TY - JOUR T1 - Transport receptor occupancy in Nuclear Pore Complex mimics JF - bioRxiv DO - 10.1101/2021.12.22.473839 SP - 2021.12.22.473839 AU - Alessio Fragasso AU - Hendrik W. de Vries AU - John Andersson AU - Eli O. van der Sluis AU - Erik van der Giessen AU - Patrick R. Onck AU - Cees Dekker Y1 - 2021/01/01 UR - http://biorxiv.org/content/early/2021/12/23/2021.12.22.473839.abstract N2 - Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess ‘Kap-centric’ NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.Competing Interest StatementThe authors have declared no competing interest. ER -