Abstract
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.
Highlights
Nuclear pore complexes (NPCs) [1] are intracellular transport hubs that mediate the rapid bidirectional traffic of hundreds of proteins, ribonucleoproteins, and metabolites across the nuclear envelope [2]
The movement of larger molecules is impaired by a permeability barrier generated by ~200 intrinsically disordered phenylalanine-glycine (FG)-rich nucleoporins (FG Nups) that are tethered to the NPC transport channel surface
surface plasmon resonance (SPR) measures the binding and release of analytes from surface-tethered ligands. We previously extended this technique to show that noninteracting bovine serum albumin (BSA) molecules could be used to determine the average height h of a surface layer
Summary
Nuclear pore complexes (NPCs) [1] are intracellular transport hubs that mediate the rapid bidirectional traffic of hundreds of proteins, ribonucleoproteins, and metabolites across the nuclear envelope [2]. Each NPC contains a 50nm-diameter central channel [3] through which only molecules smaller than ~40 kDa [4] or ~5 nm in size [5] can diffuse passively [6]. The movement of larger molecules is impaired by a permeability barrier generated by ~200 intrinsically disordered phenylalanine-glycine (FG)-rich nucleoporins (FG Nups) that are tethered to the NPC transport channel surface. The translocation of selective cargoes through NPCs is mediated by a range of soluble nuclear transport receptors (NTRs) [13]. These include members of the karyopherin family (Kaps) [14], such as the 97 kDa import receptor karyopherinb (Kapb or importinb) [15], which recognizes specific cargoes either directly or via an adaptor Kapa
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