The Department of Molecular and Cell Biology, University of California, Berkeley, MC 3200 LSA, Berkeley, California 94720-3200, United States.
ACS Chem Biol. 2011 Jul 15;6(7):700-8. doi: 10.1021/cb2000296. Epub 2011 Apr 21.
During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. A similar mechanism operates in mitosis to generate a gradient of active spindle assembly factors around mitotic chromosomes. Importin-β and RanGTP have been implicated in additional cellular processes, but the precise roles of the Ran/importin-β pathway throughout the cell cycle remain poorly understood. We implemented a FRET-based, high-throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin-β and identified importazole, a 2,4-diaminoquinazoline. Importazole specifically blocks importin-β-mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. When added during mitosis, importazole impairs the release of an importin-β cargo FRET probe and causes both predicted and novel defects in spindle assembly. Together, these results indicate that importazole specifically inhibits the function of importin-β, likely by altering its interaction with RanGTP. Importazole is a valuable tool to evaluate the function of the importin-β/RanGTP pathway at specific stages during the cell cycle.
在间期，转运受体 importin-β 将 cargo 带入细胞核，RanGTP 将其释放。在有丝分裂中，类似的机制用于在有丝分裂染色体周围产生活性纺锤体组装因子的梯度。Importin-β 和 RanGTP 已被牵连到其他细胞过程中，但 Ran/importin-β 途径在整个细胞周期中的精确作用仍知之甚少。我们实施了基于 FRET 的高通量小分子筛选，以寻找干扰 RanGTP 和 importin-β 相互作用的化合物，并鉴定出 importazole，一种 2,4-二氨基喹唑啉。Importazole 特异性地阻断了 Xenopus 卵提取物和培养细胞中 importin-β 介导的核输入，而不会破坏 transportin 介导的核输入或 CRM1 介导的核输出。在有丝分裂期间添加时，importazole 会干扰 importin-β 货物 FRET 探针的释放，并导致纺锤体组装出现预测到的和新的缺陷。总之，这些结果表明 importazole 特异性抑制了 importin-β 的功能，可能通过改变其与 RanGTP 的相互作用。Importazole 是一种评估细胞周期特定阶段 importin-β/RanGTP 途径功能的有价值工具。
