Nucleocytoplasmic transport is sustained by karyopherins (Kaps) and a Ran guanosine triphosphate (RanGTP) gradient that imports nuclear localization signal (NLS)-specific cargoes (NLS-cargoes) into the nucleus. However, how nuclear pore complex (NPC) barrier selectivity, Kap traffic, and NLS-cargo release are systematically linked and simultaneously regulated remains incoherent. In this study, we show that Kapα facilitates Kapβ1 turnover and occupancy at the NPC in a RanGTP-dependent manner that is directly coupled to NLS-cargo release and NPC barrier function. This is underpinned by the binding affinity of Kapβ1 to phenylalanine-glycine nucleoporins (FG Nups), which is comparable with RanGTP·Kapβ1, but stronger for Kapα·Kapβ1. On this basis, RanGTP is ineffective at releasing standalone Kapβ1 from NPCs. Depleting Kapα·Kapβ1 by RanGTP further abrogates NPC barrier function, whereas adding back Kapβ1 rescues it while Kapβ1 turnover softens it. Therefore, the FG Nups are necessary but insufficient for NPC barrier function. We conclude that Kaps constitute integral constituents of the NPC whose barrier, transport, and cargo release functionalities establish a continuum under a mechanism of Kap-centric control.

Nuclear pore complexes (NPCs) discriminate nonspecific macromolecules from importin and exportin receptors, collectively termed "karyopherins" (Kaps), that mediate nucleocytoplasmic transport. This selective barrier function is attributed to the behavior of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups) that guard the NPC channel. However, NPCs in vivo are typically enriched with different Kaps, and how they impact the NPC barrier remains unknown. Here, we show that two major Kaps, importinβ1/karyopherinβ1 (Kapβ1) and exportin 1/chromosomal maintenance 1 (CRM1), are required to fortify NPC barrier function in vivo. Their enrichment at the NPC is sustained by promiscuous binding interactions with the FG Nups, which enable CRM1 to compensate for the loss of Kapβ1 as a means to maintain NPC barrier function. However, such a compensatory mechanism is constrained by the cellular abundances and different binding kinetics for each respective Kap, as evidenced for importin-5. Consequently, we find that NPC malfunction and nucleocytoplasmic leakage result from poor Kap enrichment.

The transport channel of nuclear pore complexes (NPCs) contains a high density of intrinsically disordered proteins that are rich in phenylalanine-glycine (FG)-repeat motifs (FG Nups). The FG Nups interact promiscuously with various nuclear transport receptors (NTRs), such as karyopherins (Kaps), that mediate the trafficking of nucleocytoplasmic cargoes while also generating a selectively permeable barrier against other macromolecules. Although the binding of NTRs to FG Nups increases molecular crowding in the NPC transport channel, it is unclear how this impacts FG Nup barrier function or the movement of other molecules, such as the Ran importer NTF2. Here, we use surface plasmon resonance to evaluate FG Nup conformation, binding equilibria, and interaction kinetics associated with the multivalent binding of NTF2 and karyopherinβ1 (Kapβ1) to Nsp1p molecular brushes. NTF2 and Kapβ1 show different long- and short-lived binding characteristics that emerge from varying degrees of molecular retention and FG repeat binding avidity within the Nsp1p brush. Physiological concentrations of NTF2 produce a collapse of Nsp1p brushes, whereas Kapβ1 binding generates brush extension. However, the presence of prebound Kapβ1 inhibits Nsp1p brush collapse during NTF2 binding, which is dominated by weak, short-lived interactions that derive from steric hindrance and diminished avidity with Nsp1p. This suggests that binding promiscuity confers kinetic advantages to NTF2 by expediting its facilitated diffusion and reinforces the proposal that Kapβ1 contributes to the integral barrier function of the NPC.