Quantitative analysis of nuclear localization signal (NLS)-importin α interaction through fluorescence depolarization -: Evidence for auto-inhibitory regulation of NLS binding
Quantitative analysis of nuclear localization signal (NLS)-importin α interaction through fluorescence depolarization -: Evidence for auto-inhibitory regulation of NLS binding
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DOI：
10.1074/jbc.m002217200
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发表时间：
2000-07-14
影响因子：
4.8
通讯作者：
Hodel, AE
中科院分区：
文献类型：
--
作者：
Fanara, P;Hodel, MR;Hodel, AE
我们开发了一种定量体外稳态荧光去极化测定，用于测量核定位信号 (NLS) 底物与其受体的相互作用。该测定依赖于与绿色荧光蛋白融合的 NLS 在与受体结合后荧光去极化的变化。在没有功能性 NLS 的情况下没有观察到结合，并且测量的结合亲和力与之前的 NLS 功能体内研究相关。我们使用此测定法来测试 NLS 与 NLS 受体复合物相互作用的自抑制模型。该模型表明，NLS 与输入蛋白 α 的结合受到输入蛋白 α N 末端内的自动抑制序列的调节，该序列被输入蛋白 β 结合所取代。与该模型一致，NLS 底物与缺乏自抑制结构域的 N 端截短的导入蛋白 α 紧密结合（K-d 类似于 10 nM），但仅在添加导入蛋白 β 后才观察到与全长导入蛋白 α 的可测量结合。我们的定量结果支持自抑制模型，并提出了一种在细胞质高亲和力和细胞核低亲和力之间切换的机制。 NLS 受体。这种预测的相互作用模式将促进细胞质中底物的结合及其随后释放到细胞核中。
We have developed a quantitative in vitro steady-state fluorescence depolarization assay to measure the interaction of a nuclear localization signal (NLS) substrate with its receptors, This assay relies on the change in fluorescence depolarization of an NLS fused to the green fluorescent protein upon binding to receptor. No binding is observed in the absence of a functional NLS, and binding affinities measured correlate with previous in vivo studies of NLS function. We have used this assay to test an auto-inhibitory model for the interaction of an NLS with the NLS receptor complex. This model suggests that NLS binding to importin alpha is modulated by an auto-inhibitory sequence within the N terminus of importin alpha, which is displaced by importin beta binding. Consistent with this model, NLS substrates bind tightly to an N-terminally truncated importin alpha lacking the autoinhibitory domain (K-d similar to 10 nM), but measurable binding to full-length importin alpha is only observed upon addition of importin beta, Our quantitative results support the autoinhibitory model and suggest a mechanism for a switch between a cytoplasmic, high affinity and a nuclear, low affinity NLS receptor. This predicted mode of interaction would facilitate binding of substrate in the cytoplasm and its subsequent release into the nucleus.