Molecular architecture of the yeast nuclear pore complex:: Localization of Nsp1p subcomplexes
Molecular architecture of the yeast nuclear pore complex:: Localization of Nsp1p subcomplexes
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DOI：
10.1083/jcb.143.3.577
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发表时间：
1998-11-02
影响因子：
7.8
通讯作者：
Panté, N
中科院分区：
文献类型：
--
作者：
Fahrenkrog, B;Hurt, EC;Panté, N
核孔复合物（NPC）是类似于100种不同蛋白质（核孔蛋白）的超分子组装体，介导分子在细胞质和细胞核之间的双向运输。广泛的结构研究已经揭示了非洲爪蟾NPC的三维（3D）结构，和12个克隆和表征的脊椎动物核孔蛋白中的8个已经定位在NPC内。由于酵母遗传学的力量，最近已经克隆了30种酵母核孔蛋白，并在分子水平上进行了表征。然而，这些核孔蛋白在NPC的3D结构内的定位仍然是难以捉摸的，主要是由于制备用于电子显微镜（EM）的酵母细胞的限制。我们已经开发了一种新的协议，准备酵母细胞EM产生结构保存完好的酵母NPC。酵母和爪蟾NPC的直接比较显示NPC结构在进化上是保守的，尽管酵母NPC在线性尺寸上小15%。利用该制备方案和表达蛋白A标记的核孔蛋白的酵母菌株，我们通过免疫EM定位了Nsp1p及其相互作用伙伴Nup49p、Nup57p、Nup82p和Nic96p。因此，Nsp1p驻留在三个不同的亚复合物，位于中央门控通道的入口和出口处，并在核篮的终端环。
The nuclear pore complex (NPC), a supramolecular assembly of similar to 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus. Extensive structural studies have revealed the three-dimensional (3D) architecture of Xenopus NPCs, and eight of the similar to 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC. Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level. However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM). We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs. A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions. With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM. Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.