The permeability of reconstituted nuclear pores provides direct evidence for the selective phase model

Nuclear pore complexes (NPCs) maintain a permeability barrier between the nucleus and the cytoplasm through FG-repeat-containing nucleoporins (Nups). We previously proposed a "selective phase model" in which the FG repeats interact with one another to form a sieve-like barrier that can be locally?disrupted by the binding of nuclear transport receptors (NTRs), but not by inert macromolecules, allowing selective passage of NTRs and associated cargo. Here, we provide direct evidence for this model in a physiological context. By using NPCs reconstituted from Xenopus laevis egg extracts, we show that Nup98 is essential for maintaining the permeability barrier. Specifically, the multivalent cohesion between FG repeats is required, including cohesive FG repeats close to the anchorage point to the NPC scaffold. Our data exclude alternative models that are based solely on an interaction between the FG repeats and NTRs and indicate that the barrier is formed by a sieve-like FG hydrogel.     

FG/FxFG as well as GLFG repeats form a selective permeability barrier with self‐healing properties

The permeability barrier of nuclear pore complexes (NPCs) controls all nucleo‐cytoplasmic exchange. It is freely permeable for small molecules. Objects larger than ≈30 kDa can efficiently cross this barrier only when bound to nuclear transport receptors (NTRs) that confer translocation‐promoting properties. We had shown earlier that the permeability barrier can be reconstituted in the form of a saturated FG/FxFG repeat hydrogel. We now show that GLFG repeats, the other major FG repeat type, can also form highly selective hydrogels. While supporting massive, reversible importin‐mediated cargo influx, FG/FxFG, GLFG or mixed hydrogels remained firm barriers towards inert objects that lacked nuclear transport signals. This indicates that FG hydrogels immediately reseal behind a translocating species and thus possess ‘self‐healing’ properties. NTRs not only left the barrier intact, they even tightened it against passive influx, pointing to a role for NTRs in establishing and maintaining the permeability barrier of NPCs.     

Atomic structure of the nuclear pore complex targeting domain of a Nup116 homologue from the yeast, Candida glabrata

The nuclear pore complex (NPC), embedded in the nuclear envelope, is a large, dynamic molecular assembly that facilitates exchange of macromolecules between the nucleus and the cytoplasm. The yeast NPC is an eightfold symmetric annular structure composed of ～456 polypeptide chains contributed by ～30 distinct proteins termed nucleoporins. Nup116, identified only in fungi, plays a central role in both protein import and mRNA export through the NPC. Nup116 is a modular protein with N‐terminal “FG” repeats containing a Gle2p‐binding sequence motif and a NPC targeting domain at its C‐terminus. We report the crystal structure of the NPC targeting domain of Candida glabrata Nup116, consisting of residues 882–1034 [CgNup116(882–1034)], at 1.94 Å resolution. The X‐ray structure of CgNup116(882–1034) is consistent with the molecular envelope determined in solution by small‐angle X‐ray scattering. Structural similarities of CgNup116(882–1034) with homologous domains from Saccharomyces cerevisiae Nup116, S. cerevisiae Nup145N, and human Nup98 are discussed. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Domain topology of nucleoporin Nup98 within the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate selective transport of macromolecules across the nuclear envelope in interphase eukaryotic cells. NPCs are composed of roughly 30 different proteins (nucleoporins) of which about one third are characterized by the presence of phenylalanine-glycine (FG) repeat domains that allow the association of soluble nuclear transport receptors with the NPC. Two types of FG (FG/FxFG and FG/GLFG) domains are found in nucleoporins and Nup98 is the sole vertebrate nucleoporin harboring the GLFG-type repeats. By immuno-electron microscopy using isolated nuclei from Xenopus oocytes we show here the localization of distinct domains of Nup98. We examined the localization of the C- and N-terminal domain of Nup98 by immunogold-labeling using domain-specific antibodies against Nup98 and by expressing epitope tagged versions of Nup98. Our studies revealed that anchorage of Nup98 to NPCs through its C-terminal autoproteolytic domain occurs in the center of the NPC, whereas its N-terminal GLFG domain is more flexible and is detected at multiple locations within the NPC. Additionally, we have confirmed the central localization of Nup98 within the NPC using super resolution structured illumination fluorescence microscopy (SIM) to position Nup98 domains relative to markers of cytoplasmic filaments and the nuclear basket. Our data support the notion that Nup98 is a major determinant of the permeability barrier of NPCs.     

Facilitated aggregation of FG nucleoporins under molecular crowding conditions

Intrinsically disordered and phenylalanine–glycine‐rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high‐resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.     

Nucleoporin domain topology is linked to the transport status of the nuclear pore complex

Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of approximately 30 different proteins (nucleoporins), of which around one third contain phenylalanine-glycine (FG)-repeat domains that are thought to mediate the main interaction between the NPC and soluble transport receptors. We have recently shown that the FG-repeat domain of Nup153 is flexible within the NPC, although this nucleoporin is anchored to the nuclear side of the NPC. By using domain-specific antibodies, we have now mapped the domain topology of Nup214 in Xenopus oocytes and in human somatic cells by immuno-EM. We have found that whereas Nup214 is anchored to the cytoplasmic side of the NPC via its N-terminal and central domain, its FG-repeat domain appears flexible, residing on both sides of the NPC. Moreover, the spatial distribution of the FG-repeat domains of both Nup153 and Nup214 shifts in a transport-dependent manner, suggesting that the location of FG-repeat domains within the NPC correlates with cargo/receptor interactions and that they concomitantly move with cargo through the central pore of the NPC.     

Role of Nup98 in nuclear entry of human immunodeficiency virus type 1 cDNA

Human immunodeficiency virus type 1 (HIV-1), like other lentiviruses, can infect non-dividing cells. The lentiviruses are most likely to have evolved a nuclear import strategy to import HIV-1 cDNA and viral protein complex through the nuclear pore complex (NPC) formed by nucleoporin proteins (Nup). In this study, we found that synthesis of integrated and 2LTR but not full-length form of HIV-1 cDNA was clearly impaired in culture via transduction of vesicular stomatitis virus matrix protein (VSV M), an inhibitor protein, through binding to the phenylalanine-glycine (FG) repeat region of Nup98. The impairment of synthesis of integrated and 2LTR DNA with VSV M was restored by ectopic overexpression of Nup98. A series of experiments using Nup98-depleted NPC by the small interfering RNA (siRNA) technique showed specific impairment of NPC structure and some functions, including nuclear import of HIV-1 cDNA. Our results suggest that Nup98 on the NPC specifically participates in the nuclear entry of HIV-1 cDNA following HIV-1 entry.     

A gradient of affinity for the karyopherin Kap95p along the yeast nuclear pore complex

Karyopherins (Kaps) transport cargo across the nuclear pore complex (NPC) by interacting with nucleoporins that contain phenylalanine-glycine (FG) peptide repeats (FG Nups). As a test of the "affinity gradient" model for Kap translocation, we measured the apparent affinity of Kap95p to FG Nups representing three distinct regions of the S. cerevisiae NPC. We find that the affinity of Kap95p-Kap60p-cargo complexes to Nup1p (a nuclear basket Nup) is 225-fold higher than to Nup100p (a central scaffold Nup) and 4000-fold higher than to Nup42p (a cytoplasmic filament Nup), revealing a steep gradient of affinity for Kap95p complexes along the yeast NPC. A high affinity binding site for a Kap95p import complex was mapped to the C terminus of Nup1p, and, surprisingly, deletion of all FG repeats in that region did not eliminate binding of the complex. Instead, a 36-amino acid truncation of the C terminus of Nup1p reduced its affinity for the Kap95p import complex by 450-fold. Mutant yeast that express Nup1pDelta36 instead of full-length Nup1p display specific defects in Kap95p localization and Kap95p-mediated nuclear import. We conclude that a high affinity binding site for Kap95p at the nuclear basket increases the translocation efficiency of Kap95p import complexes across the NPC.     

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine‐glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino‐acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non‐specific, small, and pliant interaction network between cargo and FG repeats.     

Large cargo transport by nuclear pores: implications for the spatial organization of FG‐nucleoporins

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine‐glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non‐uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG‐NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.     

Promiscuous Binding of Karyopherinβ1 Modulates FG Nucleoporin Barrier Function and Expedites NTF2 Transport Kinetics

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.     

A Highlights from MBoC Selection: The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution

The nuclear pore complex (NPC) regulates transport between the nucleus and cytoplasm. Soluble cargo-protein complexes navigate through the pore by binding to phenylalanine-glycine (FG)-repeat proteins attached to the channel walls. The Nup62 complex contains the FG-repeat proteins Nup62, Nup54, and Nup58 and is located in the center of the NPC. The three proteins bind each other via conserved coiled-coil segments. To determine the stoichiometry of the Nup62 complex, we undertook an in vitro study using gel filtration and analytical ultracentrifugation. Our results reveal a 1:1:1 stoichiometry of the Nup62 complex, where Nup54 is central with direct binding to Nup62 and Nup58. At high protein concentration, the complex forms larger assemblies while maintaining the Nup62:Nup54:Nup58 ratio. For the homologous Nsp1 complex from Saccharomyces cerevisiae, we determine the same stoichiometry, indicating evolutionary conservation. Furthermore, we observe that eliminating one binding partner can result in the formation of complexes with noncanonical stoichiometry, presumably because unpaired coiled-coil elements tend to find a promiscuous binding partner. We suggest that these noncanonical stoichiometries observed in vitro are unlikely to be physiologically relevant.     

Mutations in tap uncouple RNA export activity from translocation through the nuclear pore complex

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA- and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo.     

Characterisation of the passive permeability barrier of nuclear pore complexes

Nuclear pore complexes (NPCs) restrict uncontrolled nucleocytoplasmic fluxes of inert macromolecules but permit facilitated translocation of nuclear transport receptors and their cargo complexes. We probed the passive barrier of NPCs and observed sieve‐like properties with a dominating mesh or channel radius of 2.6 nm, which is narrower than proposed earlier. A small fraction of diffusion channels has a wider opening, explaining the very slow passage of larger molecules. The observed dominant passive diameter approximates the distance of adjacent hydrophobic clusters of FG repeats, supporting the model that the barrier is made of FG repeat domains cross‐linked with a spacing of an FG repeat unit length. Wheat germ agglutinin and the dominant‐negative importin β^45‐462 fragment were previously regarded as selective inhibitors of facilitated NPC passage. We now observed that they do not distinguish between the passive and the facilitated mode. Instead, their inhibitory effect correlates with the size of the NPC‐passing molecule. They have little effect on small species, inhibit the passage of green fluorescent protein‐sized objects >10‐fold and virtually block the translocation of larger ones. This suggests that passive and facilitated NPC passage proceed through one and the same permeability barrier.     

Dissecting the roles of Cse1 and Nup2 in classical NLS‐cargo release in vivo

The importin α/β transport machinery mediates the nuclear import of cargo proteins that bear a classical nuclear localization sequence (cNLS). These cargo proteins are linked to the major nuclear protein import factor, importin‐β, by the importin‐α adapter, after which cargo/carrier complexes enter the nucleus through nuclear pores. In the nucleus, cargo is released by the action of RanGTP and the nuclear pore protein Nup2, after which the importins are recycled to the cytoplasm for further transport cycles. The nuclear export of importin‐α is mediated by Cse1/CAS. Here, we exploit structures of functionally important complexes to identify residues that are critical for these interactions and provide insight into how cycles of protein import and recycling of importin‐α occur in vivo using a Saccharomyces cerevisiae model. We examine how these molecular interactions impact protein localization, cargo import, function and complex formation. We show that reversing the charge of key residues in importin‐α (Arg44) or Cse1 (Asp220) results in loss of function of the respective proteins and impairs complex formation both in vitro and in vivo. To extend these results, we show that basic residues in the Nup2 N‐terminus are required for both Nup2 interaction with importin‐α and Nup2 function. These results provide a more comprehensive mechanistic model of how Cse1, RanGTP and Nup2 function in concert to mediate cNLS‐cargo release in the nucleus.     

Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry

Disassembly of nuclear pore complexes (NPCs) is a decisive event during mitotic entry in cells undergoing open mitosis, yet the molecular mechanisms underlying NPC disassembly are unknown. Using chemical inhibition and depletion experiments we show that NPC disassembly is a phosphorylation-driven process, dependent on CDK1 activity and supported by members of the NIMA-related kinase (Nek) family. We identify phosphorylation of the GLFG-repeat nucleoporin Nup98 as an important step in mitotic NPC disassembly. Mitotic hyperphosphorylation of Nup98 is accomplished by multiple kinases, including CDK1 and Neks. Nuclei carrying a phosphodeficient mutant of Nup98 undergo nuclear envelope breakdown slowly, such that both the dissociation of Nup98 from NPCs and the permeabilization of the nuclear envelope are delayed. Together, our data provide evidence for a phosphorylation-dependent mechanism underlying disintegration of NPCs during prophase. Moreover, we identify mitotic phosphorylation of Nup98 as a rate-limiting step in mitotic NPC disassembly.     

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA‐dependent DEAD‐box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP₆)‐bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure‐function analysis in S. cerevisiae and human (h) cells, and find that the high‐affinity Nup42‐Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy‐terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1‐Dbp5/hDDX19B interaction site. A nup42‐CTD/gle1‐CTD/Dbp5 trimeric complex forms in the presence of IP₆. Deletion of NUP42 abrogates Gle1‐Dbp5 interaction, and disruption of the Nup42 or IP₆ binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42‐CTD and IP₆ stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42‐hGle1B interaction.      

Changes in nucleoporin domain topology in response to chemical effectors

Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca2+ and 2 mM Mg2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.     

Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures-one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.