembly events.

中文翻译：

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Exportins 可以抑制体外的主要有丝分裂组装事件：膜融合、核孔形成和纺锤体组装

摘要 非洲爪蟾卵提取物是一种强大的体外工具，用于研究复杂的生物过程，包括核重建、核膜和孔组装以及纺锤体组装。提取物已被进一步用于证明核输入受体或输入蛋白对这些细胞周期组装事件的临时调节作用。在这里，我们表明 exportins 也可以在这些事件中发挥作用。添加 Crm1、Exportin-t 或 Exportin-5 可减少体外核孔组装。RanQ69L-GTP 是 RanGTP 的组成型活性形式，可改善抑制作用。Crm1 和 Exportin-t 均抑制核膜融合，再次被 RanQ69L-GTP 抵消。在有丝分裂提取物中，Crm1 和 Exportin-t 对纺锤体组装产生负面影响。使用 Crm1- 或 Exportin-t-beads 从提取物中提取的下拉显示已知核孔蛋白对核孔和纺锤体组装至关重要，RanQ69L-GTP 减少了这些目标相互作用的一个子集。这项研究提出了一个模型，其中出口蛋白，如进口蛋白，可以调节主要的有丝分裂组装事件。