Choreography of Importin α/CAS Complex Assembly and Disassembly at the Nuclear Pore Complex

Abstract

Nuclear pore complexes (NPCs) mediate the exchange of proteins and RNAs between the cytoplasm and the nucleoplasm of eukaryotic cells. Signal-dependent transport cargos must assemble with soluble nuclear transport receptors to form transport complexes, which must then be disassembled after transport. The assembly and disassembly of transport complexes is promoted by proteins near the cytoplasmic and nucleoplasmic exit sides of the NPC. Here we report the use of single molecule fluorescence resonance energy transfer (smFRET) and particle tracking to investigate the choreography of importin alpha/CAS complex assembly and disassembly in permeabilized cells. Importin alpha acts with importin beta to transport cargos into the nucleus. We directly show that importin alpha/CAS complexes form in the nuclear basket region of the NPC, at the termination of the nuclear import process. These newly formed importin alpha/CAS complexes are preferentially released into the nucleus rather than immediately exported to the cytoplasm. The rapid dissociation of some importin alpha/CAS complexes at the NPC is consistent with the formation of a transient (2-4 ms lifetime) cargo/importin alpha/CAS complex, which disassembles either by losing a CAS or a cargo molecule. These data indicate that assembly and disassembly reactions occur concomitantly and stochastically. Importin alpha mutants confirm that the nucleoporin Nup50 promotes importin alpha/CAS complex assembly and that the smFRET signals observed at the NPC result from specific interactions rather than chance encounters. The disassembly of importin alpha/CAS complexes occurs after export in the cytoplasmic filament region of the NPC. This disassembly reaction is promoted by RanGTPase activating factors, indicating that GTP hydrolysis catalyzes the dissociation process. Thus, we clearly demonstrate the power of single particle tracking and smFRET for monitoring molecular interactions with high time resolution (2 ms) in a complex system.