Nup93, a vertebrate homologue of yeast Nic96p, forms a complex with a novel 205-kDa protein and is required for correct nuclear pore assembly
Nup93, a vertebrate homologue of yeast Nic96p, forms a complex with a novel 205-kDa protein and is required for correct nuclear pore assembly
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DOI：
10.1091/mbc.8.10.2017
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发表时间：
1997-10-01
影响因子：
3.3
通讯作者：
Hurt, E
中科院分区：
文献类型：
--
作者：
Grandi, P;Dang, T;Hurt, E
通过电子显微镜观察，酵母和脊椎动物的核孔在形态上具有显著的相似性，但相应蛋白质之间的序列相似性却更难观察到。在此，我们在人类和非洲爪蟾中都鉴定出一种脊椎动物核孔蛋白Nup93，它已被证明是酵母核孔蛋白Nic96p的进化相关同源物。针对人类Nup93的多克隆抗血清可在人类、大鼠和非洲爪蟾细胞中检测到相应的蛋白质。免疫荧光和免疫电子显微镜将脊椎动物Nup93定位在核篮以及核孔门控通道的核入口处或其附近。从哺乳动物和非洲爪蟾细胞提取物中进行的免疫沉淀表明，一小部分Nup93与核孔蛋白p62发生物理相互作用，就像酵母Nic96p与酵母p62同源物相互作用一样。然而，大部分脊椎动物Nup93是从核孔中提取出来的，并且在非洲爪蟾卵提取物中也与一种新发现的205 kDa蛋白质形成复合物。对人类205 kDa蛋白质进行的质谱测序表明，该蛋白质由人类数据库中存在的一个开放阅读框KIAO225编码。推测的205 kDa人类核孔蛋白在秀丽隐杆线虫和酿酒酵母中具有相关的序列同源物。为了分析Nup93复合物在核孔中的作用，在对非洲爪蟾核重建提取物进行免疫耗竭后组装了缺乏Nup93复合物的细胞核。缺乏Nup93复合物的细胞核在正确的核孔组装方面明显存在缺陷。从这些实验中，我们得出结论：脊椎动物和酵母的核孔在其功能重要的核心部分具有显著的同源性，并且随着Nup93和205 kDa蛋白质的鉴定，我们扩展了对酵母和脊椎动物中该核心的近邻相互作用的认识。
Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAO225, present in the human database. The putative human nucleoporin of 205kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex-depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.