Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, Texas A&M University, College Station, TX 77843, USA.
Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):E1584-93. doi: 10.1073/pnas.1220610110. Epub 2013 Apr 8.
Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the cytoplasm and the nucleoplasm. Soluble nuclear transport receptors bind signal-dependent cargos to form transport complexes that diffuse through the NPC and are then disassembled. Although transport receptors enable the NPC's permeability barrier to be overcome, directionality is established by complex assembly and disassembly. Here, we delineate the choreography of importin-α/CAS complex assembly and disassembly in permeabilized cells, using single-molecule fluorescence resonance energy transfer and particle tracking. Monitoring interaction sequences in intact NPCs ensures spatiotemporal preservation of structures and interactions critical for activity in vivo. We show that key interactions between components are reversible, multiple outcomes are often possible, and the assembly and disassembly of complexes are precisely controlled to occur at the appropriate place and time. Importin-α mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-α/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-α/CAS complexes after export occurs in the cytoplasmic filament region of the NPC. Mathematical models derived from our data emphasize the intimate connection between transport and the coordinated assembly and disassembly of importin-α/CAS complexes for generating productive transport cycles.
核孔复合体（NPC）介导细胞质和核质之间的大分子交换。可溶性核转运受体结合信号依赖性货物，形成扩散穿过 NPC 的转运复合物，然后复合物被拆开。虽然转运受体使 NPC 的渗透屏障得以克服，但方向性是通过复合物的组装和拆卸来建立的。在这里，我们使用单分子荧光共振能量转移和颗粒追踪，描绘了在通透细胞中导入蛋白-α/衔接蛋白复合物的组装和拆卸的顺序。在完整的 NPC 中监测相互作用序列确保了结构和对体内活性至关重要的相互作用的时空保存。我们表明，组件之间的关键相互作用是可逆的，通常有多种结果，并且复合物的组装和拆卸受到精确控制，以在适当的位置和时间发生。与细胞质 Ran GTP 酶激活因子一起使用，在核导入过程中损害相互作用的导入蛋白-α突变体，证明导入蛋白-α/衔接蛋白复合物在核篮区域形成，在蛋白质导入结束时，并且在 NPC 的细胞质丝区域发生出口后，导入蛋白-α/衔接蛋白复合物的拆卸。从我们的数据得出的数学模型强调了运输与导入蛋白-α/衔接蛋白复合物的协调组装和拆卸之间的密切联系，以产生有效的运输循环。
