A Ran-Dependent Importin-Beta/Nup153 Barrier In The Nuclear Pore Complex

The nuclear pore complex (NPC) is a large multi-protein assembly that mediates the selective transport of molecules between the cytoplasm and nucleus in eukaryotic cells. While the factors involved in various transport pathways are largely known, the physical mechanism by which the NPC supports both efficient and selective molecular translocation remains unclear. We have identified a component of the NPC selectivity barrier consisting of the transport receptor importin-β and the nucleoporin Nup153 which can be modulated by the GTPase Ran. We have investigated importin-β and Nup153's effect on the kinetic rates of both passive and active nuclear import and its regulation by Ran. Furthermore, using photobleach step counting and super-resolution imaging, we have characterized the numbers and spatial distributions of importin-β within the pore suggesting that this transport receptor is a functional and surprisingly stable component of the NPC. Finally, we demonstrate in vitro with fluorescence fluctuation spectroscopy that importin-β and Nup153 bind to form large cross-linked complexes which can be dispersed by RanGTP. Our results suggest that importin-β in conjunction with Nup153 can constitute a selective 'gate' in the NPC whose permeability towards both inert and transport-competent cargos is sensitive to RanGTP.