Transport receptor occupancy in nuclear pore complex mimics

Abstract

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.

Self-regulation of the nuclear pore complex enables clogging-free crowded transport

“…We found that the decay of the number of NTRs remaining in the pore did not follow mono-exponential decay (Figure 7A), indicating the presence of more than one escape timescale. Instead, the decay is well approximated as a bi-exponential, consistent with the experimental reports of two populations of NTRs with “slow” and “fast” release timescales [32,36–40]. The apparent clear separation between the “slow” and the “fast” regimes occurs because the deepening of the effective potential due to competition-induced release (Figure 7B) proceeds at a non-uniform rate: rapidly initially (as many NTRs quickly escape in the initial stages of decay), and then very slowly (as it takes the remaining NTRs longer and longer to escape from the pore).…”

“…Furthermore, the mechanism of competition-induced release allows us to explain previous observations of apparent “fast” and “slow” fractions of NTRs within the NPC [36, 37] and in vitro FG nup assemblies and artificial NPC mimics [32, 40]. As NTRs escape from an FG nup assembly, reduced competition between the remaining NTRs deepens the effective potential within the assembly, leading to very long escape times.…”

“…The puzzle of transport times that decrease with crowding has been hypothesized to be connected to another long-standing puzzle in the field: the presence of a population of NTRs in FG nup assemblies with residence times much longer (minutes to hours) than typical transport times (milliseconds) [32, 36, 37, 40]. It has been suggested that the “slow” and the “fast” NTR phases have distinct functional roles whereby the strongly bound “slow” NTRs strengthen the FG nup barrier, while the “fast” NTRs rapidly transport cargoes through the NPC [32].…”

“…An additional longstanding experimental puzzle is the observation of a long-lived fraction of endogenous NTRs within perme-abilized cells, which leave the NPC at timescales (minutes and hours) that are much greater than expected from single molecule transport times (milliseconds) [36–39]. This phenomenon has been reproduced in in vitro assemblies of surface grafted FG nups and artificial nanopores [32, 40], leading to the proposal of a “kap-centric model”, which suggests that NTRs (known also as karyo-pherins or “kaps”) actively control the kinetic properties of nucleocytoplasmic transport. It has been suggested that the observations of clogging-free transport [26, 31] are examples of NTR-mediated control of transport [36].…”

Liquid-liquid phase separation of intrinsically disordered FG-Nups is driven by highly-dynamic hydrophobic FG-motifs

“…Here, we investigate LLPS of FG-Nups by carrying out coarse-grained molecular dynamics (CGMD) simulations using a one-bead-per-amino-acid (1-BPA) MD model [38][39][40]. This model has been used previously to study nuclear transport through the yeast NPC [39,[41][42][43][44][45][46] and biomimetic nanopores [47][48][49][50], and to analyze the LLPS of dipeptide repeat proteins [51]. Here, we show that several intrinsically disordered FG-Nups of the yeast NPC can phase separate into liquid-like condensates and that this phase transition is mainly driven by the highly dynamic hydrophobic FGmotifs that line the FG-Nup sequences.…”