Choreography of importin-/CAS complex assembly and disassembly at nuclear pores.
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abstract
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-/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- mutants that impair interactions during nuclear import were used together with cytoplasmic Ran GTPase-activating factors to demonstrate that importin-/CAS complexes form in the nuclear basket region, at the termination of protein import, and disassembly of importin-/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-/CAS complexes for generating productive transport cycles.
