Characterization of the nuclear import pathway for BLM protein
Characterization of the nuclear import pathway for BLM protein
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BLM 蛋白核输入途径的表征
DOI：
10.1016/j.abb.2017.09.019
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发表时间：
2017-11-15
影响因子：
3.9
通讯作者：
Xiong, Jianming
中科院分区：
文献类型：
--
作者：
Duan, Zhiqiang;Zhao, Jiafu;Xiong, Jianming
BLM蛋白是RecQ解旋酶的一个成员，其核定位信号（nuclear localization signal，NLS）介导的核定位对DNA重组、复制和转录至关重要，但BLM蛋白进入核的机制尚不清楚。在这项研究中，BLM的核输入途径进行了研究。我们发现，BLM的核输入受到输入蛋白β 1和NTF 2/E42 K两种显性负突变体的抑制，它们分别缺乏结合Ran和RanGDP的能力，但不受Ran/Q69 L的抑制，Ran/Q69 L缺乏GTP水解。进一步的研究表明，在毛地黄皂苷透化的HeLa细胞中，使用importin β 1，RanGDP和NTF 2重建BLM的核输入。此外，BLM通过其NLS结构域与importin β 1的14-16个HEAT重复序列直接结合importin β 1。此外，通过siRNA去除importin（31）、Ran或NTF 2破坏了BLM蛋白在细胞核中的积累。这些结果表明，BLM通过importin β 1，RanGDP和NTF 2依赖的途径进入细胞核，首次证明了DNA解旋酶的核运输机制。(C)2017爱思唯尔公司All rights reserved.
Numerous studies have shown that nuclear localization of BLM protein, a member of the RecQ helicases, mediated by nuclear localization signal (NLS) is critical for DNA recombination, replication and transcription, but the mechanism by which BLM protein is imported into the nucleus remains unknown. In this study, the nuclear import pathway for BLM was investigated. We found that nuclear import of BLM was inhibited by two dominant-negative mutants of importin beta 1 and NTF2/E42K, which lacks the ability to bind Ran and RanGDP, respectively, but was not inhibited by the Ran/Q69L, which is deficient in GTP hydrolysis. Further studies revealed that nuclear import of BLM was reconstituted using importin beta 1, RanGDP and NTF2 in digitonin-permeabilized HeLa cells. Moreover, BLM had direct binding to importin beta 1 through its NLS domain with the 14-16 HEAT repeats of importin beta 1. Furthermore, importin (31, Ran or NTF2 depletion by siRNA disrupted the accumulation of BLM protein in the nucleus. These results showed that BLM enters the nucleus via the importin beta 1, RanGDP and NTF2 dependent pathway, demonstrating for the first time the nuclear trafficking mechanism of a DNA helicase. (C) 2017 Elsevier Inc. All rights reserved.