Abstract
Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a ~50 MDa complex consisting of ~30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ~40 kDa must be aided across by transport factors, most of which belong to a related family of proteins termed karyopherins (Kaps). These transport factors bind to the disordered phenylalanine-glycine (FG) repeat domains in a family of NPC proteins termed FG nups, and this specific binding allows the transport factors to cross the NPC. However, we still know little in terms of the molecular and kinetic details regarding how this binding translates to selective passage of transport factors across the NPC. Here we show that the specific interactions between Kaps and FG nups are strongly modulated by the presence of a cellular milieu whose proteins appear to act as very weak competitors that nevertheless collectively can reduce Kap/FG nup affinities by several orders of magnitude. Without such modulation, the avidities between Kaps and FG nups measured in vitro are too tight to be compatible with the rapid transport kinetics observed in vivo. We modeled the multivalent interactions between the disordered repeat binding sites in the FG nups and multiple cognate binding sites on Kap, showing that they should indeed be sensitive to even weakly binding competitors; the introduction of such competition reduces the availability of these binding sites, dramatically lowering the avidity of their specific interactions and allowing rapid nuclear transport.
Highlights
Eukaryotic cells segregate their genetic material with a double-layered nuclear envelope, which protects the genomic DNA of the cell and enables highly regulated gene expression and creates a barrier that a wide range of biomolecules must cross to maintain cell viability
All known transport in and out of the nucleus occurs through nuclear pore complexes (NPCs)1 embedded in the nuclear envelope
The selectively permeable barrier of the NPC is formed by a family of proteins containing characteristic disordered phenylalanine-glycine (FG) repeats, termed FG nups, which surround and line the central channel of the NPC (21, 22) and which have been the focus of much study (2, 9, 13, 17, 23–28)
Summary
Eukaryotic cells segregate their genetic material with a double-layered nuclear envelope, which protects the genomic DNA of the cell and enables highly regulated gene expression and creates a barrier that a wide range of biomolecules must cross to maintain cell viability. The selectively permeable barrier of the NPC is formed by a family of proteins containing characteristic disordered phenylalanine-glycine (FG) repeats, termed FG nups 16 –20), which surround and line the central channel of the NPC (21, 22) and which have been the focus of much study (2, 9, 13, 17, 23–28) These proteins interact with Kaps and other transport factors, through multivalent interactions between the FG repeats and multiple binding sites along each transport factor (29 – 47). 52 and 57– 61), which are often involved in interactions with multiple partners that require high specificity and low affinity (i.e. fast off-rates), such as those involved in signaling (52) This family of proteins is well suited for the job of gating transport across the NPC, a process that requires numerous. Measurements typically probe the avidity resulting from the combined binding strengths of these multiple simultaneous interaction sites rather than the affinity between individual phenylalanines and the Kap binding pockets
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