Nucleocytoplasmic Transport: A Role for Nonspecific Competition in Karyopherin-Nucleoporin Interactions

Abstract

Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), which in yeast is a ϳ50 MDa complex consisting of ϳ30 different proteins. Small molecules can freely exchange through the NPC, but macromolecules larger than ϳ40 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. 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 but also 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. Although NPCs can be permeable to molecules below ϳ9 nm in diameter (1), macromolecules and macromolecular complexes are typically actively carried across by transport factors. One family of such transport factors is the karyopherins (Kaps). Kaps can shuttle through the NPC on their own (2) or laden with cargo to be imported or exported (3-12) (reviewed in Refs. 13-15). Kaps interact with their cargo in a RanGTP-dependent manner, binding and releasing transported molecules on the appropriate side of the nuclear envelope (3-12) (reviewed in Refs. 13-15). The selectively permeable barrier of the NPC is formed by a family of proteins containing characteristic disordered phenylalanine-glycine (FG) repeats, termed FG nups (reviewed in Refs. 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)(24)(25)(26)(27)(28). These proteins interact specifically with Kaps and other transport factors, through multivalent interactions between the FG repeats and multiple binding sites al...

How to operate a nuclear pore complex by Kap-centric control

“…Hence, Kap-FG Nup binding is characterized by highly multivalent interactions, 26,27 which are recognized to enhance stability and specificity due to strong binding avidity. 28 As raised by Tetenbaum-Novatt et al, 29 the known sub-mM Kapb1-FG domain binding affinities 11,[30][31][32] may "ensure" NPC transport selectivity but contradict the rapid »5 ms dwell times of nucleocytoplasmic transport cargoes in vivo 33 and in vitro. [34][35][36] It is intriguing that even FG Nup-coated nanopores are able to recapitulate both the selectivity and speed of Kapb1 translocation with a dwell time of »2.5 ms, 37 In fact, the NPC problem conflates specific binding, translocation speed and nanoscale spatial constraints.…”

“…47 Fourth, even non-specific molecules could modulate and weaken Kap-FG Nup binding. 29 Together these intersecting lines of evidence reveal the inherent sensitivity of the FG Nups to microenvironmental factors that can impact on NPC selectivity and transport kinetics.…”

Nuclear transport receptor binding avidity triggers a self-healing collapse transition in FG-nucleoporin molecular brushes

“…One example pertains to the collapse of Nup153 FG domain brushes upon binding Kapβ1 at picomolar concentrations (6), which has been interpreted (10) to imply a substantially stronger binding affinity over reported K D values (approximately 10 nM) (27). Indeed, the incompatibility of in vitro-obtained Kapβ1-FG domain binding affinities (27)(28)(29) to describe in vivo transport rates questions even the relevance of known K D measurements (30).In this work, we sought to correlate the conformational changes of surface-tethered FG domains directly to multivalent Kapβ1-FG binding interactions (i.e., binding avidity) using a surface plasmon resonance (SPR)-based assay that we developed for this purpose. Importantly, this allows for an in situ measure of FG domain surface density, conformational height change, and Kap-FG binding activity.…”

The nuclear pore complex: structure and function

“…Crystal structures have shown that the interaction between FG repeats and transport receptors mainly involves the Phe residues of the FG repeats, together with the flanking Gly residues that provide conformational flexibility, and hydrophobic residues of the receptor (Quimby et al, 2001;Fribourg et al, 2001;Bayliss et al, 2002a;Bayliss et al, 2002b;Liu and Stewart, 2005;Vognsen et al, 2013). Karyopherins possess several FG-binding sites and their translocation through the NPC is accomplished due to multiple and rapid binding events to the FG-nucleoporins (Rexach and Blobel, 1995;Kutay et al, 1997;Bayliss et al, 2000;Allen et al, 2001;Gilchrist et al, 2002;Tetenbaum-Novatt et al, 2012). While originally it was assumed that an "affinity gradient" between karyopherins and nucleoporins determines translocation from the cytoplasmic filaments to the nuclear basket (Ben-Efraim and Gerace, 2001;Pyhtila and Rexach, 2003), it has now been shown that only FG-nucleoporins that are symmetrically localized to both sides of the NPC are essential for translocation and cell viability, whereas the asymmetric FG-nucleoporins are dispensable for transport (Strawn et al, 2004 (Rout et al, 2000;Rout et al, 2003;Lim et al, 2006;Lim et al, 2007), the selective phase/hydrogel model (Ribbeck and Görlich, 2002;Frey et al, 2006;Frey et al, 2007), the reduction of dimensionality model (Peters, 2005;MoussaviBaygi et al, 2011), and the forest model (Yamada et al, 2010).…”

“…Cytokinesis, the final step of mitosis, is also hampered by siRNA-mediated depletion of Nup153 (Mackay et al, 2009;Lussi et al, 2010). Nup153 depletion causes mislocalization of Aurora B kinase, which is regulating many mitotic processes (see review van der Waal et al, 2012), during cytokinesis. This mislocalization of Aurora B leads to the activation of the so-called abscission checkpoint and consequently abscission delay and an increased number of cells in cytokinesis (Mackay et al, 2009;Mackay et al, 2010;Lussi et al, 2010).…”