The Experts below are selected from a list of 2139 Experts worldwide ranked by ideXlab platform
Günter Blobel - One of the best experts on this subject based on the ideXlab platform.
-
Karyopherin mediated import of integral inner nuclear membrane proteins
Nature, 2006Co-Authors: Megan C King, C Lusk, Günter BlobelAbstract:Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble ‘classical’ nuclear localization signals. These sequences can mediate direct binding to Karyopherin-α and are essential for the passage of integral membrane proteins to the INM. Furthermore, Karyopherin-α, Karyopherin-β1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane. For proteins to carry out their role in cells, they must be located in the right cellular compartment. This positioning is usually achieved via discrete signal sequences in proteins that target them to the correct location. Many of these signal sequences have been fully characterized, but those that destine a protein to the inner nuclear membrane were a notable exception. A new study shows that targeting of integral inner nuclear membrane proteins involves signal sequences similar to those that target soluble proteins into the nucleus. This result is surprising, and opens up the study of a whole class of inner nuclear membrane proteins that have a role in gene regulation, and are linked to various human diseases. Sequences responsible for targeting integral membrane proteins to the inner nuclear membrane (INM) resemble classical nuclear localization signals. Recognition of these signals by the import receptor Karyopherin-alpha promotes nuclear import; targeting of INM proteins is dependent on the Ran GTPase system and specific nuclear pore complex proteins.
-
Karyopherins and nuclear import
Current Opinion in Structural Biology, 2001Co-Authors: Min Chook Yuh, Günter 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.
-
Structure of the nuclear transport complex Karyopherin-beta2-Ran x GppNHp.
Nature, 1999Co-Authors: Yuh Min Chook, Günter 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.
-
structure of the nuclear transport complex Karyopherin beta2 ran x gppnhp
Nature, 1999Co-Authors: Yuh Min Chook, Günter 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.
-
crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor Karyopherin α
Cell, 1998Co-Authors: Elena Conti, Günter 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.
Junona Moroianu - One of the best experts on this subject based on the ideXlab platform.
-
nuclear import and dna binding of human papillomavirus type 45 l1 capsid protein
Journal of Cellular Biochemistry, 2000Co-Authors: Lisa M Nelson, Robert C. Rose, Lucia Leroux, Christophore Lane, Kate Bruya, Junona MoroianuAbstract:During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high-risk HPV45 that when digitonin-permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor-mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic Karyopherin α2β1 heterodimers. HPV45 L1 bound strongly to Karyopherin α2, and weakly to Karyopherin β1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST-NLSHPV45L1 fusion protein was efficiently mediated by Karyopherin α2β1 heterodimers, and only weakly by Karyopherin β1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by Karyopherin α2β1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV-DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence. J. Cell. Biochem. 79:225–238, 2000. © 2000 Wiley-Liss, Inc.
-
Nuclear import of HPV11 L1 capsid protein is mediated by Karyopherin α2β1 heterodimers
Journal of Cellular Biochemistry, 1999Co-Authors: Eric Merle, Robert C. Rose, Lucia Leroux, Junona MoroianuAbstract:L1 major capsid proteins of human papillomaviruses (HPVs) enter the nuclei of host cells at two times during the viral life cycle: 1) after infection and 2) later during the productive phase, when they assemble the replicated HPV genomic DNA into infectious virions. L1 proteins are stable in two oligomeric configurations: as homopentameric capsomers, and as capsids composed of 72 capsomers. We found that intact L1 capsids of HPV type 11 cannot enter the nucleus, suggesting that capsid disassembly may be required for HPV11 L1 nuclear import. We established that HPV11 L1 is imported in a receptor-mediated manner into the nuclei of digitonin-permeabilized HeLa cells. HPV11 L1 docked at the nuclear pore complexes via Karyopherin α2β1 heterodimers. Anti-Karyopherin-β1 and anti-Karyopherin α2 antibodies specifically inhibited nuclear import of HPV11 L1. Moreover, nuclear import of HPV11 L1 could be reconstituted using Karyopherin α2, β1, RanGDP and p10. In agreement with the docking and import data, we found that HPV11 L1 binds to Karyopherin α2 and that this interaction is inhibited by a peptide representing the classical nuclear localization signal of SV40 T antigen. These results strongly suggest that HPV11 L1 enters the nucleus of the infected host cell via the Karyopherin α2β1 pathway. J. Cell. Biochem. 74:628–637, 1999. © 1999 Wiley-Liss, Inc.
-
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.
-
RanGTP-mediated nuclear export of Karyopherin α involves its interaction with the nucleoporin Nup153
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Junona Moroianu, Günter Blobel, Aurelian RaduAbstract:Abstract Using binding assays, we discovered an interaction between Karyopherin α2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of Karyopherin β1, termed β1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear Karyopherin α from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-Karyopherin α antibodies, we found that Karyopherin α export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of Karyopherin α was inhibited by peptides representing the interacting domains of Nup153 and Karyopherin α2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, β1*, although it inhibited import, did not inhibit export of Karyopherin α. Hence, Karyopherin α import into and export from nuclei are asymmetric processes.
-
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, Günter 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.
Aurelian Radu - One of the best experts on this subject based on the ideXlab platform.
-
RanGTP-mediated nuclear export of Karyopherin α involves its interaction with the nucleoporin Nup153
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Junona Moroianu, Günter Blobel, Aurelian RaduAbstract:Abstract Using binding assays, we discovered an interaction between Karyopherin α2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of Karyopherin β1, termed β1*, that contains a determinant for binding to the peptide repeat containing nucleoporin Nup98. In an in vitro assay in which export of endogenous nuclear Karyopherin α from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-Karyopherin α antibodies, we found that Karyopherin α export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of Karyopherin α was inhibited by peptides representing the interacting domains of Nup153 and Karyopherin α2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, β1*, although it inhibited import, did not inhibit export of Karyopherin α. Hence, Karyopherin α import into and export from nuclei are asymmetric processes.
-
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, Günter 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.
-
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, Günter 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.
-
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, Günter 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.
-
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, Günter 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.
Christopher F. Basler - One of the best experts on this subject based on the ideXlab platform.
-
Ebola Virus VP24 Proteins Inhibit the Interaction of NPI-1 Subfamily Karyopherin α Proteins with Activated STAT1
Journal of Virology, 2007Co-Authors: St. Patrick Reid, Charalampos Valmas, Osvaldo Martinez, Freddy Mauricio Sanchez, Christopher F. BaslerAbstract:The Zaire ebolavirus protein VP24 was previously demonstrated to inhibit alpha/beta interferon (IFN-α/β)- and IFN-γ-induced nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1) and to inhibit IFN-α/β- and IFN-γ-induced gene expression. These properties correlated with the ability of VP24 to interact with the nuclear localization signal receptor for PY-STAT1, Karyopherin α1. Here, VP24 is demonstrated to interact not only with overexpressed but also with endogenous Karyopherin α1. Mutational analysis demonstrated that VP24 binds within the PY-STAT1 binding region located in the C terminus of Karyopherin α1. In addition, VP24 was found to inhibit PY-STAT1 binding to both overexpressed and endogenous Karyopherin α1. We assessed the binding of both PY-STAT1 and the VP24 proteins from Zaire, mouse-adapted Zaire, and Reston Ebola viruses for interaction with all six members of the human Karyopherin α family. We found, in contrast to previous studies, that PY-STAT1 can interact not only with Karyopherin α1 but also with Karyopherins α5 and α6, which together comprise the NPI-1 subfamily of Karyopherin αs. Similarly, all three VP24s bound and inhibited PY-STAT1 interaction with Karyopherins α1, α5, and α6. Consistent with their ability to inhibit the Karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN-β-induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with Karyopherins α1, α5, or α6 by binding within the PY-STAT1 binding region of the Karyopherins and that this function is conserved among the VP24 proteins of different Ebola virus species.
-
Ebola virus VP24 proteins inhibit the interaction of NPI-1 subfamily Karyopherin alpha proteins with activated STAT1
2007Co-Authors: St. Patrick Reid, Charalampos Valmas, Osvaldo Martinez, Freddy Mauricio Sanchez, Christopher F. BaslerAbstract:The Zaire ebolavirus protein VP24 was previously demonstrated to inhibit alpha/beta interferon (IFN-/)-and IFN--induced nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1) and to inhibit IFN-/- and IFN--induced gene expression. These properties correlated with the ability of VP24 to interact with the nuclear localization signal receptor for PY-STAT1, Karyopherin 1. Here, VP24 is demonstrated to interact not only with overexpressed but also with endogenous Karyopherin 1. Mutational analysis demon-strated that VP24 binds within the PY-STAT1 binding region located in the C terminus of Karyopherin 1. In addition, VP24 was found to inhibit PY-STAT1 binding to both overexpressed and endogenous Karyopherin 1. We assessed the binding of both PY-STAT1 and the VP24 proteins from Zaire, mouse-adapted Zaire, and Reston Ebola viruses for interaction with all six members of the human Karyopherin family. We found, in contrast to previous studies, that PY-STAT1 can interact not only with Karyopherin 1 but also with karyo-pherins 5 and 6, which together comprise the NPI-1 subfamily of Karyopherin s. Similarly, all three VP24s bound and inhibited PY-STAT1 interaction with Karyopherins 1, 5, and 6. Consistent with their ability to inhibit the Karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN--induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with Karyopherins 1, 5, or 6 by binding withi
-
Ebola Virus VP24 Binds Karyopherin α1 and Blocks STAT1 Nuclear Accumulation
Journal of Virology, 2006Co-Authors: St. Patrick Reid, Osvaldo Martinez, Lawrence W. Leung, Amy L. Hartman, Megan L. Shaw, Caroline Carbonnelle, Viktor E. Volchkov, Stuart T. Nichol, Christopher F. BaslerAbstract:Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-α/β) and the ability of cells to respond to IFN-α/β or IFN-γ. The EBOV VP35 protein has previously been identified as an EBOV-encoded inhibitor of IFN-α/β production. However, the mechanism by which EBOV infection inhibits responses to IFNs has not previously been defined. Here we demonstrate that the EBOV VP24 protein functions as an inhibitor of IFN-α/β and IFN-γ signaling. Expression of VP24 results in an inhibition of IFN-induced gene expression and an inability of IFNs to induce an antiviral state. The VP24-mediated inhibition of cellular responses to IFNs correlates with the impaired nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1), a key step in both IFN-α/β and IFN-γ signaling. Consistent with this proposed function for VP24, infection of cells with EBOV also confers a block to the IFN-induced nuclear accumulation of PY-STAT1. Further, VP24 is found to specifically interact with Karyopherin α1, the nuclear localization signal receptor for PY-STAT1, but not with Karyopherin α2, α3, or α4. Overexpression of VP24 results in a loss of Karyopherin α1-PY-STAT1 interaction, indicating that the VP24-Karyopherin α1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.
Mary Shannon Moore - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
the nuclear import of rcc1 requires a specific nuclear localization sequence receptor Karyopherin alpha3 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.
-
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, Günter 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.