Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr.
Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr.
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DOI：
10.1083/jcb.141.1.31
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发表时间：
1998-04-06
期刊：
影响因子：
--
通讯作者：
Forbes D
中科院分区：
文献类型：
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作者：
Shah S;Tugendreich S;Forbes D
核进口中的一个主要问题是核孔蛋白(S)在穿过核孔时与核定位序列(NLS)受体及其货物相互作用的身份。配基印迹和分离蛋白的溶液结合研究试图获得这些核孔蛋白身份的线索，但这些研究没有必要探索与活体环境相去甚远的结合事件。在这里，我们问的是什么结合事件发生在更生理的非洲爪哇卵子提取液，它含有处于组装能力状态的核孔子复合体。然后，我们根据组装的核孔本身对我们的结论进行了评估。我们已经使用免疫沉淀来鉴定与生理相关的核孔蛋白和进口蛋白亚基的复合体。同时，我们已经证明了核孔蛋白免疫荧光定位于核孔及其相关结构的亚区是可能的。通过免疫沉淀，我们发现核孔蛋白Nup153和与孔相关的细丝蛋白Tpr存在于非洲爪哇卵提取液中与Importinα和β形成的稳定的亚复合体中，它们分别位于组装孔的核内侧。无论是在鸡蛋提取物中还是在组装的核孔提取物中，Importin亚基都不与核孔蛋白Nup62、Nup93、Nup98或Nup214/Can形成稳定的复合体。在对Nup153复合体的表征中，我们发现Nup153可以与包含Importinα，β和NLS底物的完整进口复合体结合，这与该核孔蛋白参与核进口的末端步骤是一致的。Importinβ与Nup153直接结合，在体外可以在Nup153 FXFG重复区域的多个位置结合。Tpr没有FXFG重复序列，可与β和α/β异源二聚体结合，但只与那些不携带NLS底物的异源二聚体结合。Tpr与Importinβ的复合体与Nup153的复合体从根本上不同于Nup153，另外发现重组的β或β45-462片段与内源性Importinβ/Nup153复合体自由交换，但不能取代TPR复合体中的内源性Importinβ。然而，GTP类似物GMP-PNP能够分解Nup153-和Tpr-Importinβ复合体。重要的是，对分离的核提取液的分析表明，在组装的核孔中存在Nup153-和Tpr-Importinβ复合体。因此，Nup153和Tpr是Importinβ的主要生理结合部位。讨论了这些相互作用所起作用的模型。
A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin α and β in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin α, β, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin β binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin β and to importin α/β heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin β is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant β or β45–462 fragment freely exchanges with the endogenous importin β/Nup153 complex, but cannot displace endogenous importin β from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153– and Tpr–importin β complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153– and Tpr–importin β complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin β. Models for the roles of these interactions are discussed.