Steady-state nuclear localization of exportin-t involves RanGTP binding and two distinct nuclear pore complex interaction domains

Vertebrate tRNA export receptor exportin-t (Xpo-t) binds to RanGTP and mature tRNAs cooperatively to form a nuclear export complex. Xpo-t shuttles bidirectionally through nuclear pore complexes (NPCs) but is mainly nuclear at steady state. The steady-state distribution of Xpo-t is shown to depend on its interaction with RanGTP. Two distinct Xpo-t NPC interaction domains that bind differentially to peripherally localized nucleoporins in vitro are identified. The N terminus binds to both Nup153 and RanBP2/Nup358 in a RanGTP-dependent manner, while the C terminus binds to CAN/Nup214 independently of Ran. We propose that these interactions increase the concentration of tRNA export complexes and of empty Xpo-t in the vicinity of NPCs and thus increase the efficiency of the Xpo-t transport cycle.     

Molecular mechanism of the nuclear protein import cycle

The nuclear import of proteins through nuclear pore complexes (NPCs) illustrates how a complex biological function can be generated by a spatially and temporally organized cycle of interactions between cargoes, carriers and the Ran GTPase. Recent work has given considerable insight into this process, especially about how interactions are coordinated and the basis for the molecular recognition that underlies the process. Although considerable progress has been made in identifying and characterizing the molecular interactions in the soluble phase that drive the nuclear protein import cycle, understanding the precise mechanism of translocation through NPCs remains a major challenge.     

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.     

NTF2 mediates nuclear import of Ran.

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.     

The SUMO-specific isopeptidase SENP2 associates dynamically with nuclear pore complexes through interactions with karyopherins and the Nup107-160 nucleoporin subcomplex

The association of small, ubiquitin-related modifier-specific isopeptidases (also known as sentrin-specific proteases, or SENPs) with nuclear pore complexes (NPCs) is conserved in eukaryotic organisms ranging from yeast to mammals. However, the functional significance of this association remains poorly understood, particularly in mammalian cells. In this study, we have characterized the molecular basis for interactions between SENP2 and NPCs in human cells. Using fluorescence recovery after photobleaching, we demonstrate that SENP2, although concentrated at the nuclear basket, is dynamically associated with NPCs. This association is mediated by multiple targeting elements within the N-terminus of SENP2 that function cooperatively to mediate NPC localization. One of these elements consists of a high-affinity nuclear localization signal that mediates indirect tethering to FG-repeat-containing nucleoporins through karyopherins. A second element mediates interactions with the Nup107-160 nucleoporin subcomplex. A third element consists of a nuclear export signal. Collectively, our findings reveal that SENP2 is tethered to NPCs through a complex interplay of interactions with nuclear import and export receptors and nucleoporins. Disruption of these interactions enhances SENP2 substrate accessibility, suggesting an important regulatory node in the SUMO pathway.     

Arx1 functions as an unorthodox nuclear export receptor for the 60S preribosomal subunit

Shuttling transport receptors carry cargo through nuclear pore complexes (NPCs) via transient interactions with Phe-Gly (FG)-rich nucleoporins. Here, we identify Arx1, a factor associated with a late 60S preribosomal particle in the nucleus, as an unconventional export receptor. Arx1 binds directly to FG nucleoporins and exhibits facilitated translocation through NPCs. Moreover, Arx1 functionally overlaps with the other 60S export receptors, Xpo1 and Mex67-Mtr2, and is genetically linked to nucleoporins. Unexpectedly, Arx1 is structurally unrelated to known shuttling transport receptors but homologous to methionine aminopeptidases (MetAPs), however, without enzymatic activity. Typically, the MetAP fold creates a central cavity that binds the methionine. In contrast, the predicted central cavity of Arx1 is involved in the interaction with FG repeat nucleoporins and 60S subunit export. Thus, an ancient enzyme fold has been adopted by Arx1 to function as a nuclear export receptor.     

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.     

The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs.     

Ran-dependent nuclear export mediators: a structural perspective

Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP-exportin-cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions.     

Molecular mechanism of translocation through nuclear pore complexes during nuclear protein import.

The trafficking of macromolecules between cytoplasm and nucleus through nuclear pore complexes is mediated by specific carrier molecules such as members of the importin-beta family. Nuclear pore proteins (nucleoporins) frequently contain sequence repeats based on FG cores and carriers appear to move their cargo through the pores by hopping between successive FG cores. A major question is why some macromolecules are transported while others are not. This selectivity may be generated by the ability to bind FG repeats, a local concentration of carrier-cargo complexes near the entrance to the pore channel, and steric hindrance produced by high concentrations of nucleoporins in the channel.     

From the trap to the basket: getting to the bottom of the nuclear pore complex

Nuclear pore complexes (NPCs) are large supramolecular assemblies that perforate the double-membraned nuclear envelope and serve as the sole gateways of molecular exchange between the cytoplasm and the nucleus in interphase cells. Combining novel specimen preparation regimes with innovative use of high-resolution scanning electron microscopy, Hans Ris produced in the late eighties stereo images of the NPC with unparalleled clarity and structural detail, thereby setting new standards in the field. Since that time, efforts undertaken to resolve the molecular structure and architecture, and the numerous interactions that occur between NPC proteins (nucleoporins), soluble transport receptors, and the small GTPase Ran, have led to a deeper understanding of the functional role of NPCs in nucleocytoplasmic transport. In spite of these breakthroughs, getting to the bottom of the actual cargo translocation mechanism through the NPC remains elusive and controversial. Here, we review recent insights into NPC function by correlating structural findings with biochemical data. By introducing new experimental and computational results, we reexamine how NPCs can discriminate between receptor-mediated and passive cargo to promote vectorial translocation in a highly regulated manner. Moreover, we comment on the importance and potential benefits of identifying and experimenting with individual key components implicated in the translocation mechanism. We conclude by dwelling on questions that we feel are pertinent to a more rational understanding of the physical aspects governing NPC mechanics. Last but not least, we substantiate these uncertainties by boldly suggesting a new direction in NPC research as a means to verify such novel concepts, for example, a de novo designed ‘minimalist’ NPC. This article is dedicated to the memory of Hans Ris.     

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 pores: from yeast to higher eukaryotes

In eukaryotes, bidirectional transport of macro-molecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs), whose overall architecture has been evolutionary conserved from yeast to vertebrates. In recent years, fast progress in characterizing the NPCs components (the nucleoporins or Nups) has been made in the yeast S. cerevisiae, and to a lesser extent in vertebrates. In addition, despite the low homology between most yeast and vertebrate nucleoporins, their organization and their topological mapping within the NPC substructures have been broadly conserved during evolution.     

Nucleoporins: leaving the nuclear pore complex for a successful mitosis

The nuclear envelope (NE) separates the cytoplasm and the cell nucleus of interphase eukaryotic cells and nuclear pore complexes (NPCs) mediate the macromolecular exchange between these two compartments. The NE and the NPCs of vertebrate cells disassemble during prophase and the nuclear pore proteins (nucleoporins) are distributed within the mitotic cytoplasm. For an increasing number of them active mitotic functions have been assigned over the past few years. Nucleoporins are participating in spindle assembly, kinetochore organisation, and the spindle assembly checkpoint, all processes that control chromosome segregation and are important for maintenance of genome integrity. But nucleoporins are also engaged in early and late mitotic events, such as centrosome positioning and cytokinesis. Here we will highlight recent progress in deciphering the roles for nucleoporins in the distinct steps of mitosis.     

The conserved transmembrane nucleoporin NDC1 is required for nuclear pore complex assembly in vertebrate cells

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in the nuclear envelope (NE), through which exchange of molecules between the nucleus and cytosol occurs. Biogenesis of NPCs is complex and poorly understood. In particular, almost nothing is known about how NPCs are anchored in the NE. Here, we characterize vertebrate NDC1--a transmembrane nucleoporin conserved between yeast and metazoans. We show by RNA interference (RNAi) and biochemical depletion that NDC1 plays an important role in NPC and NE assembly in vivo and in vitro. RNAi experiments suggest a functional link between NDC1 and the soluble nucleoporins Nup93, Nup53, and Nup205. Importantly, NDC1 interacts with Nup53 in vitro. This suggests that NDC1 function involves forming a link between the NE membrane and soluble nucleoporins, thereby anchoring the NPC in the membrane.     

The role of nuclear pores in gene regulation,development and disease

Nuclear‐pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000–4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well‐characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport‐dependent and transport‐independent roles of NPCs in the regulation of nuclear function and gene expression.     

The yeast nuclear pore complex and transport through it

Exchange of macromolecules between the nucleus and cytoplasm is a key regulatory event in the expression of a cell's genome. This exchange requires a dedicated transport system: (1) nuclear pore complexes (NPCs), embedded in the nuclear envelope and composed of proteins termed nucleoporins (or "Nups"), and (2) nuclear transport factors that recognize the cargoes to be transported and ferry them across the NPCs. This transport is regulated at multiple levels, and the NPC itself also plays a key regulatory role in gene expression by influencing nuclear architecture and acting as a point of control for various nuclear processes. Here we summarize how the yeast Saccharomyces has been used extensively as a model system to understand the fundamental and highly conserved features of this transport system, revealing the structure and function of the NPC; the NPC's role in the regulation of gene expression; and the interactions of transport factors with their cargoes, regulatory factors, and specific nucleoporins.     

Nuclear pore complex-a coat specifically tailored for the nuclear envelope

Nuclear pore complexes (NPCs) are highly selective transport gates that enable the bi-directional traffic of macromolecules across the nuclear envelope (NE). NPCs are located at the fusion pores between the inner and outer membranes of the NE and are built from a common set of ～30 different proteins, nucleoporins. Remarkably, recent proteomic, bioinformatic, and structural studies have provided firm evidence that key structural nucleoporins share common ancestry with elements of coated vesicles, indicating an evolutionary link between these structures. This has provided novel insight into the origin of NPCs and may help us to better functionally characterize these fundamental components of eukaryotic cells.     

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.     

Annulate lamellae play only a minor role in the storage of excess nucleoporins in Drosophila embryos

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleo-cytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.