Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex
Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex
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DOI：
10.1128/mcb.23.20.7271-7284.2003
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发表时间：
2003-10-01
影响因子：
5.3
通讯作者：
Fontoura, BMA
中科院分区：
文献类型：
--
作者：
Enninga, J;Levay, A;Fontoura, BMA
Sec 13是内质网和核孔复合体（NPC）的组成部分。在内质网中，Sec 13参与COPII包被囊泡的生物发生，而在NPC中，其功能尚不清楚。我们在这里显示，通过酵母双杂交筛选和生化测定，在人核孔复合物蛋白Nup 96的氨基末端的区域与WD（色氨酸-天冬氨酸）重复区的人Sec 13相互作用。通过免疫荧光、共聚焦显微镜和免疫电镜观察，我们发现，在间期，Sec 13和Nup 96定位于NPC的两侧以及其他细胞内位点。在有丝分裂中，Sec 13被发现分散在整个细胞，而Nup 96池与纺锤体装置共定位。光漂白实验表明，Sec 13穿梭于核内位点和细胞质之间，并且一部分Sec 13与NPC稳定结合。Sec 13和Nup 96的Sec 13结合位点的共转染减少了Sec的移动的库][3，证明了Sec 13和Nup 96在体内的相互作用。靶向研究表明，Sec 13被主动转运到细胞核中，并含有核定位信号。这些结果表明，Sec 13与Nup 96在NPC间期稳定相互作用，并且Sec 13在细胞核和细胞质之间的穿梭可能偶联并调节这两个隔室之间的功能。
Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec][3, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.