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...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.展开更多
基金funded by NWO-I programme“Projectruimte”,Grant No.16PR3242-1.
文摘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.