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Mary Shannon Moore - One of the best experts on this subject based on the ideXlab platform.
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the nuclear import of rcc1 requires a specific nuclear localization sequence receptor karyopherin α3 qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:Abstract RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopusovarian cytosol nor a mixture of recombinant import factors (karyopherin α2, karyopherin β1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathioneS-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin α3 (Kapα3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapα1 and Kapα2, would not. Accordingly, in vitrobinding studies revealed that only Kapα3 showed significant binding to RCC1 (unlike Kapα1 and Kapα2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapα3, we found that the nuclear import of pA-RCC1 also required both karyopherin β1 and Ran.
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The Nuclear Import of RCC1 Requires a Specific Nuclear Localization Sequence Receptor, Karyopherin α3/Qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
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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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gene YDR395w/
2016Co-Authors: Jonathan S. Rosenblum, Lucy F. Pemberton, Gunter BlobelAbstract:A limited number of transport factors, or Karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccha-romyces cerevisia
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Karyopherins and nuclear import
Current Opinion in Structural Biology, 2001Co-Authors: Min Chook Yuh, Gunter BlobelAbstract:Proteins of the karyopherin α and karyopherin β families play a central role in nucleocytoplasmic transport. Recently, crystal structures of karyopherin α and its complexes with nuclear localization signal peptides, a karyopherin β2-Ran complex and complexes of full-length and fragments of karyopherin β1 with import substrates, Ran and nucleoporins have been solved. These karyopherin structures provide valuable insights into understanding the molecular mechanism of nuclear import, especially substrate recognition, substrate release by GTPase and interactions with the nuclear pore complex.
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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-β (also called importin-β) family mediate the movement of macromolecules in nuclear–cytoplasmic transport pathways. Karyopherin-β2 (transportin) binds a cognate import substrate and targets it to the nuclear pore complex. In the nucleus, Ran˙GTP binds karyopherin-β2 and dissociates the substrate. Here we present the 3.0 A structure of the karyopherin-β2–Ran˙GppNHp complex where GppNHp is a non-hydrolysable GTP analogue. Karyopherin-β2 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˙GppNHp in the complex shows extensive structural rearrangement, compared to Ran˙GDP, in regions contacting karyopherin-β2. This provides a structural basis for the specificity of the karyopherin-β 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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Nuclear Import and the Evolution of a Multifunctional RNA-binding Protein
Journal of Cell Biology, 1998Co-Authors: Jonathan S. Rosenblum, Lucy F. Pemberton, Neris Bonifaci, Gunter BlobelAbstract:La (SS-B) is a highly expressed protein that is able to bind 3′-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La, Lhp1p. The soluble transport factor, or karyopherin, that mediates the import of Lhp1p is Kap108p/Sxm1p. We have now determined a 113-amino acid domain of Lhp1p that is brought to the nucleus by Kap108p. Unexpectedly, this domain does not coincide with the previously identified nuclear localization signal of human La. Furthermore, when expressed in Saccharomyces cerevisiae, the nuclear localization of Schizosaccharomyces pombe, Drosophila, and human La proteins are independent of Kap108p. We have been able to reconstitute the nuclear import of human La into permeabilized HeLa cells using the recombinant human factors karyopherin α2, karyopherin β1, Ran, and p10. As such, the yeast and human La proteins are imported using different sequence motifs and dissimilar Karyopherins. Our results are consistent with an intermingling of the nuclear import and evolution of La.
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crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin α
Cell, 1998Co-Authors: Elena Conti, Gunter Blobel, Marc Uy, L Leighton, John KuriyanAbstract:Abstract Selective nuclear import is mediated by nuclear localization signals (NLSs) and cognate transport factors known as Karyopherins or importins. Karyopherin α recognizes "classical" monopartite and bipartite NLSs. We report the crystal structure of a 50 kDa fragment of the 60 kDa yeast karyopherin α, in the absence and presence of a monopartite NLS peptide at 2.2 A and 2.8 A resolution, respectively. The structure shows a tandem array of ten armadillo repeats, organized in a right-handed superhelix of helices. Binding of the NLS peptide occurs at two sites within a helical surface groove that is lined by conserved residues. The structure reveals the determinants of NLS specificity and suggests a model for the recognition of bipartite NLSs.
Bradford Talcott - One of the best experts on this subject based on the ideXlab platform.
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the nuclear import of rcc1 requires a specific nuclear localization sequence receptor karyopherin α3 qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:Abstract RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopusovarian cytosol nor a mixture of recombinant import factors (karyopherin α2, karyopherin β1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathioneS-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin α3 (Kapα3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapα1 and Kapα2, would not. Accordingly, in vitrobinding studies revealed that only Kapα3 showed significant binding to RCC1 (unlike Kapα1 and Kapα2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapα3, we found that the nuclear import of pA-RCC1 also required both karyopherin β1 and Ran.
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The Nuclear Import of RCC1 Requires a Specific Nuclear Localization Sequence Receptor, Karyopherin α3/Qip
Journal of Biological Chemistry, 2000Co-Authors: Bradford Talcott, Mary Shannon MooreAbstract:RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.
Derek R. Duckett - One of the best experts on this subject based on the ideXlab platform.
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Abstract 4193: Identification of a novel SIRT1/FEN1 interaction and their regulation by Karyopherins
Cancer Research, 2011Co-Authors: Daniel Feurstein, Jessica Anderson, Derek R. DuckettAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The structure-specific flap endonuclease 1 (FEN-1) is a multifunctional enzyme that participates in various activities such as DNA replication, DNA repair and apoptotic DNA fragmentation. Several mechanisms, including protein-protein interactions, sub-cellular compartmentalization and posttranslational modifications have been proposed to explain how FEN1 accomplishes these various activities. For example, the C-terminus of FEN1 has been shown to be acetylated by the histone acetyl transferase protein (HAT) p300 which is known to result in a significant reduction in the DNA binding and nuclease activities of FEN1. We now demonstrate that the NAD-dependent class III histone deacetylase SIRT1 physically interacts with FEN1 and that the opposing deacetylation by SIRT1 subsequently restores the endonuclease activity of FEN1. Moreover, clonogenicity experiments suggest that genetic knockdown of FEN1 or SIRT1 in cancer cells sensitizes those cells to irradiation treatment. This is of significance as it has been observed that increased SIRT1 levels protect mice from irradiation-induced cancer. Furthermore, we have also identified two nuclear shuttling proteins, Karyopherin α2 and Karyopherin β1 as interacting partners of both SIRT1 and FEN1. Using mass spectrometry, immunoprecipitation combined with subsequent western-blot analysis and confocal microscopy we demonstrate that, under basal conditions, a significant amount of both SIRT1 and FEN1 is located in the cytoplasm of cells. However, after induction of DNA damage by the methylating agent MMS, Karyopherin α2 and Karyopherin β1 mediate the translocation of SIRT1 and FEN1 into the nucleus in a time dependent manner. In summary we describe a novel interaction between SIRT1 and FEN1, demonstrate that acetylation/deacetylation regulates FEN1 activity and that the Karyopherins α2 and β1 mediate the nuclear import of SIRT1 and FEN1 following DNA damage. Our results suggest that inhibiting the nuclear import or activation of FEN1 or SIRT1 could represent potential anticancer strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2011-4193
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abstract 4193 identification of a novel sirt1 fen1 interaction and their regulation by Karyopherins
Cancer Research, 2011Co-Authors: Daniel Feurstein, Jessica Anderson, Derek R. DuckettAbstract:Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL The structure-specific flap endonuclease 1 (FEN-1) is a multifunctional enzyme that participates in various activities such as DNA replication, DNA repair and apoptotic DNA fragmentation. Several mechanisms, including protein-protein interactions, sub-cellular compartmentalization and posttranslational modifications have been proposed to explain how FEN1 accomplishes these various activities. For example, the C-terminus of FEN1 has been shown to be acetylated by the histone acetyl transferase protein (HAT) p300 which is known to result in a significant reduction in the DNA binding and nuclease activities of FEN1. We now demonstrate that the NAD-dependent class III histone deacetylase SIRT1 physically interacts with FEN1 and that the opposing deacetylation by SIRT1 subsequently restores the endonuclease activity of FEN1. Moreover, clonogenicity experiments suggest that genetic knockdown of FEN1 or SIRT1 in cancer cells sensitizes those cells to irradiation treatment. This is of significance as it has been observed that increased SIRT1 levels protect mice from irradiation-induced cancer. Furthermore, we have also identified two nuclear shuttling proteins, Karyopherin α2 and Karyopherin β1 as interacting partners of both SIRT1 and FEN1. Using mass spectrometry, immunoprecipitation combined with subsequent western-blot analysis and confocal microscopy we demonstrate that, under basal conditions, a significant amount of both SIRT1 and FEN1 is located in the cytoplasm of cells. However, after induction of DNA damage by the methylating agent MMS, Karyopherin α2 and Karyopherin β1 mediate the translocation of SIRT1 and FEN1 into the nucleus in a time dependent manner. In summary we describe a novel interaction between SIRT1 and FEN1, demonstrate that acetylation/deacetylation regulates FEN1 activity and that the Karyopherins α2 and β1 mediate the nuclear import of SIRT1 and FEN1 following DNA damage. Our results suggest that inhibiting the nuclear import or activation of FEN1 or SIRT1 could represent potential anticancer strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4193. doi:10.1158/1538-7445.AM2011-4193
Yuh Min Chook - One of the best experts on this subject based on the ideXlab platform.
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Karyopherins and condensates.
Current Opinion in Cell Biology, 2020Co-Authors: Charis E. Springhower, Michael K. Rosen, Yuh Min ChookAbstract:Abstract Several aggregation-prone RNA-binding proteins, including FUS, EWS, TAF15, hnRNP A1, hnRNP A2, and TDP-43, are mutated in neurodegenerative diseases. The nuclear–cytoplasmic distribution of these proteins is controlled by proteins in the karyopherin family of nuclear transport factors (Kaps). Recent studies have shown that Kaps not only transport these proteins but also inhibit their self-association/aggregation, acting as molecular chaperones. This chaperone activity is impaired for disease-causing mutants of the RNA-binding proteins. Here, we review physical data on the mechanisms of self-association of several disease-associated RNA-binding proteins, through liquid–liquid phase separation and amyloid fiber formation. In each case, we relate these data to biophysical, biochemical, and cell biological data on the inhibition of self-association by Kaps. Our analyses suggest that Kaps may be effective chaperones because they contain large surfaces with diverse physical properties that enable them to engage multiple different regions of their cargo proteins, blocking self-association.
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Karyopherins in cancer
Current Opinion in Cell Biology, 2018Co-Authors: Tolga Cagatay, Yuh Min ChookAbstract:Malfunction of nuclear–cytoplasmic transport contributes to many diseases including cancer. Defective nuclear transport leads to changes in both the physiological levels and temporal-spatial location of tumor suppressors, proto-oncogenes and other macromolecules that in turn affect the tumorigenesis process and drug sensitivity of cancer cells. In addition to their nuclear transport functions in interphase, Karyopherin nuclear transport receptors also have important roles in mitosis and chromosomal integrity. Therefore, alterations in the expressions or regular functions of Karyopherins may have substantial effects on the course and outcome of diseases.
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Nuclear localization signals for four distinct karyopherin-β nuclear import systems
Biochemical Journal, 2015Co-Authors: Michael M. Soniat, Yuh Min ChookAbstract:The Karyopherin-β family of proteins mediates nuclear transport of macromolecules. Nuclear versus cytoplasmic localization of proteins is often suggested by the presence of NLSs (nuclear localization signals) or NESs (nuclear export signals). Import-Karyopherin-βs or Importins bind to NLSs in their protein cargos to transport them through nuclear pore complexes into the nucleus. Until recently, only two classes of NLS had been biochemically and structurally characterized: the classical NLS, which is recognized by the Importin-α/β heterodimer and the PY-NLS (proline–tyrosine NLS), which is recognized by Karyopherin-β2 or Transportin-1. Structures of two other Karyopherin-βs, Kap121 and Transportin-SR2, in complex with their respective cargos were reported for the first time recently, revealing two new distinct classes of NLSs. The present paper briefly describes the classical NLS, reviews recent literature on the PY-NLS and provides in-depth reviews of the two newly discovered classes of NLSs that bind Kap121p and Transportin-SR respectively.
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nuclear import by karyopherin βs recognition and inhibition
Biochimica et Biophysica Acta, 2011Co-Authors: Yuh Min Chook, Katherine E SuelAbstract:Proteins in the Karyopherinβ family mediate the majority of macromolecular transport between the nucleus and the cytoplasm. Eleven of the 19 known human Karyopherinβs and 10 of the 14 S. cerevisiae Karyopherinβs mediate nuclear import through recognition of nuclear localization signals or NLSs in their cargos. This receptor-mediated process is essential to cellular viability as proteins are translated in the cytoplasm but many have functional roles in the nucleus. Many known Karyopherinβ-cargo interactions were discovered through studies of the individual cargos rather than the Karyopherins, and this information is thus widely scattered in the literature. We consolidate information about cargos that are directly recognized by import-Karyopherinβs and review common characteristics or lack thereof among cargos of different import pathways. Knowledge of Karyopherinβ-cargo interactions is also critical for the development of nuclear import inhibitors and the understanding of their mechanisms of inhibition.
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nuclear import by karyopherin βs recognition and inhibition
Biochimica et Biophysica Acta, 2011Co-Authors: Yuh Min Chook, Katherine E SuelAbstract:Proteins in the Karyopherinβ family mediate the majority of macromolecular transport between the nucleus and the cytoplasm. Eleven of the 19 known human Karyopherinβs and 10 of the 14 S. cerevisiae Karyopherinβs mediate nuclear import through recognition of nuclear localization signals or NLSs in their cargos. This receptor-mediated process is essential to cellular viability as proteins are translated in the cytoplasm but many have functional roles in the nucleus. Many known Karyopherinβ-cargo interactions were discovered through studies of the individual cargos rather than the Karyopherins, and this information is thus widely scattered in the literature. We consolidate information about cargos that are directly recognized by import-Karyopherinβs and review common characteristics or lack thereof among cargos of different import pathways. Knowledge of Karyopherinβ-cargo interactions is also critical for the development of nuclear import inhibitors and the understanding of their mechanisms of inhibition.