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Aurelian Radu - One of the best experts on this subject based on the ideXlab platform.
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Nuclear protein import: Ran-GTP dissociates the Karyopherin alphaBeta heterodimer by displacing alpha from an overlapping binding site on Beta
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract The alpha subunit of the Karyopherin heterodimer functions in recognition of the protein import substrate and the Beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the Karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of Karyopherin Beta's binding sites both for Ran-GTP and for Karyopherin alpha. We discovered that Karyopherin Beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and Karyopherin alpha bind to overlapping sites on Karyopherin Beta. Having a higher affinity to the overlapping site, Ran-GTP displaces Karyopherin alpha and binds to Karyopherin Beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for Karyopherin Beta to a cluster of basic residues analogous to those previously shown to constitute Karyopherin alpha's binding site to Karyopherin Beta.
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The binding site of Karyopherin alpha for Karyopherin Beta overlaps with a nuclear localization sequence
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of Karyopherin alpha. We found that the C-terminal region of Karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds Karyopherin Beta. Surprisingly, Karyopherin alpha also contains an NLS. Thus, Karyopherin alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of Karyopherin alpha overlaps with the binding site of Karyopherin alpha for Karyopherin Beta. Hence, binding of Karyopherin Beta to Karyopherin alpha covers the NLS of Karyopherin alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of Karyopherin alpha to a heterologous NLS protein in the presence of Karyopherin Beta.
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Mammalian Karyopherin alpha 1 Beta and alpha 2 Beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and Beta subunit interacts with peptide repeat-containing nucleoporins.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Junona Moroianu, Gunter Blobel, Makoto Hijikata, Aurelian RaduAbstract:Although only 44% identical to human Karyopherin alpha 1, human Karyopherin alpha 2 (Rch1 protein) substituted for human Karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and Karyopherin Beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, Karyopherin Beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous Karyopherin Beta. Recombinant Karyopherin Beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant Karyopherin alpha 1 or alpha 2 did not bind unless Karyopherin Beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (Karyopherin alpha 1 or alpha 2, Karyopherin Beta, Ran, and p10) import depended on Karyopherin Beta. Localization of the exogenously added transport factors after a 30-min import reaction showed Karyopherin Beta at the nuclear envelope and Karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled Karyopherin Beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.
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previously identified protein of uncertain function is Karyopherin alpha and together with Karyopherin Beta docks import substrate at nuclear pore complexes
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract Previously, we had purified a cytosolic protein complex, termed Karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa Beta subunit. We now report that the Karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant Karyopherin alpha or Beta subunit, we showed that neither Karyopherin alpha nor Karyopherin Beta alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant Karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized Karyopherin alpha and Beta subunits and import substrate as a ligand, we found that only Karyopherin alpha bound ligand. We suggest that Karyopherin Beta functions as an adaptor that binds both to Karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of Karyopherin alpha-substrate across the NPC.
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identification of a protein complex that is required for nuclear protein import and mediates docking of import substrate to distinct nucleoporins
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Aurelian Radu, Gunter Blobel, Mary Shannon MooreAbstract:Abstract We have identified and characterized a 9S protein complex from a Xenopus ovary cytosolic subfraction (fraction A) that constitutes this fraction's activity in recognizing a model nuclear import substrate and docking it at the nuclear pore complex. Because of its function, the complex is termed Karyopherin. The 54- and 56-kDa subunits of the complex are termed alpha 1 and alpha 2, respectively, and the 97-kDa subunit is termed Beta. In an alternative approach we have identified Karyopherin Beta from a rat liver cytosolic subfraction A by using immobilized rat nucleoporin Nup98 in a single, affinity-based enrichment step. We have molecularly cloned and sequenced rat Karyopherin Beta. Comparison with protein sequence data banks showed no significant similarity to other known proteins. Using nitrocellulose-immobilized rat liver nuclear envelope proteins and nuclear import substrate as a ligand, we found Xenopus fraction A-dependent binding to at least three bona fide nucleoporins (Nup214, Nup153, and Nup98) and to a candidate nucleoporin with an estimated molecular mass of 270 kDa. We propose that these nucleoporins function as docking proteins for Karyopherin-mediated binding of substrate in a nuclear import/export pathway across the nuclear pore complex.
Gunter Blobel - One of the best experts on this subject based on the ideXlab platform.
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structure of the nuclear transport complex Karyopherin Beta2 ran x gppnhp
Nature, 1999Co-Authors: Yuh Min Chook, Gunter BlobelAbstract:Transport factors in the Karyopherin-Beta (also called importin-Beta) family mediate the movement of macromolecules in nuclear-cytoplasmic transport pathways. Karyopherin-Beta2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran x GTP binds Karyopherin-Beta2 and dissociates the substrate. Here we present the 3.0 A structure of the Karyopherin-Beta2-Ran x GppNHp complex where GppNHp is a non-hydrolysable GTP analogue. Karyopherin-Beta2 contains eighteen HEAT repeats arranged into two continuous orthogonal arches. Ran is clamped in the amino-terminal arch and substrate-binding activity is mapped to the carboxy-terminal arch. A large loop in HEAT repeat 7 spans both arches. Interactions of the loop with Ran and the C-terminal arch implicate it in GTPase-mediated dissociation of the import-substrate. Ran x GppNHp in the complex shows extensive structural rearrangement, compared to Ran GDP, in regions contacting Karyopherin-Beta2. This provides a structural basis for the specificity of the Karyopherin-Beta family for the GTP-bound state of Ran, as well as a rationale for interactions of the Karyopherin-Ran complex with the regulatory proteins ranGAP, ranGEF and ranBP1.
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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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Nuclear protein import: Ran-GTP dissociates the Karyopherin alphaBeta heterodimer by displacing alpha from an overlapping binding site on Beta
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract The alpha subunit of the Karyopherin heterodimer functions in recognition of the protein import substrate and the Beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the Karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of Karyopherin Beta's binding sites both for Ran-GTP and for Karyopherin alpha. We discovered that Karyopherin Beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and Karyopherin alpha bind to overlapping sites on Karyopherin Beta. Having a higher affinity to the overlapping site, Ran-GTP displaces Karyopherin alpha and binds to Karyopherin Beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for Karyopherin Beta to a cluster of basic residues analogous to those previously shown to constitute Karyopherin alpha's binding site to Karyopherin Beta.
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The binding site of Karyopherin alpha for Karyopherin Beta overlaps with a nuclear localization sequence
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of Karyopherin alpha. We found that the C-terminal region of Karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds Karyopherin Beta. Surprisingly, Karyopherin alpha also contains an NLS. Thus, Karyopherin alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of Karyopherin alpha overlaps with the binding site of Karyopherin alpha for Karyopherin Beta. Hence, binding of Karyopherin Beta to Karyopherin alpha covers the NLS of Karyopherin alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of Karyopherin alpha to a heterologous NLS protein in the presence of Karyopherin Beta.
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Mammalian Karyopherin alpha 1 Beta and alpha 2 Beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and Beta subunit interacts with peptide repeat-containing nucleoporins.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Junona Moroianu, Gunter Blobel, Makoto Hijikata, Aurelian RaduAbstract:Although only 44% identical to human Karyopherin alpha 1, human Karyopherin alpha 2 (Rch1 protein) substituted for human Karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and Karyopherin Beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, Karyopherin Beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous Karyopherin Beta. Recombinant Karyopherin Beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant Karyopherin alpha 1 or alpha 2 did not bind unless Karyopherin Beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (Karyopherin alpha 1 or alpha 2, Karyopherin Beta, Ran, and p10) import depended on Karyopherin Beta. Localization of the exogenously added transport factors after a 30-min import reaction showed Karyopherin Beta at the nuclear envelope and Karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled Karyopherin Beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.
Junona Moroianu - One of the best experts on this subject based on the ideXlab platform.
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distinct nuclear import and export pathways mediated by members of the Karyopherin Beta family
Journal of Cellular Biochemistry, 1998Co-Authors: Junona MoroianuAbstract:Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of Karyopherin alpha and Beta1, that docks via Beta1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the Karyopherin Beta family have been discovered. Karyopherin Beta2 mediates import of mRNA binding proteins, whereas Karyopherin Beta3 and Beta4 mediate import of a set of ribosomal proteins. Two other Beta Karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of Karyopherin alpha, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of Karyopherin Beta family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the Karyopherin Beta superfamily and to characterize the molecular mechanisms of translocation of Karyopherins together with their cargoes through the NPC.
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Nuclear protein import: Ran-GTP dissociates the Karyopherin alphaBeta heterodimer by displacing alpha from an overlapping binding site on Beta
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract The alpha subunit of the Karyopherin heterodimer functions in recognition of the protein import substrate and the Beta subunit serves to dock the trimeric complex to one of many sites on nuclear pore complex fibers. The small GTPase Ran and the Ran interactive protein, p10, function in the release of the docked complex. Repeated cycles of docking and release are thought to concentrate the transport substrate for subsequent diffusion into the nucleus. Ran-GTP dissociates the Karyopherin heterodimer and forms a stoichiometric complex with Ran-GTP. Here we report the mapping of Karyopherin Beta's binding sites both for Ran-GTP and for Karyopherin alpha. We discovered that Karyopherin Beta's binding site for Ran-GTP shows a striking sequence similarity to the cytoplasmic Ran-GTP binding protein, RanBP1. Moreover, we found that Ran-GTP and Karyopherin alpha bind to overlapping sites on Karyopherin Beta. Having a higher affinity to the overlapping site, Ran-GTP displaces Karyopherin alpha and binds to Karyopherin Beta. Competition for overlapping binding sites may be the mechanism by which GTP bound forms of other small GTPases function in corresponding dissociation-association reactions. We also mapped Ran's binding site for Karyopherin Beta to a cluster of basic residues analogous to those previously shown to constitute Karyopherin alpha's binding site to Karyopherin Beta.
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The binding site of Karyopherin alpha for Karyopherin Beta overlaps with a nuclear localization sequence
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract By using proteolysis, recombinant mutant proteins, or synthetic peptides and by testing these reagents in liquid phase binding or nuclear import assays, we have mapped binding regions of Karyopherin alpha. We found that the C-terminal region of Karyopherin alpha recognizes the nuclear localization sequence (NLS), whereas its N-terminal region binds Karyopherin Beta. Surprisingly, Karyopherin alpha also contains an NLS. Thus, Karyopherin alpha belongs to a group of proteins that contain both a ligand (NLS) and a cognate receptor (NLS recognition site) in one molecule with a potential for autologous ligand-receptor interactions. The NLS of Karyopherin alpha overlaps with the binding site of Karyopherin alpha for Karyopherin Beta. Hence, binding of Karyopherin Beta to Karyopherin alpha covers the NLS of Karyopherin alpha. This prevents autologous ligand receptor interactions and explains the observed cooperative binding of Karyopherin alpha to a heterologous NLS protein in the presence of Karyopherin Beta.
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Mammalian Karyopherin alpha 1 Beta and alpha 2 Beta heterodimers: alpha 1 or alpha 2 subunit binds nuclear localization signal and Beta subunit interacts with peptide repeat-containing nucleoporins.
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Junona Moroianu, Gunter Blobel, Makoto Hijikata, Aurelian RaduAbstract:Although only 44% identical to human Karyopherin alpha 1, human Karyopherin alpha 2 (Rch1 protein) substituted for human Karyopherin alpha 1 (hSRP-1/NPI-1) in recognizing a standard nuclear localization sequence and Karyopherin Beta-dependent targeting to the nuclear envelope of digitonin-permeabilized cells. By immunofluorescence microscopy of methanol-fixed cells, Karyopherin Beta was localized to the cytoplasm and the nuclear envelope and was absent from the nuclear interior. Digitonin permeabilization of buffalo rat liver cells depleted their endogenous Karyopherin Beta. Recombinant Karyopherin Beta can bind directly to the nuclear envelope of digitonin-permeabilized cells at 0 degree C (docking reaction). In contrast, recombinant Karyopherin alpha 1 or alpha 2 did not bind unless Karyopherin Beta was present. Likewise, in an import reaction (at 20 degrees C) with all recombinant transport factors (Karyopherin alpha 1 or alpha 2, Karyopherin Beta, Ran, and p10) import depended on Karyopherin Beta. Localization of the exogenously added transport factors after a 30-min import reaction showed Karyopherin Beta at the nuclear envelope and Karyopherin alpha 1 or alpha 2, Ran, and p10 in the nuclear interior. In an overlay assay with SDS/PAGE-resolved and nitrocellulose-transferred proteins of the nuclear envelope, 35S-labeled Karyopherin Beta bound to at least four peptide repeat-containing nucleoporins--Nup358, Nup214, Nup153, and Nup98.
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previously identified protein of uncertain function is Karyopherin alpha and together with Karyopherin Beta docks import substrate at nuclear pore complexes
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Junona Moroianu, Gunter Blobel, Aurelian RaduAbstract:Abstract Previously, we had purified a cytosolic protein complex, termed Karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa Beta subunit. We now report that the Karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant Karyopherin alpha or Beta subunit, we showed that neither Karyopherin alpha nor Karyopherin Beta alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant Karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized Karyopherin alpha and Beta subunits and import substrate as a ligand, we found that only Karyopherin alpha bound ligand. We suggest that Karyopherin Beta functions as an adaptor that binds both to Karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of Karyopherin alpha-substrate across the NPC.
Ian G. Macara - One of the best experts on this subject based on the ideXlab platform.
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Ran-binding protein 1 (RanBP1) forms a ternary complex with Ran and Karyopherin Beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by Karyopherin Beta.
Journal of Biological Chemistry, 1997Co-Authors: Karen M. Lounsbury, Ian G. MacaraAbstract:The nuclear accumulation of proteins containing nuclear localization signals requires the Ran GTPase and a complex of proteins assembled at the nuclear pore. RanBP1 is a cytosolic Ran-binding protein that inhibits RCC1-stimulated release of GTP from Ran. RanBP1 also promotes the binding of Ran to Karyopherin Beta (also called importin Beta and p97) and is a co-stimulator of RanGAP activity. Yeast Karyopherin Beta inhibits the GTP hydrolysis by Ran catalyzed by RanGAP. To further define the roles of RanBP1 and Karyopherin Beta in Ran function, we explored the effects of RanBP1 and Karyopherin Beta on mammalian proteins known to regulate Ran. Like RanBP1, Karyopherin Beta prevented the release of GTP from Ran stimulated by RCC1 or EDTA. As with the yeast protein, mammalian Karyopherin Beta completely blocked RanGAP activity. However, the addition of RanBP1 to this assay partially rescued the inhibited RanGAP activity. Kinetic analysis of the effects on RanGAP activity by Karyopherin Beta and RanBP1 revealed a combination of competitive and noncompetitive interactions. Solution binding assays confirmed the ability of RanBP1 to associate with Ran and Karyopherin Beta in a ternary complex, and RanBP1 binding was not competed out by the addition of Karyopherin Beta. These results demonstrate that RanBP1 and Karyopherin Beta interact with distinct sites of Ran and suggest that RanBP1 plays an essential role in nuclear transport by permitting RanGAP-mediated hydrolysis of GTP on Ran complexed to Karyopherin Beta.
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ran binding protein 1 ranbp1 forms a ternary complex with ran and Karyopherin Beta and reduces ran gtpase activating protein rangap inhibition by Karyopherin Beta
Journal of Biological Chemistry, 1997Co-Authors: Karen M. Lounsbury, Ian G. MacaraAbstract:The nuclear accumulation of proteins containing nuclear localization signals requires the Ran GTPase and a complex of proteins assembled at the nuclear pore. RanBP1 is a cytosolic Ran-binding protein that inhibits RCC1-stimulated release of GTP from Ran. RanBP1 also promotes the binding of Ran to Karyopherin β (also called importin β and p97) and is a co-stimulator of RanGAP activity. Yeast Karyopherin β inhibits the GTP hydrolysis by Ran catalyzed by RanGAP. To further define the roles of RanBP1 and Karyopherin β in Ran function, we explored the effects of RanBP1 and Karyopherin β on mammalian proteins known to regulate Ran. Like RanBP1, Karyopherin β prevented the release of GTP from Ran stimulated by RCC1 or EDTA. As with the yeast protein, mammalian Karyopherin β completely blocked RanGAP activity. However, the addition of RanBP1 to this assay partially rescued the inhibited RanGAP activity. Kinetic analysis of the effects on RanGAP activity by Karyopherin β and RanBP1 revealed a combination of competitive and noncompetitive interactions. Solution binding assays confirmed the ability of RanBP1 to associate with Ran and Karyopherin β in a ternary complex, and RanBP1 binding was not competed out by the addition of Karyopherin β. These results demonstrate that RanBP1 and Karyopherin β interact with distinct sites of Ran and suggest that RanBP1 plays an essential role in nuclear transport by permitting RanGAP-mediated hydrolysis of GTP on Ran complexed to Karyopherin β.
Paul R Clarke - One of the best experts on this subject based on the ideXlab platform.
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xenopus ran binding protein 1 molecular interactions and effects on nuclear assembly in xenopus egg extracts
Journal of Cell Science, 1997Co-Authors: Francisco J Nicolas, Chuanmao Zhang, Michael Hughes, Martin W Goldberg, Sandra J Watton, Paul R ClarkeAbstract:Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-Beta/Karyopherin-Beta and importin-alpha/Karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/Karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.