Functional characterization of importin α8 as a classical nuclear localization signal receptor
Functional characterization of importin α8 as a classical nuclear localization signal receptor
复制标题
DOI：
10.1016/j.bbamcr.2015.07.017
复制
发表时间：
2015-10-01
影响因子：
5.1
通讯作者：
Yoneda, Yoshihiro
中科院分区：
文献类型：
--
作者：
Kimoto, Chihiro;Moriyama, Tetsuji;Yoneda, Yoshihiro
最近，基于输入蛋白 α 8 的一级结构以及输入蛋白 β 1 和几种亲核蛋白的结合能力，输入蛋白 α 8 被鉴定为输入蛋白家族成员。然而，尚无实验证据表明 importin α 8 实际上在含有经典核定位信号 (cNLS) 的货物的核运输中发挥作用。在这里，使用体外转运测定，我们证明纯化的重组导入蛋白 a8 可以与导入蛋白 β 1 一起将含有 SV40T 抗原 cNLS 的货物转运到 HeLa 细胞核中。Pull-down 测定和随后的质谱分析，鉴定了 179 个推定的导入蛋白 α 8 结合蛋白，其中只有 62 个与最接近的重要 α 家族成员导入蛋白 α 1 的蛋白重叠。在输入蛋白 α 8 结合候选者中，我们发现 DNA 损伤结合蛋白 2 (DDB2) 实际上是通过输入蛋白 α 8/β 1 途径转运到细胞核中的。此外，我们发现 importin α 的其他亚型（也被鉴定为 importin α 8 结合候选者）确实与 importin α 8 形成异二聚体。值得注意的是，我们发现这些包含 importin α 8 的异二聚体在 cNLS 底物存在下比包含 importin α 1 的异二聚体更稳定。根据这些发现，我们提出 importin α 8 作为具有不同货物特异性的 cNLS 受体发挥作用，并且除了 importin beta 结合域的自身抑制调节之外，importin α 8 的异二聚化是 cNLS 结合的一种新型调节模式。 (C) 2015 Elsevier B.V. 保留所有权利。
Importin alpha 8 has recently been identified as an importin a family member based on its primary structure and binding ability to importin beta 1 and to several karyophilic proteins. However, there has been no experimental evidence that importin alpha 8 actually functions in the nuclear transport of classical nuclear localization signal (cNLS)-containing cargo. Here, using an in vitro transport assay, we demonstrate that purified recombinant importin a8 can transport SV40T antigen cNLS-containing cargo into the nucleus of HeLa cells, in conjunction with importin beta 1. Pull-down assays, followed by mass spectrometry analysis, identified 179 putative importin alpha 8-binding proteins, only 62 of which overlap with those of importin alpha 1, the closest impordn alpha family member. Among the importin alpha 8-binding candidates, we showed that DNA damage-binding protein 2 (DDB2) was actually transported into the nucleus via the importin alpha 8/beta 1 pathway. Furthermore, we found that the other subtypes of importin alpha, which were also identified as importin alpha 8-binding candidates, indeed form heterodimers with importin alpha 8. Notably, we found that these importin alpha 8-containing heterodimers were more stable in the presence of cNLS-substrates than heterodimers containing importin alpha 1. From these findings, we propose that importin alpha 8 functions as a cNLS receptor with distinct cargo specificity, and that heterodimerization by importin alpha 8 is a novel regulatory mode of cNLS binding, in addition to the autoinhibitory regulation by the importin beta binding domain. (C) 2015 Elsevier B.V. All rights reserved.