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At least 19 recordsLinked to original sources
PubMed1996

The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence.

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.

Amino Acid Sequence↗
PubMed1995

Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.

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.

Animals↗
PubMed1997

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.

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.

Binding, Competitive↗
PubMed2000

Epstein-barr virus nuclear antigen-1 binds to nuclear transporter karyopherin alpha1/NPI-1 in addition to karyopherin alpha2/Rch1.

We searched for cellular proteins that interact with Epstein-Barr (EBV) virus nuclear antigen-1, which is a latent EBV origin-binding protein detected in all EBV latently infected cells and essential for maintenance of the latent EBV genome, by a yeast two-hybrid screening of a B lymphocyte cDNA library in this study. Interaction of polypeptides synthesized from three selected cDNA clones with EBNA-1 proteins was confirmed in vitro using their glutathione-S-transferase-fusion polypeptides and by coimmunoprecipitation analyses of B cell extracts with anti-EBNA-1 monoclonal antibodies and monospecific antibodies against cellular proteins of interest. We report the following: (i) Karyopherin alpha (karyopherin alpha1, hSRP1, and NPI-1), an adaptor subunit of nuclear localization signal receptors, which direct proteins to the nuclear pore, interacted with EBNA-1. (ii) EBNA-1 proteins endogenous in the B cell line Raji of Burkitt lymphoma origin bound to another adaptor protein, karyopherin alpha2 (hSRP1alpha, hRch1), interactions of which to recombinant EBNA-1 polypeptides were previously reported. (iii) Nearly 90% of all the cDNA clones examined was p32 (SF2-associated P32, p32/TAP, and gC1q-R), and endogenous EBNA-1 proteins in the Raji cells bound to p32, a potential of which to affect localization of EBNA-1 in transfected Vero cells has been recently suggested. These results suggest that EBNA-1, which has the unique NLS containing Lys-Arg and overlapping with one of the phosphorylation domains, is recognized and transported to the nuclei by these two distinct karyopherin alpha proteins, which are differentially expressed in different cell types, implying a regulatory localization system for EBNA-1.

B-Lymphocytes↗
PubMed1996

Expression in yeast of binding regions of karyopherins alpha and beta inhibits nuclear import and cell growth.

Using truncated forms of recombinant yeast karyopherins alpha and beta in in vitro binding assays, we mapped the regions of karyopherin alpha that bind to karyopherin beta and the regions of karyopherin beta that interact with karyopherin alpha and with Ran-GTP. Karyopherin alpha's binding region for karyopherin beta was localized to its N-terminal domain, which contains several clusters of basic residues, whereas karyopherin beta's binding region for karyopherin alpha was localized to an internal region containing two clusters of acidic residues. Karyopherin beta's binding region for Ran-GTP overlaps with that for karyopherin alpha and comprises at least one of the two acidic clusters required for karyopherin alpha binding in addition to further downstream determinants not required for karyopherin alpha binding. Overexpression in yeast of fragments containing either karyopherin beta's binding region for alpha and Ran-GTP or karyopherin alpha's binding region for beta resulted in sequestration of most of the cytosolic karyopherin alpha or karyopherin beta, respectively, in complexes containing the truncated proteins. As these binding region-containing fragments lack other domains required for function of the corresponding protein, the overexpression of either fragment also inhibited in vivo nuclear import of a model reporter protein as well as cell growth.

Animals↗
PubMed1995

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.

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.

Animals↗
PubMed1996

Nuclear protein import: Ran-GTP dissociates the karyopherin alphabeta heterodimer by displacing alpha from an overlapping binding site on beta.

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.

Amino Acid Sequence↗
PubMed1996

The nuclear transport factor karyopherin beta binds stoichiometrically to Ran-GTP and inhibits the Ran GTPase activating protein.

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 beta (Rexach, M., and Blobel, G. (1995) Cell 83, 683-692). We show here, using all recombinant yeast proteins expressed in Escherichia coli, that karyopherin beta 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 beta was dependent on karyopherin beta concentration relative to Ran-GTP. Complete inhibition of RanGAP was observed at karyopherin beta concentrations that were equimolar to Ran-GTP. In gel filtration experiments, we found Ran-GTP and karyopherin beta to form a stoichiometric complex. Ran-GDP bound only weakly to karyopherin beta. We propose that stoichiometric complex formation between karyopherin beta and Ran-GTP renders Ran-GTP inaccessible to RanGAP.

Base Sequence↗
PubMed1997

Disassembly of RanGTP-karyopherin beta complex, an intermediate in nuclear protein import.

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.

Guanosine Diphosphate↗
PubMed1998

Distinct nuclear import and export pathways mediated by members of the karyopherin beta family.

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.

Animals↗
PubMed1997

RanGTP-mediated nuclear export of karyopherin alpha involves its interaction with the nucleoporin Nup153.

Using binding assays, we discovered an interaction between karyopherin alpha2 and the nucleoporin Nup153 and mapped their interacting domains. We also isolated a 15-kDa tryptic fragment of karyopherin beta1, termed beta1*, 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 alpha from nuclei of digitonin-permeabilized cells was quantitatively monitored by indirect immunofluorescence with anti-karyopherin alpha antibodies, we found that karyopherin alpha export was stimulated by added GTPase Ran, required GTP hydrolysis, and was inhibited by wheat germ agglutinin. RanGTP-mediated export of karyopherin alpha was inhibited by peptides representing the interacting domains of Nup153 and karyopherin alpha2, indicating that the binding reactions detected in vitro are physiologically relevant and verifying our mapping data. Moreover, beta1*, although it inhibited import, did not inhibit export of karyopherin alpha. Hence, karyopherin alpha import into and export from nuclei are asymmetric processes.

Amino Acid Sequence↗
PubMed1999

Structure of the nuclear transport complex karyopherin-beta2-Ran x GppNHp.

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.

Amino Acid Sequence↗
PubMed2002

Karyopherin alpha2: a control step of glucose-sensitive gene expression in hepatic cells.

Glucose is required for an efficient expression of the glucose transporter GLUT2 and other genes. We have shown previously that the intracytoplasmic loop of GLUT2 can divert a signal, resulting in the stimulation of glucose-sensitive gene transcription. In the present study, by interaction with the GLUT2 loop, we have cloned the rat karyopherin alpha2, a receptor involved in nuclear import. The specificity of the binding was restricted to GLUT2, and not GLUT1 or GLUT4, and to karyopherin alpha2, not alpha1. When rendered irreversible by a cross-linking agent, this transitory interaction was detected in vivo in hepatocytes. A role for karyopherin alpha2 in the transcription of two glucose-sensitive genes was investigated by transfection of native and inactive green fluorescent protein-karyopherin alpha2 in GLUT2-expressing hepatoma cells. The amount of inactive karyopherin alpha2 receptor reduced, in a dose-dependent manner, the GLUT2 and liver pyruvate kinase mRNA levels by competition with endogenous active receptor. In contrast, the overexpression of karyopherin alpha2 did not significantly stimulate GLUT2 and liver pyruvate kinase mRNA accumulation in green fluorescent protein-sorted cells. The present study suggests that, in concert with glucose metabolism, karyopherin alpha2 transmits a signal to the nucleus to regulate glucose-sensitive gene expression. The transitory tethering of karyopherin alpha2 to GLUT2 at the plasma membrane might indicate that the receptor can load the cargo to be imported locally.

Animals↗
PubMed1997

Epstein-Barr virus nuclear antigen 1 forms a complex with the nuclear transporter karyopherin alpha2.

The Epstein-Barr virus (EBV) is implicated in the induction of several malignancies. The nuclear antigen 1 (EBNA1) is the only viral protein that is expressed consistently in all EBV-associated tumors. EBNA1 is involved in the replication and maintenance of the viral episome in the infected cell and exhibits oncogenic activity in transgenic mice. Here we report the identification of the nuclear transporter karyopherin alpha2 as a cellular partner of EBNA1 using the yeast "two-hybrid system." Karyopherin alpha2 is also called importin alpha or Rch1. The binding to karyopherin alpha2 was mediated through a C-terminal region of EBNA1 encompassing the nuclear localization signal, whereas clones of EBNA1 devoid of the nuclear localization signal failed to bind to karyopherin alpha2. The interaction was biochemically confirmed by far-Western analysis using bacterially expressed karyopherin alpha2 and karyopherin alpha2-specific monoclonal antibodies. The nuclear transport of EBNA1 was impaired by expression of N-terminally truncated karyopherin alpha2. Zone velocity sedimentation in a sucrose gradient indicated that: (i) EBNA1 and Rch1 colocalize; and (ii) the association of karyopherin alpha2 with high molecular weight protein complexes might be impeded by the presence of EBNA1.

Animals↗
PubMed2004

A karyopherin alpha2 nuclear transport pathway is regulated by glucose in hepatic and pancreatic cells.

We studied the role of the karyopherin alpha2 nuclear import carrier (also known as importin alpha2) in glucose signaling. In mhAT3F hepatoma cells, GFP-karyopherin alpha2 accumulated massively in the cytoplasm within minutes of glucose extracellular addition and returned to the nucleus after glucose removal. In contrast, GFP-karyopherin alpha1 distribution was unaffected regardless of glucose concentration. Glucose increased GFP-karyopherin alpha2 nuclear efflux by a factor 80 and its shuttling by a factor 4. These glucose-induced movements were not due to glycolytic ATP production. The mechanism involved was leptomycin B-insensitive, but phosphatase- and energy-dependent. HepG2 and COS-7 cells displayed no glucose-induced GFP-karyopherin alpha2 movements. In pancreatic MIN-6 cells, the glucose-induced movements of karyopherin alpha2 and the stimulation of glucose-induced gene transcription were simultaneously lost between passages 28 and 33. Thus, extracellular glucose regulates a nuclear transport pathway by increasing the nuclear efflux and shuttling of karyopherin alpha2 in cells in which glucose can stimulate the transcription of sugar-responsive genes.

Active Transport, Cell Nucleus↗
PubMed1997

Cloning and characterization of human karyopherin beta3.

Nuclear import of classical nuclear localization sequence-bearing proteins is mediated by karyopherin alpha/beta1 heterodimers. A second nuclear import pathway, mediated by karyopherin beta2 (transportin), recently was described for mRNA-binding proteins. Here we report the cloning and characterization of human karyopherin beta3, which may be involved in a third pathway for nuclear import. Karyopherin beta3 was localized mainly to the cytosol and the nucleus, particularly the nuclear rim. It bound to several of the repeat-containing nucleoporins (Nup358, Nup214, Nup153, Nup98, and p62) in overlay and solution-binding assays and was competed away by karyopherin beta1. For Nup98, we localized this binding to the peptide repeat-containing region. Karyopherin beta3 contains two putative Ran-binding homology regions and bound to Ran-GTP in a solution-binding assay with much higher affinity than to Ran-GDP. Furthermore, it interacted with two ribosomal proteins in an overlay assay. We suggest that karyopherin beta3 is a nuclear transport factor that may mediate the import of some ribosomal proteins into the nucleus.

Amino Acid Sequence↗
PubMed1995

Identification of a yeast karyopherin heterodimer that targets import substrate to mammalian nuclear pore complexes.

Targeting of import substrate to nuclear pore complexes of permeabilized vertebrate cells was previously shown to require a protein complex composed of two subunits, termed karyopherin. Yeast contain a homologue of karyopherin alpha named Srp1p, which was initially identified as a genetic suppressor of mutations in a subunit of RNA polymerase I. To determine whether yeast contain a karyopherin complex that includes Srp1p as the karyopherin alpha homologue, we genetically replaced Srp1p with a Srp1-Protein A chimera. Cytosol from this strain contained a complex, composed of the chimera and a protein of 95 kDa, that was purified using affinity chromatography on IgG Sepharose. Microsequence analysis showed that the 95-kDa protein was identical with a yeast protein encoded by gene L8300.15 on chromosome XII. Sequence comparison revealed that the L8300.15 gene product is the closest structural homologue of vertebrate karyopherin beta. The yeast alpha and beta karyopherin subunits were expressed in Escherichia coli and were purified. When combined, they formed a heterodimeric complex and were active in targeting import substrate to nuclear envelopes of mammalian cells. We propose that all karyopherins function as alpha/beta heterodimers.

Amino Acid Sequence↗