The mRNA export factor human Gle1 interacts with the nuclear pore complex protein Nup155

The protein Gle1 is required for export of mRNAs from the nucleus to the cytoplasm in both lower and higher eukaryotic cells. In human (h) cells, shuttling of hGle1 between the nucleus and cytoplasm is essential for bulk mRNA export. To date, no hGle1-interacting proteins have been reported and the mechanism by which hGle1 interacts with the nuclear pore complex (NPC) and mediates export is unknown. To identify proteins that can interact with hGle1, a genome-wide yeast two-hybrid screen was performed. Three potential hGle1-interacting partners were isolated, including clones encoding the C-terminal region of the NPC protein hNup155. This interaction between hGle1 and full-length hNup155 was confirmed in vitro, and deletion analysis identified the N-terminal 29 residues of hGle1 as the hNup155-binding domain. Experiments in HeLa cells confirmed that the nuclear rim localization of the major hGle1 protein variant (hGle1B) was dependent on the presence of these 29 N-terminal residues. This suggests that this domain of hGle1 is necessary for targeting to the NPC. This work also characterizes the first domain in hNup155, a 177 C-terminal amino acid span that binds to hGle1. The mutual interaction between hGle1 and the symmetrically distributed nuclear pore protein Nup155 suggests a model in which hGle1's association with hNup155 may represent a step in the Gle1-mediated mRNA export pathway.     

The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm

Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport.     

The yeast nucleoporin Nup188p interacts genetically and physically with the core structures of the nuclear pore complex

We have isolated a major protein constituent from a highly enriched fraction of yeast nuclear pore complexes (NPCs). The gene encoding this protein, Nup188p, was cloned, sequenced, and found to be nonessential upon deletion. Nup188p cofractionates with yeast NPCs and gives an immunofluorescent staining pattern typical of nucleoporins. Using immunoelectron microscopy, Nup188p was shown to localize to both the cytoplasmic and nucleoplasmic faces of the NPC core. There, Nup188p interacts with an integral protein of the pore membrane domain, Pom152p, and another abundant nucleoporin, Nic96p. The effects of various mutations in the NUP188 gene on the structure of the nuclear envelope and the function of the NPC were examined. While null mutants of NUP188 appear normal, other mutants allelic to NUP188 exhibit a dominant effect leading to the formation of NPC-associated nuclear envelope herniations and growth inhibition at 37 degrees C. In addition, depletion of the interacting protein Pom152p in cells lacking Nup188p resulted in severe deformations of the nuclear envelope. We suggest that Nup188p is one of a group of proteins that form the octagonal core structure of the NPC and thus functions in the structural organization of the NPC and nuclear envelope.     

Nic96p is required for nuclear pore formation and functionally interacts with a novel nucleoporin, Nup188p

The amino-terminal domain of Nic96p physically interacts with the Nsp1p complex which is involved in nucleocytoplasmic transport. Here we show that thermosensitive mutations mapping in the central domain of Nic96p inhibit nuclear pore formation at the nonpermissive temperature. Furthermore, the carboxyterminal domain of Nic96p functionally interacts with a novel nucleoporin Nup188p in an allele-specific fashion, and when ProtA-Nup188p was affinity purified, a fraction of Nic96p was found in physical interaction. Although NUP188 is not essential for viability, a null mutant exhibits striking abnormalities in nuclear envelope and nuclear pore morphology. We propose that Nic96p is a multivalent protein of the nuclear pore complex linked to several nuclear pore proteins via its different domains.     

A yeast RNA-binding protein shuttles between the nucleus and the cytoplasm.

RNA-binding proteins have been suggested to move in association with RNA as it leaves the nucleus. The NPL3 gene of the yeast Saccharomyces cerevisiae encodes in nuclear protein with consensus RNA-binding motifs and similarity to heterogeneous nuclear ribonucleoproteins and members of the S/R protein family. We show that although Npl3 is located in the nucleus, it can shuttle between nuclei in yeast heterokaryons. In contrast, other nucleus-targeted proteins do not leave the nucleus under similar conditions. Mutants missing the RNA-binding motifs or the N terminus are still capable of shuttling in and out of the nucleus. Npl3 mutants missing the C terminus fail to localize to the nucleus. Overproduction of Npl3 in wild-type cells shows cell growth. This toxicity depends on the presence of series of unique repeats in the N terminus and localization to the nucleus. We suggest that the properties of Npl3 are consistent with it being involved in export of RNAs from the nucleus.     

Versatility at the nuclear pore complex: lessons learned from the nucleoporin Nup153

The vertebrate pore protein Nup153 plays pivotal roles in nuclear pore function. In addition to being important to pore architecture, Nup153 is a key participant in both import and export. The scope of Nup153 function also extends beyond the canonical view of the pore as a trafficking gateway. During the transition into mitosis, Nup153 directs proteins involved in membrane remodeling to the nuclear envelope. As cells exit mitosis, Nup153 is recruited to the chromosomal surface, where nuclear pores are formed anew in a complicated process still under much experimental scrutiny. In addition, Nup153 is targeted for protease cleavage during apoptosis and in response to certain viral infections, providing molecular insight into pore reconfiguration during cell response. Overall, the versatile nature of Nup153 underscores an emerging view of the nuclear pore at the nexus of many key cellular processes.     

Human cytomegalovirus UL84 protein contains two nuclear export signals and shuttles between the nucleus and the cytoplasm

Previous studies defined pUL84 of human cytomegalovirus as an essential regulatory protein with nuclear localization that was proposed to act during initiation of viral-DNA synthesis. Recently, we demonstrated that a complex domain of 282 amino acids within pUL84 functions as a nonconventional nuclear localization signal. Sequence inspection of this domain revealed the presence of motifs with homology to leucine-rich nuclear export signals. Here, we report the identification of two functional, autonomous nuclear export signals and show that pUL84 acts as a CRM-1-dependent nucleocytoplasmic shuttling protein. This suggests an unexpected cytoplasmic role for this essential viral regulatory protein.     

Nup53 is required for nuclear envelope and nuclear pore complex assembly

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53-Nup155 complex plays a critical role in the processes of NPC and NE assembly.     

Fat mass and obesity-related (FTO) shuttles between the nucleus and cytoplasm

SNPs (single nucleotide polymorphisms) on a chromosome 16 locus encompassing FTO, as well as IRX3, 5, 6, FTM and FTL are robustly associated with human obesity. FTO catalyses the Fe(II)- and 2OG-dependent demethylation of RNA and is an AA (amino acid) sensor that couples AA levels to mTORC1 (mammalian target of rapamycin complex 1) signalling, thereby playing a key role in regulating growth and translation. However, the cellular compartment in which FTO primarily resides to perform its biochemical role is unclear. Here, we undertake live cell imaging of GFP (green fluorescent protein)-FTO, and demonstrate that FTO resides in both the nucleus and cytoplasm. We show using ‘FLIP’ (fluorescence loss in photobleaching) that a mobile FTO fraction shuttles between both compartments. We performed a proteomic study and identified XPO2 (Exportin 2), one of a family of proteins that mediates the shuttling of proteins between the nucleus and the cytoplasm, as a binding partner of FTO. Finally, using deletion studies, we show that the N-terminus of FTO is required for its ability to shuttle between the nucleus and cytoplasm. In conclusion, FTO is present in both the nucleus and cytoplasm, with a mobile fraction that shuttles between both cellular compartments, possibly by interaction with XPO2.     

Genetic and physical interactions between Srp1p and nuclear pore complex proteins Nup1p and Nup2p

Nup1p is a yeast nuclear pore complex protein (nucleoporin) required for nuclear protein import, mRNA export and maintenance of normal nuclear architecture. We have used a genetic approach to identify other proteins that interact functionally with Nup1p. Here we describe the isolation of seventeen mutants that confer a requirement for Nup1p in a background in which this protein is normally not essential. Some of the mutants require wild-type Nup1p, while others are viable in combination with specific nup1 alleles. Several of the mutants show nonallelic noncomplementation, suggesting that the products may be part of a hetero-oligomeric complex. One is allelic to srp1 which, although it was identified in an unrelated screen, was shown to encode a protein that is localized to the nuclear envelope (Yano, R., M. Oakes, M. Yamaghishi, J. A. Dodd, and M. Nomura. 1992. Mol. Cell. Biol. 12:5640- 5651). We have used immunoprecipitation and fusion protein precipitation to show that Srp1p forms distinct complexes with both Nup1p and the related nucleoporin Nup2p, indicating that Srp1p is a component of the nuclear pore complex. The distant sequence similarity between Srp1p and the beta-catenin/desmoplakin family, coupled with the altered structure of the nuclear envelope in nup1 mutants, suggests that Srp1p may function in attachment of the nuclear pore complex to an underlying nuclear skeleton.     

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.     

Vertebrate Nup53 interacts with the nuclear lamina and is required for the assembly of a Nup93-containing complex

The nuclear pore complex (NPC) is an evolutionarily conserved structure that mediates exchange of macromolecules across the nuclear envelope (NE). It is comprised of approximately 30 proteins termed nucleoporins that are each present in multiple copies. We have investigated the function of the human nucleoporin Nup53, the ortholog of Saccharomyces cerevisiae Nup53p. Both cell fractionation and in vitro binding data suggest that Nup53 is tightly associated with the NE membrane and the lamina where it interacts with lamin B. We have also shown that Nup53 is capable of physically interacting with a group of nucleoporins including Nup93, Nup155, and Nup205. Consistent with this observation, depletion of Nup53 using small interfering RNAs causes a decrease in the cellular levels of these nucleoporins as well as the spindle checkpoint protein Mad1, likely due to destabilization of Nup53-containing complexes. The cellular depletion of this group of nucleoporins, induced by depleting either Nup53 or Nup93, severely alters nuclear morphology producing phenotypes similar to that previously observed in cells depleted of lamin A and Mad1. On basis of these data, we propose a model in which Nup53 is positioned near the pore membrane and the lamina where it anchors an NPC subcomplex containing Nup93, Nup155, and Nup205.     

The La antigen shuttles between the nucleus and the cytoplasm in CV-1 cells

Summary Recently we established a monoclonal antibody against the La-protein (Bachmannet al., Proc. Natl. Acad. Sci. USA, 83, 7770, 1986). The antibody gives a nuclear speckled type staining and, in addition, a perinuclear cytoplasmic staining on cultured cells in immunofluorescence microscopy. After inhibition of RNA synthesis the La-protein is transported into the cytoplasm. After prolonged inhibition it returns into the nucleus forming large growing speckles. The transport into the nucleus apparently depends on glycosylation.Abbreviations FITC Fluorescein Isothiocyanate - RITC Rhodamine Isothiocyanate - scRNP small cytoplasmic Ribonucleoprotein - snRNP Small nuclear Ribonucleoprotein - mab Monoclonal antibody - Ig Immunoglobulin - BSA Bovine Serum Albumin - PBS Phosphate Buffered Saline - PMSF Phenylmethanesulfonyl Fluoride - SDS Sodium Dodecyl Sulfate - EGTA Ethyleneglycol bis(-aminoethyl ether)-N,N,N,N-tetraacetic Acid - MCTD Mixed Connective Tissue Disease - SLE Systemic Lupus Erythematosus     

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.     

The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.     

Herpes simplex virus ICP27 protein directly interacts with the nuclear pore complex through Nup62, inhibiting host nucleocytoplasmic transport pathways

The herpes simplex virus ICP27 protein is important for the expression and nuclear export of viral mRNAs. Although several binding sites have been mapped along the ICP27 sequence for various RNA and protein partners, including the transport receptor TAP of the host cell nuclear transport machinery, several aspects of ICP27 trafficking through the nuclear pore complex remain unclear. We investigated if ICP27 could interact directly with the nuclear pore complex itself, finding that ICP27 directly binds the core nucleoporin Nup62. This is confirmed through co-immunoprecipitation and in vitro binding assays with purified components. Mapping with ICP27 deletion and point mutants further shows that the interaction requires sequences in both the N and C termini of ICP27. Expression of wild type ICP27 protein inhibited both classical, importin α/β-dependent and transportin-dependent nuclear import. In contrast, an ICP27 point mutant that does not interact with Nup62 had no such inhibitory effect. We suggest that ICP27 association with Nup62 provides additional binding sites at the nuclear pore for ICP27 shuttling, thus supporting ICP27-mediated transport. We propose that ICP27 competes with some host cell transport receptors for binding, resulting in inhibition of those host transport pathways.     

Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs.     

Stat5B shuttles between cytoplasm and nucleus in a cytokine-dependent and -independent manner

In response to cytokine stimuli, Stats are phosphorylated and translocated to the nucleus to activate target genes. Then, most are dephosphorylated and returned to the cytoplasm. Using Ba/F3 cells, we found that the nuclear export of Stat5B by cytokine depletion was inhibited by leptomycin B (LMB), a specific inhibitor of nuclear export receptor chromosome region maintenance 1. Interestingly, LMB treatment in the absence of cytokine led to the accumulation of Stat5B in the nucleus, suggesting that Stat5B shuttles between the nucleus and the cytoplasm as a monomer without cytokine stimulation. This notion is supported by the observation that LMB-induced accumulation of Stat5B in the nucleus was also observed with Stat5B having a mutated tyrosine 699, which is essential for dimer formation. Using a series of mutant Stat5Bs, we identified a part of the coiled coil domain to be a critical region for monomer nuclear import and a more N-terminal region to be critical for the cytokine stimulation dependent import of Stat5B. Taken together, we propose a model in which Stat5B shuttles between the nucleus and cytoplasm by two different mechanisms, one being a factor-independent constitutive shuttling by monomeric form, and the other, a factor stimulation-dependent one regulated by tyrosine phosphorylation and subsequent dimerization.     

Domain-specific antibodies reveal multiple-site topology of Nup153 within the nuclear pore complex

Nup153, one of the best characterized nuclear pore complex proteins (nucleoporins), plays a critical role in the import of proteins into the nucleus as well as in the export of RNAs and proteins from the nucleus. Initially an epitope of Nup153 was found to reside at the distal ring of the NPC, whereas more recently another epitope was localized to the nuclear ring moiety of the NPC. In an effort to more definitively determine the location of Nup153 within the 3-D architecture of the NPC we have generated domain-specific antibodies against distinct domains of Xenopus Nup153. With this approach we have found that the N-terminal domain is exposed at the nuclear ring of the NPC, whereas the zinc-finger domain of Nup153 is exposed at the distal ring of the NPC. In contrast, the C-terminal domain of Nup153 is not restricted to one particular subdomain of the NPC but rather appears to be highly flexible. Exogenous epitope-tagged hNup153 incorporated into Xenopus oocyte NPCs further underscored these findings. Our data illustrate that multiple domain-specific antibodies are essential to understanding the topology of a nucleoporin within the context of the NPC. Moreover, this approach has revealed new clues to the mechanisms by which Nup153 may contribute to nucleocytoplasmic transport.