FG Domains Research Articles

In vivo analysis of human nucleoporin repeat domain interactions

The nuclear pore complex (NPC), assembled from ∼30 proteins termed nucleoporins (Nups), mediates selective nucleocytoplasmic trafficking. A subset of nucleoporins bear a domain with multiple phenylalanine-glycine (FG) motifs. As binding sites for transport receptors, FG Nups are critical in translocation through the NPC. Certain FG Nups are believed to associate via low-affinity, cohesive interactions to form the permeability barrier of the pore, although the form and composition of this functional barrier are debated. We used green fluorescent protein-Nup98/HoxA9 constructs with various numbers of repeats and also substituted FG domains from other nucleoporins for the Nup98 domain to directly compare cohesive interactions in live cells by fluorescence recovery after photobleaching (FRAP). We find that cohesion is a function of both number and type of FG repeats. Glycine-leucine-FG (GLFG) repeat domains are the most cohesive. FG domains from several human nucleoporins showed no interactions in this assay; however, Nup214, with numerous VFG motifs, displayed measurable cohesion by FRAP. The cohesive nature of a human nucleoporin did not necessarily correlate with that of its yeast orthologue. The Nup98 GLFG domain also functions in pore targeting through binding to Nup93, positioning the GLFG domain in the center of the NPC and supporting a role for this nucleoporin in the permeability barrier.

Conserved Spatial Organization of FG Domains in the Nuclear Pore Complex

Selective transport through the nuclear pore complex (NPC) requires nucleoporins containing natively unfolded phenylalanine-glycine (FG) domains. Several differing models for their dynamics within the pore have been proposed. We characterize the behavior of the FG nucleoporins in vivo using polarized fluorescence microscopy. Using nucleoporins tagged with green fluorescent protein along their FG domains, we show that some of these proteins are ordered, indicating an overall orientational organization within the NPC. This orientational ordering of the FG domains depends on their specific context within the NPC, but is independent of active transport and cargo load. For most nups, behavior does not depend on the FG motifs. These data support a model whereby local geometry constrains the orientational organization of the FG nups. Intriguingly, homologous yeast and mammalian proteins show conserved behavior, suggesting functional relevance. Our findings have implications for mechanistic models of NPC transport.

Peptide Ordering within Nuclear Pores in Living Cells

It is a profound belief of biologists, based upon decades of structural biochemistry, that understanding the three-dimensional structure of individual proteins and protein complexes illuminates the basis of protein function. X-ray crystallography and other structural techniques have revealed remarkable high-resolution maps of even large, multimolecular structures. However, the approaches which give such spectacular information in vitro are unable to probe structures of proteins that are functioning in their natural milieu in living cells. This frontier between structural biology and cellular biology is only beginning to be addressed. An example of the progress is work from the laboratory of Sanford Simon (Rockefeller University, New York). Earlier works by Mattheyses et al. (1) and Kampman et al. (2), and a new article by Atkinson et al. (3) (this issue) describe the elegant application of fluorescence polarization to probe the orientation of specific protein components in functioning nuclear pore complexes in living cells. Nuclear pores are an essential component of eukaryotic cells that are responsible for the highly regulated, bidirectional transport of proteins and RNA between the cytoplasm and nucleus. They are very large structures that contain a large aqueous channel that spans the double membrane of the nuclear envelope. Pores are composed of ∼30 different proteins (nucleoporins) present in multiple copies, with several thousand proteins comprising a single nuclear pore. Crystallography has provided structural information about isolated nucleoporins and their interaction sites (4). Electron microscopy of intact nuclear pore complexes has revealed a structure with octagonal symmetry (5,6). Simon and colleagues have taken advantage of the random distribution of nuclear pores around the approximately circular cross section of the nuclear envelope. Nuclear pore proteins labeled with green fluorescent protein were introduced into cells by molecular biology techniques. By illumination of the sample with polarized light and collection of the emission in two orthogonally polarized channels, the fluorescence anisotropies of nuclear pores with their varying orientations around the nuclear envelope cross section were determined. Thus, the intrinsic geometry of the positions of the nuclear pores in the nuclear envelope enabled investigation of the anisotropy at various angles. Channel nucleoporins have a predicted α-helical region and an unfolded domain containing phenylalaine-glycine (FG) repeats. The ensemble of FG domains from multiple nucleoporins in a channel is thought to function as a diffusion barrier to regulate transport of large macromolecules (Fig. 1 (7)). This article addresses the question of whether FG regions in different nucleoporins are oriented relative to the central axis of functioning nuclear pores in living cells. The findings were significant: some of the FG domains of specific nucleoporins were ordered. Ordering was more likely for nucleoporins lining the center of the nuclear pore complex than for those at the cytoplasmic or nuclear entrances. Ordering was not altered by active transport or attachment of cargo. Although one might have predicted (wished?) a change with function, the time resolution of anisotropy measurements (2 s) was likely insufficient to capture fluctuations due to individual transport events. Figure 1 Schematic of nuclear pore. Adapted from Fig. 12-10 in Alberts et al. (7). These studies are a hopeful beginning, demonstrating the possibility of asking structural questions within living cells. We can look forward to improved techniques, including faster time resolution approaches and the application of single-molecule fluorescence imaging, that will enable the detection of function-related structural changes in proteins in their normal, intracellular milieu. These types of biophysical techniques should be applicable to numerous other dynamic protein structures within cells.

Nuclear Transport: Beginning to Gel?

The massive nuclear pore complex mediates nucleocytoplasmic traffic ranging from a single histone to a viral genome. To date, dissecting mechanism has been more an exercise in prediction than biochemical certainty. A recent study combines recombinant proteins with nuclei reconstituted in vitro to test predictions in a startlingly productive manner.

Systematic analysis of barrier-forming FG hydrogels from Xenopus nuclear pore complexes

Nuclear pore complexes (NPCs) control the traffic between cell nucleus and cytoplasm. While facilitating translocation of nuclear transport receptors (NTRs) and NTR·cargo complexes, they suppress passive passage of macromolecules ⩾30 kDa. Previously, we reconstituted the NPC barrier as hydrogels comprising S. cerevisiae FG domains. We now studied FG domains from 10 Xenopus nucleoporins and found that all of them form hydrogels. Related domains with low FG motif density also substantially contribute to the NPC's hydrogel mass. We characterized all these hydrogels and observed the strictest sieving effect for the Nup98-derived hydrogel. It fully blocks entry of GFP-sized inert objects, permits facilitated entry of the small NTR NTF2, but arrests importin β-type NTRs at its surface. O-GlcNAc modification of the Nup98 FG domain prevented this arrest and allowed also large NTR·cargo complexes to enter. Solid-state NMR spectroscopy revealed that the O-GlcNAc-modified Nup98 gel lacks amyloid-like β-structures that dominate the rigid regions in the S. cerevisiae Nsp1 FG hydrogel. This suggests that FG hydrogels can assemble through different structural principles and yet acquire the same NPC-like permeability.

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

Conformational changes at supramolecular interfaces are fundamentally coupled to binding activity, yet it remains a challenge to probe this relationship directly. Within the nuclear pore complex, this underlies how transport receptors known as karyopherins proceed through a tethered layer of intrinsically disordered nucleoporin domains containing Phe-Gly (FG)-rich repeats (FG domains) that otherwise hinder passive transport. Here, we use nonspecific proteins (i.e., BSA) as innate molecular probes to explore FG domain conformational changes by surface plasmon resonance. This mathematically diminishes the surface plasmon resonance refractive index constraint, thereby providing the means to acquire and correlate height changes in a surface-tethered FG domain layer to Kap binding affinities in situ with respect to their relative spatial arrangements. Stepwise measurements show that FG domain collapse is caused by karyopherin β1 (Kapβ1) binding at low concentrations, but this gradually transitions into a reextension at higher Kapβ1 concentrations. This ability to self-heal is intimately coupled to Kapβ1-FG binding avidity that promotes the maximal incorporation of Kapβ1 into the FG domain layer. Further increasing Kapβ1 to physiological concentrations leads to a "pileup" of Kapβ1 molecules that bind weakly to unoccupied FG repeats at the top of the layer. Therefore, binding avidity does not hinder fast transport per se. Revealing the biophysical basis underlying the form-function relationship of Kapβ1-FG domain behavior results in a convergent picture in which transport and mechanistic aspects of nuclear pore complex functionality are reconciled.

Single-Molecule Analysis of the Recognition Forces Underlying Nucleo-Cytoplasmic Transport

Macromolecular exchange between the nucleus and cytoplasm of cells is gated at nuclear pores by a family of intrinsically-disordered nucleoporins (nups). These feature phenylalanine-glycine repeats in ‘cohesive’ domains (FG domains) that interact to form a physical barrier. Through unknown mechanisms, karyopherins (importins, exportins, transportins, NTRs) penetrate this barrier to facilitate the movement of large proteins and RNPs across without paying an external energetic cost, simply by interacting with FG domains. To address the molecular binding and dissociation mechanisms involved in this coupled gating-translocation process, single molecule force spectroscopy was used here to measure the interaction force between nup FG repeats, and between importin beta and nup FG repeats. As predicted, cohesive FG domains bound each other through multiple FG repeat interactions. In contrast importin bound only relaxed coil multiple FG repeats simultaneously, whereas just one FG binding site was assessed in collapsed coil multiple FG domains. Most importantly, the interaction forces and fast dissociation rate constants measured between two FG repeats, and between importin and one FG repeat, were almost identical. This suggests that the force needed to separate interactions between FG repeats of nups at the NPC (i.e. for kaps to penetrate the gate and translocate across) could be provided in full by the enthalpy gained through the formation of karyopherin-FG repeat interactions.

Effect of FG Motifs on Ordering Proteins within the Nuclear Pore Complex

Transport across the nuclear membrane occurs through the nuclear pore complex (NPC). The transport through the NPC is both selective and rapid, and involves the phenylalanine glycine repeat nucleoporins (FG nups). The FG motifs can bind cargo and may also be able to interact with each other but the mechanism of transport remains unclear. We have developed a theoretical framework which exploits the symmetry of the NPC and its organization in the nuclear envelope to resolve the order and disorder of individual protein domains within the complex. Specific domains of individual nucleoporins (nups) were tagged with GFP and examined using fluorescence polarization microscopy to determine their organization and dynamics. We characterized the degree to which the FG domains are ordered in vivo. This approach revealed different degrees of organization within a single FG domain: the tips and middle of the FG domains are less ordered than NPC-anchored domains, but still display a surprising degree of organization. We examined three mammalian nups: Nup54, Nup62, and Nup98. We observe that of these, Nup54 is the most ordered within the NPC and Nup62 is the least ordered. We then investigated the contribution of the FG repeats to the organization of these nucleoporins by mutating the FG motifs to AG. We show that these mutations have no effect on the organization of Nup62, decrease but do not eliminate the order shown by Nup54, and prevent Nup98 from localizing correctly to the NPC. We therefore propose that multiple factors contribute to the organization of FG nups in vivo, and that the influence of the FG repeats on order varies between different FG nups.

P granules extend the nuclear pore complex environment in the C. elegans germ line.

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)-like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.

Protein Domain Organization in the Nuclear Pore Complex Studied by Fluorescence Anisotropy

Fluorescence anisotropy measurements can yield information about the mobility, orientation and proximity of fluorophores and can be used to study the dynamics of proteins in vivo. The nuclear pore complex (NPC) is a large macromolecular complex, the size and complexity of which present experimental challenges. The dynamics of nucleoporins (nups) were examined using fluorescence polarization microscopy. Using a theoretical framework which exploits the symmetry of the NPC and its organization in the nuclear envelope we can resolve the order and disorder of individual protein domains within the complex. Specific domains of individual nups were tagged with GFP and examined using fluorescence polarization microscopy. We characterized the domain organization of the FG nups, which are required for transport of cargo through the NPC. This approach revealed both structured and unstructured domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. This technique allows the collection of structural information in vivo with the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nups, which are implicated in the mechanism of cargo transport.

A Bimodal Distribution of Two Distinct Categories of Intrinsically Disordered Structures with Separate Functions in FG Nucleoporins

Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two separate zones of traffic with distinct physicochemical properties.

Fluorescence Anisotropy Reveals Order and Disorder of Protein Domains in the Nuclear Pore Complex

We present a new approach for studying individual protein domains within the nuclear pore complex (NPC) using fluorescence polarization microscopy. The NPC is a large macromolecular complex, the size and complexity of which presents experimental challenges. Using fluorescence anisotropy and exploiting the symmetry of the NPC and its organization in the nuclear envelope, we have resolved order and disorder of individual protein domains. Fluorescently tagging specific domains of individual nucleoporins revealed both rigid and flexible domains: the tips of the FG domains are disordered, whereas the NPC-anchored domains are ordered. Our technique allows the collection of structural information in vivo, providing the ability to probe the organization of protein domains within the NPC. This has particular relevance for the FG domain nucleoporins, which are crucial for nucleocytoplasmic transport.

Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex

Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.

Ultrathin nucleoporin phenylalanine–glycine repeat films and their interaction with nuclear transport receptors

Nuclear pore complexes (NPCs) are highly selective gates that mediate the exchange of all proteins and nucleic acids between the cytoplasm and the nucleus. Their selectivity relies on a supramolecular assembly of natively unfolded nucleoporin domains containing phenylalanine-glycine (FG)-rich repeats (FG repeat domains), in a way that is at present poorly understood. We have developed ultrathin FG domain films that reproduce the mode of attachment and the density of FG repeats in NPCs, and that exhibit a thickness that corresponds to the nanoscopic dimensions of the native permeability barrier. By using a combination of biophysical characterization techniques, we quantified the binding of nuclear transport receptors (NTRs) to such FG domain films and analysed how this binding affects the swelling behaviour and mechanical properties of the films. The results extend our understanding of the interaction of FG domain assemblies with NTRs and contribute important information to refine the model of transport across the permeability barrier.

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 plant interaction network between cargo and FG repeats.

Translocation through the nuclear pore: Kaps pave the way

Transport through the nuclear pore complex (NPC), a keystone of the eukaryotic building plan, is known to involve a large channel and an abundance of phenylalanine-glycine (FG) protein domains serving as binding sites for soluble nuclear transport receptors and their cargo complexes. However, the conformation of the FG domains in vivo, their arrangement in relation to the transport channel and their function(s) in transport are still vividly debated. Here, we revisit a number of representative transport models-specifically Brownian affinity gating, selective phase gating, reversible FG domain collapse, and reduction of dimensionality (ROD)-in the light of new data obtained by optical single transporter recording, optical superresolution microscopy, artificial nanopores, and many other techniques. The analysis suggests that a properly adapted, simplified version of the ROD model accounts well for the available data. This has implications for nucleocytoplasmic transport in general.

Intramolecular Cohesion of Coils Mediated by Phenylalanine–Glycine Motifs in the Natively Unfolded Domain of a Nucleoporin

The nuclear pore complex (NPC) provides the sole aqueous conduit for macromolecular exchange between the nucleus and the cytoplasm of cells. Its diffusion conduit contains a size-selective gate formed by a family of NPC proteins that feature large, natively unfolded domains with phenylalanine–glycine repeats (FG domains). These domains of nucleoporins play key roles in establishing the NPC permeability barrier, but little is known about their dynamic structure. Here we used molecular modeling and biophysical techniques to characterize the dynamic ensemble of structures of a representative FG domain from the yeast nucleoporin Nup116. The results showed that its FG motifs function as intramolecular cohesion elements that impart order to the FG domain and compact its ensemble of structures into native premolten globular configurations. At the NPC, the FG motifs of nucleoporins may exert this cohesive effect intermolecularly as well as intramolecularly to form a malleable yet cohesive quaternary structure composed of highly flexible polypeptide chains. Dynamic shifts in the equilibrium or competition between intra- and intermolecular FG motif interactions could facilitate the rapid and reversible structural transitions at the NPC conduit needed to accommodate passing karyopherin–cargo complexes of various shapes and sizes while simultaneously maintaining a size-selective gate against protein diffusion.

Nuclear mRNA export requires coordinated FG‐Nup binding and DEAD‐box protein‐mediated remodeling events

Nucleocytoplasmic transport of proteins and nucleic acids is essential for proper gene expression. Using the Saccharomyces cerevisiae model, we discovered key events for directional export of messenger (m) RNA. Distinct steps were pinpointed at nuclear, central and cytoplasmic nuclear pore complex (NPC) structures. Nearly half of the ∼30 NPC proteins harbor Phe‐Gly (FG) repeat domains that potentially serve as binding sites for the shuttling mRNA export receptor Mex67. A large‐scale deletion strategy was employed to generate mutants lacking discrete combinations of FG‐domains, and specific subsets of FG domains were required for mRNA export. The FG mutants also revealed multiple functionally independent translocation routes through NPCs. mRNA export required FG domains on the nuclear NPC face (presumably for initial docking at the NPC) and in the NPC central core. At the cytoplasmic NPC face, we found that the RNA‐dependent ATPase activity of the DEAD‐box protein Dbp5 was stimulated by inositol hexakisphosphate‐bound Gle1. The ADP‐bound form of Dbp5 triggered release of the RNA‐binding protein Nab2 from RNA. This novel nucleotide‐dependent switch mechanism remodeled mRNA‐protein complexes for directional translocation. We predict that these FG‐binding and mRNP remodeling steps are coupled for spatial regulation of mRNA export through and subsequent release from the NPC.Supported by grants from the NIGMS.

Nanomechanical Basis of Selective Gating by the Nuclear Pore Complex

The nuclear pore complex regulates cargo transport between the cytoplasm and the nucleus. We set out to correlate the governing biochemical interactions to the nanoscopic responses of the phenylalanineglycine (FG)-rich nucleoporin domains, which are involved in attenuating or promoting cargo translocation. We found that binding interactions with the transport receptor karyopherin-beta1 caused the FG domains of the human nucleoporin Nup153 to collapse into compact molecular conformations. This effect was reversed by the action of Ran guanosine triphosphate, which returned the FG domains into a polymer brush-like, entropic barrier conformation. Similar effects were observed in Xenopus oocyte nuclei in situ. Thus, the reversible collapse of the FG domains may play an important role in regulating nucleocytoplasmic transport.

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.