Choreography of importin-α/CAS complex assembly and disassembly at nuclear pores

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-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 GTPaseactivating 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.FRET | nucleocytoplasmic transport

Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

“…It is established that structured regions on the FxFG Nups located at the nuclear NPC face contribute to import cargo disassembly (Gilchrist et al 2002;Gilchrist and Rexach 2003;Matsuura et al 2003;Matsuura and Stewart 2005;Sun et al 2013). These structured domains are located adjacent to the FxFG domains that bind importing Kaps (Pyhtila and Rexach 2003).…”

“…Importantly, NPC structures involved in the terminal steps of the protein import and export pathways are critical for the action of TRs in the Ran-dependent karyopherin (Kap) family. For example, in addition to their FG repeat domains that bind Kaps, Nups localized asymmetrically at the nuclear NPC face contain key binding sites for coordinated Kap-import cargo disassembly (Gilchrist et al 2002;Gilchrist and Rexach 2003;Matsuura et al 2003;Matsuura and Stewart 2005;Sun et al 2013). Additionally, at the cytoplasmic NPC face, the mammalian FG Nup358 (RanBP2) functions as a multi-subunit SUMO E3 complex that coordinates SUMO ligase activity, export cargo disassembly through binding the RanGAP, and Kap binding (Werner et al 2012).…”

Deciphering the Structure and Function of Nuclear Pores Using Single-Molecule Fluorescence Approaches

“…Notably, the smFRET experiments identified a novel CAS/Imp α/cargo complex (which presumably also includes RanGTP) that was not previously considered when analyzing nuclear import. This transient CAS/Imp α/cargo complex has a very short lifetime (< 1 ms), and it disassembles into Imp α/cargo or CAS/Imp α complexes, 88 i.e., both productive (forward) and non-productive (reverse) reactions occur. These observations clearly establish that the NPC is more than simply a sum of its parts – there are emergent properties that are not readily predicted.…”

MD Simulations and FRET Reveal an Environment-Sensitive Conformational Plasticity of Importin-β

“…However, in our simulations, the IBB domain stabilized only the C-terminal arch of Importin-b (HEAT repeats [12][13][14][15][16][17][18][19], which is in direct contact with the helical part of the IBB domain. Other domains of the molecule underwent large conformational changes, adopting an open conformation.…”

“…Importin-b is a highly versatile molecule that is able to transport a variety of cargoes by binding them directly or through an adaptor protein (13)(14)(15). Importinb:cargo complexes form in the cytosol and then cross the nuclear pore and enter the nucleus, where the complexes dissociate upon binding of the small GTPase Ran, in its GTP-bound form (16)(17)(18). The Importin-b:RanGTP complex shuttles back to the cytoplasm, where GTP hydrolysis is triggered and RanGDP is released, closing the cycle (19,20).…”