INTERACTION OF A NUCLEAR LOCATION SIGNAL WITH ISOLATED NUCLEAR ENVELOPES AND IDENTIFICATION OF SIGNAL-BINDING PROTEINS BY PHOTOAFFINITY-LABELING
INTERACTION OF A NUCLEAR LOCATION SIGNAL WITH ISOLATED NUCLEAR ENVELOPES AND IDENTIFICATION OF SIGNAL-BINDING PROTEINS BY PHOTOAFFINITY-LABELING
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DOI：
10.1073/pnas.86.23.9327
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发表时间：
1989-12-01
影响因子：
11.1
通讯作者：
RIEDEL, N
中科院分区：
文献类型：
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作者：
BENDITT, JO;MEYER, C;RIEDEL, N
核膜（NE）分隔真核细胞的两个主要区室，细胞核和细胞质。最近的研究表明，核蛋白进入细胞核的摄取是通过这些蛋白质内所含的核定位信号（NLS）与NE中的受体结合，然后通过核孔复合物易位而启动的。为了检查结合步骤而不受核内事件的干扰，我们使用了由（i）固定在载玻片上的纯化大鼠肝内斯和（ii）与非核载体蛋白缀合的原型猿猴病毒40大T抗原（SV 40 T）NLS组成的系统，并且我们通过间接免疫荧光观察了受体-配体相互作用。在该系统中，野生型SV 40 T NLS缀合物与载体蛋白孵育分离的内斯导致信号序列依赖性的结合，可以被过量的缀合和未缀合的野生型肽竞争性阻断，不需要ATP，并且不受转运抑制凝集素麦胚凝集素的影响。相反，用突变体SV 40 T NLS缀合物仅观察到最小结合。这些结果与在其他更复杂的体外系统中获得的结果一致，表明SV 40 T NLS的结合是受体介导的。结合在很大程度上废除了NE与非离子去污剂Triton X-100的提取，这表明该受体是可溶于去污剂。我们发现在Triton X-100上清液中的四个主要NLS结合蛋白的表观分子量为76，67，59，和58 kDa的光亲和标记与一个高度特异性的交联剂，叠氮-NLS。这里描述的系统的复杂性降低应该是有用的其他潜在的NLS的功能研究，用于识别和分离其结合位点，并针对这些结合位点的抗体的筛选。
The nuclear envelope (NE) separates the two major compartments of eukaryotic cells, the nucleus and the cytoplasm. Recent studies suggest that the uptake of nuclear proteins into the nucleus is initiated by binding of nuclear location signals (NLSs) contained within these proteins to receptors in the NE, followed by translocation through the nuclear pore complex. To examine the binding step without interference from intranuclear events, we have used a system consisting of (i) purified rat liver NEs fixed onto glass slides and (ii) the prototype simian virus 40 large T antigen (SV40 T) NLS conjugated to nonnuclear carrier proteins, and we have visualized the receptor-ligand interaction by indirect immunofluorescence. In this system, incubation of isolated NEs with the wild-type SV40 T NLS conjugate with carrier proteins resulted in binding that was signal sequence-dependent, could be competitively blocked with excess conjugated and unconjugated wild-type peptide, did not require ATP, and was not affected by the transport-inhibiting lectin wheat germ agglutinin. In contrast, only minimal binding was observed with a mutant SV40 T NLS conjugate. These results are consistent with those obtained in other, more complex in vitro systems and suggest that binding of the SV40 T NLS is receptor-mediated. Binding is largely abolished by extraction of the NE with the nonionic detergent Triton X-100, suggesting that the receptor is soluble in detergent. We find in the Triton X-100 supernatant four major NLS-binding proteins with apparent molecular masses of 76, 67, 59, and 58 kDa by photoaffinity labeling with a highly specific crosslinker, azido-NLS. The reduced complexity of the system described here should be useful for the functional study of other potential NLSs for the identification and isolation of their binding sites and for the screening of antibodies raised against these binding sites.