Minimal nuclear pore complexes define FG repeat domains essential for transport

Translocation through nuclear pore complexes (NPCs) requires interactions between receptor-cargo complexes and phenylalanine-glycine (FG) repeats in multiple FG domain-containing NPC proteins (FG-Nups). We have systematically deleted the FG domains of 11 Saccharomyces cerevisiae FG-Nups in various combinations. All five asymmetrically localized FG domains deleted together were non-essential. However, specific combinations of symmetrically localized FG domains were essential. Over half the total mass of FG domains could be deleted without loss of viability or the NPC's normal permeability barrier. Significantly, symmetric deletions caused mild reductions in Kap95-Kap60-mediated import rates, but virtually abolished Kap104 import. These results suggest the existence of multiple translocation pathways.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

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.     

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.     

The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.     

Calcium regulation of nucleocytoplasmic transport

Bidirectional trafficking of macromolecules between the cytoplasm and the nucleus is mediated by the nuclear pore complexes (NPCs) embedded in the nuclear envelope (NE) of eukaryotic cell. The NPC functions as the sole pathway to allow for the passive diffusion of small molecules and the facilitated translocation of larger molecules. Evidence shows that these two transport modes and the conformation of NPC can be regulated by calcium stored in the lumen of nuclear envelope and endoplasmic reticulum. However, the mechanism of calcium regulation remains poorly understood. In this review, we integrate data on the observations of calcium-regulated structure and function of the NPC over the past years. Furthermore, we highlight challenges in the measurements of dynamic conformational changes and transient transport kinetics in the NPC. Finally, an innovative imaging approach, single-molecule super-resolution fluorescence microscopy, is introduced and expected to provide more insights into the mechanism of calcium-regulated nucleocytoplasmic transport.     

Should I stay or should I go? Nucleocytoplasmic trafficking in plant innate immunity

Communication between the cytoplasm and the nucleus is a fundamental feature of eukaryotic cells. Bidirectional transport of macromolecules across the nuclear envelope is typically mediated by receptors and occurs exclusively through nuclear pore complexes (NPCs). The components and molecular mechanisms regulating nucleocytoplasmic trafficking and signalling processes are well studied in animals and yeast but are poorly understood in plants. Current work shows that components of the NPC and the nuclear import and export machinery play essential roles in plant innate immunity. Translocation of defence regulators and Resistance (R) proteins between the cytoplasm and the nucleus are recently uncovered aspects of plant defence responses against pathogens. Future studies will reveal more details on the spatial and temporal dynamics and regulation of this process.     

Nanocompartmentalization of the Nuclear Pore Lumen

The nuclear pore complex (NPC) employs the intrinsically disordered regions (IDRs) from a family of phenylalanine-glycine-rich nucleoporins (FG-Nups) to control nucleocytoplasmic transport. It has been a long-standing mystery how the IDR-mediated mass exchange can be rapid yet selective. Here, we use a computational microscope to show that nanocompartmentalization of IDR subdomains leads to a remarkably elaborate gating structure as programmed by the amino acid sequences. In particular, we reveal a heterogeneous permeability barrier that combines an inner ring barrier with two vestibular condensates. Throughout the NPC, we find a polarized electrostatic potential and a diffuse thermoreversible FG network featuring mosaic FG territories with low FG-FG pairing fraction. Our theoretical anatomy of the central transporter sheds light into the sequence-structure-function relationship of the FG-Nups and provides a picture of nucleocytoplasmic mass exchange that allows a reconciliation of transport efficiency and specificity.     

The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion

Nuclear pore complexes (NPCs) restrict the nucleocytoplasmic flux of most macromolecules, but permit facilitated passage of nuclear transport receptors and their cargo complexes. We found that a simple hydrophobic interaction column can mimic the selectivity of NPCs surprisingly well and that nuclear transport receptors appear to be the most hydrophobic soluble proteins. This suggests that surface hydrophobicity represents a major sorting criterion of NPCs. The rate of NPC passage of cargo-receptor complexes is, however, not dominated just by properties of the receptors. We found that large cargo domains drastically hinder NPC passage and require more than one receptor molecule for rapid translocation. This argues against a rigid translocation channel and instead suggests that NPC passage involves a partitioning of the entire translocating species into a hydrophobic phase, whereby the receptor:cargo ratio determines the solubility in that permeability barrier. Finally, we show that interfering with hydrophobic interactions causes a reversible collapse of the permeability barrier of NPCs, which is consistent with the assumption that the barrier is formed by phenylalanine-rich nucleoporin repeats that attract each other through hydrophobic interactions.     

Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex

Nuclear pore complexes (NPCs) form aqueous conduits in the nuclear envelope and gate the diffusion of large proteins between the cytoplasm and nucleoplasm. NPC proteins (nucleoporins) that contain phenylalanine-glycine motifs in filamentous, natively unfolded domains (FG domains) line the diffusion conduit of the NPC, but their role in the size-selective barrier is unclear. We show that deletion of individual FG domains in yeast relaxes the NPC permeability barrier. At the molecular level, the FG domains of five nucleoporins anchored at the NPC center form a cohesive meshwork of filaments through hydrophobic interactions, which involve phenylalanines in FG motifs and are dispersed by aliphatic alcohols. In contrast, the FG domains of four peripherally anchored nucleoporins are generally noncohesive. The results support a two-gate model of NPC architecture featuring a central diffusion gate formed by a meshwork of cohesive FG nucleoporin filaments and a peripheral gate formed by repulsive FG nucleoporin filaments.     

Nuclear protein accumulation by facilitated transport and intranuclear binding

Nuclear proteins are transported from the cytoplasm into the nucleus via nuclear envelope pore complexes (NPCs). At the molecular level, the mechanisms responsible for this transport remain obscure. However, it is known that, for many proteins, the process requires ATP and proceeds against formidable nucleocytoplasmic concentration gradients. Therefore, the NPC is often thought of as an active transport site. In this article, Philip Paine presents the alternative hypothesis that, on current evidence, protein translocation across the nuclear envelope and accumulation in the nucleus can equally well be explained by facilitated transport through the NPC and subsequent intranuclear binding.     

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.     

Efficiency, selectivity, and robustness of nucleocytoplasmic transport

All materials enter or exit the cell nucleus through nuclear pore complexes (NPCs), efficient transport devices that combine high selectivity and throughput. NPC-associated proteins containing phenylalanine–glycine repeats (FG nups) have large, flexible, unstructured proteinaceous regions, and line the NPC. A central feature of NPC-mediated transport is the binding of cargo-carrying soluble transport factors to the unstructured regions of FG nups. Here, we model the dynamics of nucleocytoplasmic transport as diffusion in an effective potential resulting from the interaction of the transport factors with the flexible FG nups, using a minimal number of assumptions consistent with the most well-established structural and functional properties of NPC transport. We discuss how specific binding of transport factors to the FG nups facilitates transport, and how this binding and competition between transport factors and other macromolecules for binding sites and space inside the NPC accounts for the high selectivity of transport. We also account for why transport is relatively insensitive to changes in the number and distribution of FG nups in the NPC, providing an explanation for recent experiments where up to half the total mass of the FG nups has been deleted without abolishing transport. Our results suggest strategies for the creation of artificial nanomolecular sorting devices.     

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.     

Single molecule studies of nucleocytoplasmic transport

Molecular traffic between the cytoplasm and the nucleoplasm of eukaryotic cells is mediated by nuclear pore complexes (NPCs). Hundreds, if not thousands, of molecules interact with and transit through each NPC every second. The pore is blocked by a permeability barrier, which consists of a network of intrinsically unfolded polypeptides containing thousands of phenylalanine-glycine (FG) repeat motifs. This FG-network rejects larger molecules and admits smaller molecules or cargos bound to nuclear transport receptors (NTRs). For a cargo transport complex, minimally consisting of a cargo molecule plus an NTR, access to the permeability barrier is provided by interactions between the NTR and the FG repeat motifs. Numerous models have been postulated to explain the controlled accessibility and the transport characteristics of the FG-network, but the amorphous, flexible nature of this structure has hindered characterization. A relatively recent development is the ability to monitor the real-time movement of single molecules through individual NPCs via single molecule fluorescence (SMF) microscopy. A major advantage of this approach is that it can be used to continuously monitor a series of specific molecular interactions in an active pore with millisecond time resolution, which therefore allows one to distinguish between kinetic and thermodynamic control. Novel insights and prospects for the future are outlined in this review. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Nuclear translocation of proteins and the effect of phosphatidic acid

Transport of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors called importins, typically dimmers of a cargo-binding α-subunit and a β-subunit that mediates translocation through the nuclear pore complexes (NPCs). However, how proteins without canonical NLS move into the nucleus is not well understood. Recent results indicate that phospholipids, such as phosphatidic acid, play important roles in the intracellular translocation of proteins between the nucleus and cytoplasm.     

The dynamics of karyopherin-mediated nuclear transport.

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.     

O-GlcNAc modification of nuclear pore complexes accelerates bidirectional transport

Macromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG-repeat domains in NPCs are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated transport of proteins in both directions, and decreasing modification slowed transport. Superresolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the nonspecific permeability of the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.     

Dynamics of nuclear pore complex organization through the cell cycle

In eukaryotic cells, all macromolecules that traffic between the nucleus and the cytoplasm cross the double nuclear membrane through nuclear pore complexes (NPCs). NPCs are elaborate gateways that allow efficient, yet selective, translocation of many different macromolecules. Their protein composition has been elucidated, but how exactly these nucleoporins come together to form the pore is largely unknown. Recent data suggest that NPCs are composed of an extremely stable scaffold on which more dynamic, exchangeable parts are assembled. These could be targets for molecular rearrangements that change nuclear pore transport properties and, ultimately, the state of the cell.     

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.