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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore
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Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

机译：Importinα/货物复合物在核孔处解离的单分子测量

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摘要

Macromolecules are transported between the cytoplasm and the nucleoplasm of eukaryotic cells through nuclear pore complexes (NPCs). Large (more than ≈ 40 kDa) transport cargoes imported into the nucleus typically form a complex with at least one soluble transport cofactor of the importin (Imp) β superfamily. Many cargoes require an accessory cofactor, Imp α, which binds to Imp β and to the nuclear localization sequence on the cargo. We previously reported the use of narrow-field epifluorescence microscopy to directly monitor cargoes in transit through NPCs in permeabil-ized cells. We now report an expanded approach in which single-molecule fluorescence resonance energy transfer (FRET) is used to detect the disassembly of Imp α/cargo complexes as they transit through NPCs. We found that CAS, the recycling cofactor for Imp α. and RanGTP are essential for this dissociation process. After Imp α/cargo complex dissociation, most Imp α and cargo molecules entered the nucleoplasm. In contrast, the majority of Imp α/cargo complexes that did not dissociate at the NPC in the presence of CAS and RanGTP returned to the cytoplasm. These data are consistent with a model in which Imp α/cargo complexes are dissociated on the nucleoplasmic side of the NPC, and this dissociation requires both CAS and RanGTP.
机译：大分子通过核孔复合物（NPC）在真核细胞的细胞质和核质之间转运。进口到核中的大型（大于约40 kDa）运输货物通常与进口蛋白（Imp）β超家族的至少一种可溶性运输辅因子形成复合物。许多货物需要辅助辅因子Impα，它与Impβ和货物上的核定位序列结合。我们以前曾报道过使用窄场落射荧光显微镜直接监测通透细胞中通过NPC转运的货物。我们现在报告一种扩展的方法，其中单分子荧光共振能量转移（FRET）用于检测Impα/货物配合物通过NPC时的分解。我们发现CAS是Impα的回收辅助因子。 RanGTP对于此解离过程至关重要。 Impα/货物复合物解离后，大多数Impα和货物分子进入核质。相反，在CAS和RanGTP存在的情况下，大多数在NPC上未解离的Impα/货物复合物返回到细胞质中。这些数据与其中Impα/货物复合物在NPC的核质侧解离的模型一致，并且这种解离需要CAS和RanGTP。

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