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Frauke Melchior - One of the best experts on this subject based on the ideXlab platform.
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The Ran GTPase-Activating Protein (RanGAP1) Is Critically Involved in Smooth Muscle Cell Differentiation, Proliferation and Migration following Vascular Injury: Implications for Neointima Formation and Restenosis
2016Co-Authors: Marc Vorpahl, Cornelia Michaelis, Annette Flotho, Frauke MelchiorAbstract:Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50 % induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction o
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sumoylation of the gtpase ran by the ranbp2 sumo e3 ligase complex
Journal of Biological Chemistry, 2015Co-Authors: Volkan Sakin, Sebastian M Richter, Hehsuan Hsiao, Henning Urlaub, Frauke MelchiorAbstract:The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2's E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP's affinity to RanBP2's RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2's RBDs, which is prevented by the transport factor NTF2.
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the ran gtpase activating protein RanGAP1 is critically involved in smooth muscle cell differentiation proliferation and migration following vascular injury implications for neointima formation and restenosis
PLOS ONE, 2014Co-Authors: Marc Vorpahl, Frauke Melchior, Cornelia Michaelis, Annette Flotho, Sabine Schonhofermerl, Rainer WesselyAbstract:Differentiation and dedifferentiation, accompanied by proliferation play a pivotal role for the phenotypic development of vascular proliferative diseases (VPD), such as restenosis. Increasing evidence points to an essential role of regulated nucleoporin expression in the choice between differentiation and proliferation. However, whether components of the Ran GTPase cycle, which is of pivotal importance for both nucleocytoplasmic transport and for mitotic progression, are subject to similar regulation in VPD is currently unknown. Here, we show that differentiation of human coronary artery smooth muscle cell (CASMC) to a contractile phenotype by stepwise serum depletion leads to significant reduction of RanGAP1 protein levels. The inverse event, dedifferentiation of cells, was assessed in the rat carotid artery balloon injury model, a well-accepted model for neointima formation and restenosis. As revealed by temporospatial analysis of RanGAP1 expression, neointima formation in rat carotid arteries was associated with a significant upregulation of RanGAP1 expression at 3 and 7 days after balloon injury. Of note, neointimal cells located at the luminal surface revealed persistent RanGAP1 expression, as opposed to cells in deeper layers of the neointima where RanGAP1 expression was less or not detectable at all. To gain first evidence for a direct influence of RanGAP1 levels on differentiation, we reduced RanGAP1 in human coronary artery smooth muscle cells by siRNA. Indeed, downregulation of the essential RanGAP1 protein by 50% induced a differentiated, spindle-like smooth muscle cell phenotype, accompanied by an upregulation of the differentiation marker desmin. Reduction of RanGAP1 levels also resulted in a reduction of mitogen induced cellular migration and proliferation as well as a significant upregulation of the cyclin-dependent kinase inhibitor p27KIP1, without evidence for cellular necrosis. These findings suggest that RanGAP1 plays a critical role in smooth muscle cell differentiation, migration and proliferation in vitro and in vivo. Appropriate modulation of RanGAP1 expression may thus be a strategy to modulate VPD development such as restenosis.
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Downregulation of RanGAP1 in arrested, differentiated smooth muscle cells (A).
2014Co-Authors: Marc Vorpahl, Frauke Melchior, Sabine Schönhofer-merl, Cornelia Michaelis, Annette Flotho, Rainer WesselyAbstract:To induce differentiation, CASMCs were depleted from serum. RanGAP1 expression in CASMC was assessed by Western Blotting 12(∼70 kDa) form as well as the SUMO-1 conjugated (∼90 kDa) form of the RanGAP-1 protein. Both bands revealed reduced RanGAP1 expression over time in cells entering quiescence. α-SM Actin protein, a marker of cell differentiation, is increased over time. Actin levels are displayed as loading control. Effect of posttranscriptional gene silencing of RanGAP1 by small interfering RNA (siRNA) on cell cycle and differentiation markers (B). siRNA mediated gene silencing of RanGAP1 was able to reduce the 90 kD band by 31.8±21.2% (90 kD band) and 75%±14.7% (70 kD band) 48 h post transfection, respectively (average of three different experiments). RanGAP1 depletion was associated with a strong increase of p27Kip1 expression by 60±34%. RanGAP1 deficiency was also associated with a sharp increase in desmin expression with levels even higher than in quiescent cells. CASMC denotes coronary artery smooth muscle cells; (+) denotes serum stimulated CASMC; (−) denotes quiescent CASMC (≥72 h serum depletion); “control” denotes oligofectamine transfected cells without siRNAs; siRNA-SCR denotes scrambled (control) siRNA.
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RanGAP1 expression in the rat carotid artery injury model.
2014Co-Authors: Marc Vorpahl, Frauke Melchior, Sabine Schönhofer-merl, Cornelia Michaelis, Annette Flotho, Rainer WesselyAbstract:To determine the spatiotemporal expression pattern of RanGAP1 during the course of neointima formation, the rat carotid injury model was applied. Immunohistochemical staining revealed upregulation of RanGAP1 at day 3 (b, f, j, and day 7 (c, g, k) whereas RanGAP1 expression ceased when SMC proliferation decreases at day 14 (d, h, l) subsequent to balloon injury.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
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the karyopherin kap122p pdr6p imports both subunits of the transcription factor iia into the nucleus
Journal of Cell Biology, 1999Co-Authors: Anton A. Titov, Gunter BlobelAbstract:We discovered a nuclear import pathway mediated by the product of the previously identified Saccharomyces cerevisiae gene PDR6 (pleiotropic drug resistance). This gene product functions as a karyopherin (Kap) for nuclear import. Consistent with previously proposed nomenclature, we have renamed this gene KAP122. Kap122p was localized both to the cytoplasm and the nucleus. As a prominent import substrate of Kap122p, we identified the complex of the large and small subunit (Toa1p and Toa2p, respectively) of the general transcription factor IIA (TFIIA). Recombinant GST-Kap122p formed a complex with recombinant His6-Toa1p/Toa2p. In wild-type cells, Toa1p and Toa2p were localized to the nucleus. Consistent with Kap122p being the principal Kap for import of the Toa1p–Toa2p complex, we found that deletion of KAP122 results in increased cytoplasmic localization of both Toa1p and Toa2p. Deletion of KAP122 is not lethal, although deletion of TOA1 and TOA2 is. Together these data suggest that Kap122p is the major Kap for the import of Toa1p–Toa2p into the nucleus. Like other substrate–Kap complexes, the Toa1p/Toa2p/Kap122p complex isolated from yeast cytosol or reconstituted from recombinant proteins, was dissociated by RanGTP but not RanGDP. Kap122p bound to nucleoporins, specifically, to the peptide repeat–containing fragments of Nup1p and Nup2p.
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sumo 1 modification and its role in targeting the ran gtpase activating protein RanGAP1 to the nuclear pore complex
Journal of Cell Biology, 1998Co-Authors: Michael J Matunis, Gunter BlobelAbstract:RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.
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Disassembly of RanGTP-karyopherin beta complex, an intermediate in nuclear protein import.
Journal of Biological Chemistry, 1997Co-Authors: Monique Floer, Gunter Blobel, Michael RexachAbstract:We previously showed that RanGTP forms a 1:1 complex with karyopherin beta that renders RanGTP inaccessible to RanGAP (Floer, M., and Blobel, G. (1996) J. Biol. Chem. 271, 5313-5316) and karyopherin beta functionally inactive (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). Recycling of both factors for another round of function requires dissociation of the RanGTP-karyopherin beta complex. Here we show using BIAcoreTM, a solution binding assay, and GTP hydrolysis and exchange assays, with yeast proteins, that karyopherin beta and RanGTP are recycled efficiently in a reaction that involves karyopherin alpha, RanBP1, RanGAP, and the C terminus of the nucleoporin Nup1. We find that karyopherin alpha first releases RanGTP from karyopherin beta in a reaction that does not require GTP hydrolysis. The released RanGTP is then sequestered by RanBP1, and the newly formed karyopherin alphabeta binds to the C terminus of Nup1. Finally, RanGTP is converted to RanGDP via nucleotide hydrolysis when RanGAP is present. Conversion of RanGTP to RanGDP can also occur via nucleotide exchange in the presence of RanGEF, an excess of GDP, and if RanBP1 is absent. Additional nucleoporin domains that bind karyopherin alphabeta stimulate recycling of karyopherin beta and Ran in a manner similar to the C terminus of Nup1.
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a novel ubiquitin like modification modulates the partitioning of the ran gtpase activating protein RanGAP1 between the cytosol and the nuclear pore complex
Journal of Cell Biology, 1996Co-Authors: Michael J Matunis, Elias Coutavas, Gunter BlobelAbstract:Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribnucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70-kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein. Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified from of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associated with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.
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The Nuclear Transport Factor Karyopherin Binds Stoichiometrically to Ran-GTP and Inhibits the Ran GTPase Activating Protein
Journal of Biological Chemistry, 1996Co-Authors: Monique Floer, Gunter BlobelAbstract:Abstract The heterodimeric karyopherin functions in targeting a nuclear localization sequence (NLS)-containing protein to the nuclear pore complex followed by Ran-GTP and p10-mediated translocation of the NLS protein into the nucleoplasm. It was shown recently that Ran-GTP dissociated the karyopherin heterodimer and, in doing so, associated with karyopherin β (Rexach, M., and Blobel, G.(1995) Cell 83, 683-692). We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin β binds to Ran-GTP and inhibits GTP hydrolysis stimulated by RanGAP (the Ran-specific GTPase activating protein). Inhibition of RanGAP-stimulated GTP hydrolysis by karyopherin β was dependent on karyopherin β concentration relative to Ran-GTP. Complete inhibition of RanGAP was observed at karyopherin β concentrations that were equimolar to Ran-GTP. In gel filtration experiments, we found Ran-GTP and karyopherin β to form a stoichiometric complex. Ran-GDP bound only weakly to karyopherin β. We propose that stoichiometric complex formation between karyopherin β and Ran-GTP renders Ran-GTP inaccessible to RanGAP.
Rohit Mahajan - One of the best experts on this subject based on the ideXlab platform.
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modification of ran gtpase activating protein by the small ubiquitin related modifier sumo 1 requires ubc9 an e2 type ubiquitin conjugating enzyme homologue
Journal of Biological Chemistry, 1998Co-Authors: Frauke Melchior, Rohit Mahajan, Michael J Matunis, Qingsheng Tian, Paul AndersonAbstract:Abstract Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). InXenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.
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modification of ran gtpase activating protein by the small ubiquitin related modifier sumo 1 requires ubc9 an e2 type ubiquitin conjugating enzyme homologue
Journal of Biological Chemistry, 1998Co-Authors: Gene W Lee, Rohit Mahajan, Frauke Melchior, Michael J Matunis, Qingsheng Tian, Paul AndersonAbstract:Abstract Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). InXenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.
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molecular characterization of the sumo 1 modification of RanGAP1 and its role in nuclear envelope association
Journal of Cell Biology, 1998Co-Authors: Rohit Mahajan, Larry Gerace, Frauke MelchiorAbstract:The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101– amino acid protein are proteolytically removed before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope (NE), where it was previously shown to be required for nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.
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a small ubiquitin related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein ranbp2
Cell, 1997Co-Authors: Rohit Mahajan, Larry Gerace, Christian Delphin, Tinglu Guan, Frauke MelchiorAbstract:We have found that the mammalian Ran GTPase-activating protein RanGAP1 is highly concentrated at the cytoplasmic periphery of the nuclear pore complex (NPC), where it associates with the 358-kDa Ran-GTP-binding protein RanBP2. This interaction requires the ATP-dependent posttranslational conjugation of RanGAP1 with SUMO-1 (for small ubiquitin-related modifier), a novel protein of 101 amino acids that contains low but significant homology to ubiquitin. SUMO-1 appears to represent the prototype for a novel family of ubiquitin-related protein modifiers. Inhibition of nuclear protein import resulting from antibodies directed at NPC-associated RanGAP1 cannot be overcome by soluble cytosolic RanGAP1, indicating that GTP hydrolysis by Ran at RanBP2 is required for nuclear protein import.
Michael J Matunis - One of the best experts on this subject based on the ideXlab platform.
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modification of ran gtpase activating protein by the small ubiquitin related modifier sumo 1 requires ubc9 an e2 type ubiquitin conjugating enzyme homologue
Journal of Biological Chemistry, 1998Co-Authors: Frauke Melchior, Rohit Mahajan, Michael J Matunis, Qingsheng Tian, Paul AndersonAbstract:Abstract Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). InXenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.
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modification of ran gtpase activating protein by the small ubiquitin related modifier sumo 1 requires ubc9 an e2 type ubiquitin conjugating enzyme homologue
Journal of Biological Chemistry, 1998Co-Authors: Gene W Lee, Rohit Mahajan, Frauke Melchior, Michael J Matunis, Qingsheng Tian, Paul AndersonAbstract:Abstract Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1, 2). InXenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.
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sumo 1 modification and its role in targeting the ran gtpase activating protein RanGAP1 to the nuclear pore complex
Journal of Cell Biology, 1998Co-Authors: Michael J Matunis, Gunter BlobelAbstract:RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.
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a novel ubiquitin like modification modulates the partitioning of the ran gtpase activating protein RanGAP1 between the cytosol and the nuclear pore complex
Journal of Cell Biology, 1996Co-Authors: Michael J Matunis, Elias Coutavas, Gunter BlobelAbstract:Ran is a nuclear Ras-like GTPase that is required for the bidirectional transport of proteins and ribnucleoproteins across the nuclear pore complex (NPC). A key regulator of the Ran GTP/GDP cycle is the 70-kD Ran-GTPase-activating protein RanGAP1. Here, we report the identification and localization of a novel form of RanGAP1. Using peptide sequence analysis and specific mAbs, RanGAP1 was found to be modified by conjugation to a ubiquitin-like protein. Immunoblot analysis and immunolocalization by light and EM demonstrated that the 70-kD unmodified from of RanGAP1 is exclusively cytoplasmic, whereas the 90-kD modified form of RanGAP1 is associated with the cytoplasmic fibers of the NPC. The modified form of RanGAP1 also appeared to associated with the mitotic spindle apparatus during mitosis. These findings have specific implications for Ran function and broad implications for protein regulation by ubiquitin-like modifications. Moreover, the variety and function of ubiquitin-like protein modifications in the cell may be more diverse than previously realized.
Dirk Gorlich - One of the best experts on this subject based on the ideXlab platform.
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ntf2 mediates nuclear import of ran
The EMBO Journal, 1998Co-Authors: Katharina Ribbeck, Murray Stewart, Gerd Lipowsky, Helen M Kent, Dirk GorlichAbstract:Importin β 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 β family transport receptor.
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ranbp1 is crucial for the release of rangtp from importin β related nuclear transport factors
FEBS Letters, 1997Co-Authors: Ralf F Bischoff, Dirk GorlichAbstract:Nucleocytoplasmic transport appears mediated by shuttling transport receptors that bind RanGTP as a means to regulate interactions with their cargoes. The receptor·RanGTP complexes are kinetically very stable with nucleotide exchange and GTP hydrolysis being blocked, predicting that a specific disassembly mechanism exists. Here we show in three cases receptor·RanGTP·RanBP1 complexes to be the key disassembly intermediates, where RanBP1 stimulates the off-rate at the receptor/RanGTP interface by more than two orders of magnitude. The transiently released RanGTP·RanBP1 complex is then induced by RanGAP to hydrolyse GTP, preventing the receptor to rebind RanGTP. The efficient release of importin β from RanGTP requires importin α, in addition to RanBP1.
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the asymmetric distribution of the constituents of the ran system is essential for transport into and out of the nucleus
The EMBO Journal, 1997Co-Authors: Elisa Izaurralde, Ulrike Kutay, Iain W Mattaj, Cayetano Von Kobbe, Dirk GorlichAbstract:The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.
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export of importin α from the nucleus is mediated by a specific nuclear transport factor
Cell, 1997Co-Authors: Ulrike Kutay, F R Bischoff, Susanne Kostka, Regine Kraft, Dirk GorlichAbstract:Abstract NLS proteins are transported into the nucleus by the importin α/β heterodimer. Importin α binds the NLS, while importin β mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin β and displaces importin α. Importin α must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin α re-export. CAS binds strongly to importin α only in the presence of RanGTP, forming an importin α/CAS/RanGTP complex. Importin α is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin α, explaining why import substrates stay in the nucleus.
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ran binding protein 5 ranbp5 is related to the nuclear transport factor importin beta but interacts differently with ranbp1
Molecular and Cellular Biology, 1997Co-Authors: R Deane, Dirk Gorlich, W Schafer, H P Zimmermann, L Mueller, Siegfried Prehn, Herwig Ponstingl, F R BischoffAbstract:We report the identification and characterization of a novel 124-kDa Ran binding protein, RanBP5. This protein is related to importin-beta, the key mediator of nuclear localization signal (NLS)-dependent nuclear transport. RanBP5 was identified by two independent methods: it was isolated from HeLa cells by using its interaction with RanGTP in an overlay assay to monitor enrichment, and it was also found by the yeast two-hybrid selection method with RanBP1 as bait. RanBP5 binds to RanBP1 as part of a trimeric RanBP1-Ran-RanBP5 complex. Like importin-beta, RanBP5 strongly binds the GTP-bound form of Ran, stabilizing it against both intrinsic and RanGAP1-induced GTP hydrolysis and also against nucleotide exchange. The GAP resistance of the RanBP5-RanGTP complex can be relieved by RanBP1, which might reflect an in vivo role for RanBP1. RanBP5 is a predominantly cytoplasmic protein that can bind to nuclear pore complexes. We propose that RanBP5 is a mediator of a nucleocytoplasmic transport pathway that is distinct from the importin-alpha-dependent import of proteins with a classical NLS.
