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Ralph H Kehlenbach - One of the best experts on this subject based on the ideXlab platform.
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the oncogenic fusion proteins set nup214 and sequestosome 1 sqstm1 nup214 form dynamic nuclear bodies and differentially affect nuclear protein and poly a rna export
Journal of Biological Chemistry, 2016Co-Authors: Sarah A Port, Birthe Fahrenkrog, Christiane Spillner, Adelia Mendes, Christina Valkova, Christoph Kaether, Ralph H KehlenbachAbstract:Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain Nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that Nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.
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several phenylalanine glycine motives in the nucleoporin nup214 are essential for binding of the nuclear export receptor crm1
Journal of Biological Chemistry, 2013Co-Authors: Stephanie Roloff, Christiane Spillner, Ralph H KehlenbachAbstract:Nucleoporins containing phenylalanine glycine (FG) repeats play an important role in nucleocytoplasmic transport as they bind to transport receptors and mediate translocation of transport complexes across the nuclear pore complex (NPC). Nup214/CAN, a nucleoporin that is found at the cytoplasmic side of the NPC, interacts with both import and export receptors. In functional assays, dominant-negative fragments of Nup214 inhibited CRM1-dependent nuclear export, as the export receptor became rate-limiting. Several nuclear import pathways, by contrast, were not affected by the Nup214 fragments. We now characterize the CRM1-binding region of Nup214 in detail and identify several FG motives that are required for this interaction. Our results support a model where CRM1, like other transport receptors, contacts FG-Nups via multiple binding sites.
Birthe Fahrenkrog - One of the best experts on this subject based on the ideXlab platform.
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Mitotic checkpoint protein Mad1 is required for early Nup153 recruitment to chromatin and nuclear envelope integrity.
Journal of cell science, 2020Co-Authors: Ikram Mossaid, Valérie Martinelli, Guillaume Chatel, Marcela Vaz, Birthe FahrenkrogAbstract:Nucleoporin Nup153 is a multifunctional protein and mitotic checkpoint protein Mad1 a known binding partner. The functional relevance of their interaction has remained elusive. Here, we have further dissected Nup1539s and Mad19s interface and functional interplay. By in situ proximity ligation assays, we found that the presence of a nuclear envelope (NE) is prerequisite for the Nup153-Mad1 association. Time-lapse microscopy revealed that depletion of Mad1 delayed recruitment of Nup153 to anaphase chromatin, which was often accompanied by a prolongation of anaphase. Furthermore, as seen by electron microscopic and three-dimensional structured illumination investigations, Nup153 and Mad1 depletion led to alterations in NE architecture, characterised by a change of membrane curvature at nuclear pore complexes (NPCs) and an expansion of the spacing between inner and outer nuclear membrane. Nup153 depletion, but not of Mad1, caused defects in interphase NPC assembly with partial displacement of cytoplasmic Nucleoporins and a reduction in NPC density. Together our results suggest that Nup153 has separable roles in NE and NPC formation: in post-mitotic NE reformation in concert with Mad1 and in interphase NPC assembly, independent of Mad1.
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the oncogenic fusion proteins set nup214 and sequestosome 1 sqstm1 nup214 form dynamic nuclear bodies and differentially affect nuclear protein and poly a rna export
Journal of Biological Chemistry, 2016Co-Authors: Sarah A Port, Birthe Fahrenkrog, Christiane Spillner, Adelia Mendes, Christina Valkova, Christoph Kaether, Ralph H KehlenbachAbstract:Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain Nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that Nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.
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nucleoporin domain topology is linked to the transport status of the nuclear pore complex
Journal of Molecular Biology, 2005Co-Authors: Sara M Paulillo, Maureen A. Powers, Katharine S. Ullman, Erica M Phillips, Joachim Koser, Ursula Sauder, Birthe FahrenkrogAbstract:Nuclear pore complexes (NPCs) facilitate macromolecular exchange between the nucleus and cytoplasm of eukaryotic cells. The vertebrate NPC is composed of ∼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.
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comparative spatial localization of protein a tagged and authentic yeast nuclear pore complex proteins by immunogold electron microscopy
Journal of Structural Biology, 2000Co-Authors: Birthe Fahrenkrog, John P Aris, Eduard C Hurt, Nelly Pante, Ueli AebiAbstract:The nuclear pore complex (NPC) mediates protein and RNP import in and RNA and RNP export out of the nucleus of eukaryotic cells. Due to its genetic tractability, yeast offers a versatile system for investigating the chemical composition and molecular architecture of the NPC. In this context, protein A tagging is a commonly used tool for characterizing and localizing yeast NPC proteins (Nucleoporins). By preembedding anti-protein A immunogold electron microscopy (immunogold EM), we have localized two yeast Nucleoporins, Nsp1p and Nic96p, in mutant yeast strains recombinantly expressing these Nucleoporins tagged with four (Nsp1p) or two (Nic96p) IgG binding domains of protein A (i.e., ProtA-Nsp1p and ProtA-Nic96p). We have compared the location of the recombinant fusion proteins ProtA-Nsp1p and ProtA-Nic96p (i.e., as specified by their protein A tag) to the location of authentic Nsp1p and Nic96p (i.e., as defined by the epitopes recognized by corresponding nucleoporin antibodies) and found all of them to reside at the same three NPC sites. Hence, recombinant expression and protein A tagging of the Nucleoporins Nsp1p and Nic96p have not caused any significant mislocation of the fusion proteins and thus enabled mapping of these two yeast Nucleoporins at the ultrastructural level in a faithful manner.
Michael J Matunis - One of the best experts on this subject based on the ideXlab platform.
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JCB Article Proteomic analysis of the mammalian nuclear pore complex
2013Co-Authors: J. M. Cronshaw, Wenzhu Zhang, Brian T Chait, Andrew N Krutchinsky, Michael J MatunisAbstract:As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as Nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 Nucleoporins were six previously undiscovered Nucleoporins and a novel family of WD repeat Nucleoporins. One of these WD repeat Nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function
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proteomic analysis of the mammalian nuclear pore complex
Journal of Cell Biology, 2002Co-Authors: J. M. Cronshaw, Wenzhu Zhang, Brian T Chait, Andrew N Krutchinsky, Michael J MatunisAbstract:As the sole site of nucleocytoplasmic transport, the nuclear pore complex (NPC) has a vital cellular role. Nonetheless, much remains to be learned about many fundamental aspects of NPC function. To further understand the structure and function of the mammalian NPC, we have completed a proteomic analysis to identify and classify all of its protein components. We used mass spectrometry to identify all proteins present in a biochemically purified NPC fraction. Based on previous characterization, sequence homology, and subcellular localization, 29 of these proteins were classified as Nucleoporins, and a further 18 were classified as NPC-associated proteins. Among the 29 Nucleoporins were six previously undiscovered Nucleoporins and a novel family of WD repeat Nucleoporins. One of these WD repeat Nucleoporins is ALADIN, the gene mutated in triple-A (or Allgrove) syndrome. Our analysis defines the proteome of the mammalian NPC for the first time and paves the way for a more detailed characterization of NPC structure and function.
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a conserved biogenesis pathway for Nucleoporins proteolytic processing of a 186 kilodalton precursor generates nup98 and the novel nucleoporin nup96
Journal of Cell Biology, 1999Co-Authors: Beatriz M A Fontoura, Günter Blobel, Michael J MatunisAbstract:The mammalian nuclear pore complex (NPC) is comprised of ∼50 unique proteins, collectively known as Nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel Nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two Nucleoporins: Nup98, a previously characterized GLFG-repeat containing nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.
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nup358 a cytoplasmically exposed nucleoporin with peptide repeats ran gtp binding sites zinc fingers a cyclophilin a homologous domain and a leucine rich region
Journal of Biological Chemistry, 1995Co-Authors: Michael J Matunis, Günter Blobel, Doris Kraemer, Elias CoutavasAbstract:Abstract The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for Nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.
Ed Hurt - One of the best experts on this subject based on the ideXlab platform.
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analysis of the yeast nucleoporin nup188 reveals a conserved s like structure with similarity to karyopherins
Journal of Structural Biology, 2012Co-Authors: Dirk Flemming, Stefan Amlacher, Johannes Schwarz, Malik Lutzmann, Damien P Devos, Ed HurtAbstract:Nuclear pore complexes (NPCs) embedded in the double nuclear membrane mediate the entire nucleocytoplasmic transport between the nucleus and cytoplasm. Each NPC is composed of about 30 different proteins (Nucleoporins or Nups), which exist in multiple (8, 16 or 32) copies within the NPC scaffold. Recently, we have identified and characterized the large structural Nups, Nup188 and Nup192, from the thermophilic eukaryote Chaetomium thermophilum, which exhibited superior properties for biochemical and structural studies, when compared to their mesophilic orthologs. Here, we study the large structural Nups from the model organism yeast Saccharomyces cerevisiae. Our data show that yeast Nup188 like its thermophilic orthologue ctNup188 exhibits a twisted S-like structure, which flexibly binds the linker nucleoporin Nic96 via a short conserved α-helix motif. Using bioinformatic methods, we have generated a pseudo-atomic structural model of Nup188 and its related Nup192, which further strengthens the view that the large α-solenoid structural Nups are related to karyopherins.
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the mrna export machinery requires the novel sac3p thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores
The EMBO Journal, 2002Co-Authors: Tamas Fischer, Katja Straser, Attila Racz, Susana Rodrigueznavarro, Marisa Oppizzi, Petra Ihrig, Johannes Lechner, Ed HurtAbstract:Yra1p and Sub2p are components of the TREX complex, which couples transcription elongation with nuclear export of mRNAs. Here, we report a genetic interaction between Yra1p and a conserved protein Sac3p, which previously was found to interact with Sub2p. In vivo, Sac3p forms a stable complex with Thp1p, which was reported to function in transcription elongation. In addition, Sac3p binds to the mRNA exporter Mex67p–Mtr2p and requires the nucleoporin Nup1p to dock at the nuclear side of the nuclear pore complex (NPC). Significantly, mutations in Sac3p or Thp1p lead to strong mRNA export defects. Taken together, our data suggest that the novel Sac3p–Thp1p complex functions by docking the mRNP to specific Nucleoporins at the nuclear entrance of the NPC.
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modular self assembly of a y shaped multiprotein complex from seven Nucleoporins
The EMBO Journal, 2002Co-Authors: Malik Lutzmann, Ruth Kunze, Andrea Buerer, Ueli Aebi, Ed HurtAbstract:Now that it is likely that all yeast Nucleoporins are known, one of the ultimate goals is the in vitro assembly of the entire nuclear pore complex from its ∼30 individual components. Here, we report the reconstitution of seven proteins (Nup133p, Nup145p-C, Nup120p, Nup85p, Nup84p, Seh1p and Sec13p) into a heptameric 0.5 MDa nuclear pore subcomplex. We found that double plasmid transformation combined with bi-cistronic mRNA translation allow the expression and assembly of distinct subcomplexes of up to five Nucleoporins in a single Escherichia coli cell. During the sequential reconstitution of the Nup84p complex, smaller assembly intermediates can be isolated, which exhibit modular structures determined by electron microscopy that finally make up the whole Y-shaped Nup84p complex. Importantly, a seventh subunit, Nup133p, was incorporated into the complex through its interaction with Nup84p, thereby elongating one arm of the Y-shaped assembly to an ∼40 nm long stalk. Taken together, our data document that the Nup84p–Nup133p complex self-assembles in a modular concept from distinct smaller nucleoporin construction sets.
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a novel in vivo assay reveals inhibition of ribosomal nuclear export in ran cycle and nucleoporin mutants
Journal of Cell Biology, 1999Co-Authors: Ed Hurt, David Tollervey, Stefan Hannus, Birgit Schmelzl, Denise Lau, George SimosAbstract:To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct Nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.
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mex67p a novel factor for nuclear mrna export binds to both poly a rna and nuclear pores
The EMBO Journal, 1997Co-Authors: Alexandra Segref, Kishore Sharma, Valerie Doye, Andrea Hellwig, Jochen Huber, Reinhard Luhrmann, Ed HurtAbstract:An essential cellular factor for nuclear mRNA export called Mex67p which has homologous proteins in human and Caenorhabditis elegans was identified through its genetic interaction with nucleoporin Nup85p. In the thermosensitive mex67-5 mutant, poly(A)+ RNA accumulates in intranuclear foci shortly after shift to the restrictive temperature, but NLS-mediated nuclear protein import is not inhibited. In vivo, Mex67p tagged with green fluorescent protein (GFP) is found at the nuclear pores, but mutant mex67-5-GFP accumulates in the cytoplasm. Upon purification of poly(A)+ RNA derived from of UV-irradiated yeast cells, Mex67p, but not Nucleoporins Nup85p and Nup57p, was crosslinked to mRNA. In a two-hybrid screen, a putative RNA-binding protein with RNP consensus motifs was found to interact with the Mex67p carboxy-terminal domain. Thus, Mex67p is likely to participate directly in the export of mRNA from the nucleus to the cytoplasm.
Christiane Spillner - One of the best experts on this subject based on the ideXlab platform.
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the oncogenic fusion proteins set nup214 and sequestosome 1 sqstm1 nup214 form dynamic nuclear bodies and differentially affect nuclear protein and poly a rna export
Journal of Biological Chemistry, 2016Co-Authors: Sarah A Port, Birthe Fahrenkrog, Christiane Spillner, Adelia Mendes, Christina Valkova, Christoph Kaether, Ralph H KehlenbachAbstract:Genetic rearrangements are a hallmark of several forms of leukemia and can lead to oncogenic fusion proteins. One example of an affected chromosomal region is the gene coding for Nup214, a nucleoporin that localizes to the cytoplasmic side of the nuclear pore complex (NPC). We investigated two such fusion proteins, SET-Nup214 and SQSTM1 (sequestosome)-Nup214, both containing C-terminal portions of Nup214. SET-Nup214 nuclear bodies containing the nuclear export receptor CRM1 were observed in the leukemia cell lines LOUCY and MEGAL. Overexpression of SET-Nup214 in HeLa cells leads to the formation of similar nuclear bodies that recruit CRM1, export cargo proteins, and certain Nucleoporins and concomitantly affect nuclear protein and poly(A)+ RNA export. SQSTM1-Nup214, although mostly cytoplasmic, also forms nuclear bodies and inhibits nuclear protein but not poly(A)+ RNA export. The interaction of the fusion proteins with CRM1 is RanGTP-dependent, as shown in co-immunoprecipitation experiments and binding assays. Further analysis revealed that the Nup214 parts mediate the inhibition of nuclear export, whereas the SET or SQSTM1 part determines the localization of the fusion protein and therefore the extent of the effect. SET-Nup214 nuclear bodies are highly mobile structures, which are in equilibrium with the nucleoplasm in interphase and disassemble during mitosis or upon treatment of cells with the CRM1-inhibitor leptomycin B. Strikingly, we found that Nucleoporins can be released from nuclear bodies and reintegrated into existing NPC. Our results point to nuclear bodies as a means of preventing the formation of potentially insoluble and harmful protein aggregates that also may serve as storage compartments for nuclear transport factors.
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several phenylalanine glycine motives in the nucleoporin nup214 are essential for binding of the nuclear export receptor crm1
Journal of Biological Chemistry, 2013Co-Authors: Stephanie Roloff, Christiane Spillner, Ralph H KehlenbachAbstract:Nucleoporins containing phenylalanine glycine (FG) repeats play an important role in nucleocytoplasmic transport as they bind to transport receptors and mediate translocation of transport complexes across the nuclear pore complex (NPC). Nup214/CAN, a nucleoporin that is found at the cytoplasmic side of the NPC, interacts with both import and export receptors. In functional assays, dominant-negative fragments of Nup214 inhibited CRM1-dependent nuclear export, as the export receptor became rate-limiting. Several nuclear import pathways, by contrast, were not affected by the Nup214 fragments. We now characterize the CRM1-binding region of Nup214 in detail and identify several FG motives that are required for this interaction. Our results support a model where CRM1, like other transport receptors, contacts FG-Nups via multiple binding sites.
