Abstract:
Nucleocytoplasmic transport of macromolecules is essential in eukaryotic cells. In this process, the karyopherins play a central role when they transport cargoes across the nuclear pore complex. Importin 4 belongs to the karyopherin β family. Many studies have focused on finding substrates for importin 4, but no direct mechanism studies of its precise transport function have been reported. Therefore, this paper mainly aimed to study the mechanism of nucleoporins in mediating nuclear import and export of importin 4. To address this question, we constructed shRNAs targeting Nup358, Nup153, Nup98, and Nup50. We found that depletion of Nup98 resulted in a shift in the subcellular localization of importin 4 from the cytoplasm to the nucleus. Mutational analysis demonstrated that Nup98 physically and functionally interacts with importin 4 through its N-terminal phenylalanine-glycine (FG) repeat region. Mutation of nine of these FG motifs to SG motifs significantly attenuated the binding of Nup98 to importin 4, and we further confirmed the essential role of the six FG motifs in amino acids 121-360 of Nup98 in binding with importin 4. In vitro transport assay also confirmed that VDR, the substrate of importin 4, could not be transported into the nucleus after Nup98 knockdown. Overall, our results showed that Nup98 is required for efficient importin 4-mediated transport. This is the first study to reveal the mechanism of importin 4 in transporting substrates into the nucleus.
摘要:
大分子的核质转运在真核细胞中是必需的。在这个过程中,核动力蛋白在运输货物穿过核孔复合体时发挥着核心作用。Importin4属于核转运蛋白β家族。许多研究集中在寻找importin4的底物,但尚未报道其精确转运功能的直接机制研究。因此,本文主要研究核孔蛋白在转运蛋白4核进出口中的作用机制。为了解决这个问题,我们构建了靶向Nup358、Nup153、Nup98和Nup50的shRNA。我们发现Nup98的消耗导致输入蛋白4的亚细胞定位从细胞质转移到细胞核。突变分析表明,Nup98通过其N端苯丙氨酸-甘氨酸(FG)重复区与导入蛋白4在物理和功能上相互作用。这些FG基序中的9个突变为SG基序显着减弱了Nup98与导入蛋白4的结合,我们进一步证实了Nup98氨基酸121-360中的6个FG基序在与导入蛋白4结合中的重要作用。体外转运试验也证实了VDR,在Nup98敲低后,importin4的底物不能被转运到细胞核中。总的来说,我们的结果表明,Nup98是有效的导入蛋白4介导的转运所必需的。这是首次揭示importin4将底物运输到细胞核中的机制的研究。