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At least 19 recordsLinked to original sources
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↗
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↗
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↗
PubMed2006

Rules for nuclear localization sequence recognition by karyopherin beta 2.

Karyopherinbeta (Kapbeta) proteins bind nuclear localization and export signals (NLSs and NESs) to mediate nucleocytoplasmic trafficking, a process regulated by Ran GTPase through its nucleotide cycle. Diversity and complexity of signals recognized by Kap betas have prevented prediction of new Kap beta substrates. The structure of Kap beta 2 (also known as Transportin) bound to one of its substrates, the NLS of hnRNP A1, that we report here explains the mechanism of substrate displacement by Ran GTPase. Further analyses reveal three rules for NLS recognition by Kap beta 2: NLSs are structurally disordered in free substrates, have overall basic character, and possess a central hydrophobic or basic motif followed by a C-terminal R/H/KX(2-5)PY consensus sequence. We demonstrate the predictive nature of these rules by identifying NLSs in seven previously known Kap beta 2 substrates and uncovering 81 new candidate substrates, confirming five experimentally. These studies define and validate a new NLS that could not be predicted by primary sequence analysis alone.

Active Transport, Cell Nucleus↗
PubMed1997

Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta.

Heterogeneous nuclear ribonucleoprotein A1 contains a sequence, termed M9, that functions as a potent nuclear localization signal (NLS) yet bears no similarity to the well-defined basic class of NLSs. Here, we report the identification of a novel human protein, termed MIP, that binds M9 specifically both in vivo and in vitro yet fails to interact with non-functional M9 point mutants. Of note, the 101 kDa MIP protein bears significant homology to human karyopherin/importin-beta, a protein known to mediate the function of basic NLSs. The in vitro nuclear import of a protein substrate containing the M9 NLS was found to be dependent on provision of the MIP protein in trans. Cytoplasmic microinjection of a truncated form of MIP that retains the M9 binding site blocked the in vivo nuclear import of a substrate containing the M9 NLS yet failed to affect the import of a similar substrate bearing a basic NLS. These data indicate that nuclear import of hnRNP A1 is mediated by a novel cellular import pathway that is distinct from, yet evolutionarily related to, the pathway utilized by basic NLS sequences.

Amino Acid Sequence↗
PubMed2000

Purification of protein A-tagged yeast ran reveals association with a novel karyopherin beta family member, Pdr6p.

The small GTPase Ran (encoded by GSP1 and GSP2 in yeast) plays a central role in nucleocytoplasmic transport. GSP1 and GSP2 were tagged with protein A and functionally expressed in a gsp1 null mutant. After affinity purification of protein A-tagged Gsp1p or Gsp2p by IgG-Sepharose chromatography, known karyopherin beta transport receptors (e.g. Kap121p and Kap123p) and a novel member of this protein family, Pdr6p, were found to be associated with yeast Ran. Subsequent tagging of Pdr6p with green fluorescent protein revealed association with the nuclear pore complexes in vivo. Thus, functional tagging of yeast Ran allowed the study of its in vivo distribution and interaction with known and novel Ran-binding proteins.

Cell Compartmentation↗
PubMed1996

Characterization of p92, karyopherin beta, co-purified with N-acetylglucosamine-bearing nucleoporins from rat liver nuclear envelopes.

A 92k protein (p92) was purified from the wheat germ agglutinin-Sepharose (WGA-Sepharose) bound fraction of a rat liver nuclear envelope salt-extract by DEAE-5PW and hydroxyapatite HPLCs. Partial amino acid sequence analysis of p92 revealed that it is karyopherin beta, which was found recently in the cytosolic fraction. It was shown using anti-p92 antiserum that the protein is present in the nuclear envelope and cytosolic fractions, in almost the same amounts, but not in other subcellular fractions of rat liver. p92 bound to N-acetylglucosamine bearing nucleoporins (GNPs) on WGA-Sepharose, but not directly to WGA. The amount of p92 found in the rat liver nuclear envelope fraction corresponded to about 10% of the nuclear pore complex in mass, and to as much as 140 mol of p92 per mol of nuclear pore complex. Hydrodynamic analysis of the purified p92 suggested that the molecule is present as a monomer and that it is a rod-shaped molecule. The interaction of p92 and GNPs seemed to be hydrophobic and ionic. Based on these results, the participation of nuclear envelope p92 in protein nuclear transport is discussed.

Acetylglucosamine↗
PubMed2005

Identification of karyopherin beta as an immunogenic antigen of the malaria parasite using immune mice and human sera.

A differential immunoscreening of the lambdagt11 Plasmodium falciparum genomic expression library was carried out using anti-P. yoelii sera (convalescent-phase mouse sera) and immune sera collected from healthy adults, to identify novel cross-reactive and possibly protective antigens of the parasite. One clone, with an insert size of 1132 bp that reacted strongly with both the sera was selected. The insert was found to be a part of the P. falciparum karyopherin beta (PfKbeta) homologue. RT-PCR and Northern blot analysis confirmed the expression of PfKbeta in the blood stages of the parasite. The approximately 110 kDa protein was localized in the cytoplasm at the ring and trophozoite, and in the parasitophorous vacuole at the schizont stage. Two large fragments of PfKbeta representing the N- and C-terminal halves were expressed in E. coli. The recombinant proteins were highly immunogenic in mice, and also found to be the target for immune response in natural infections of Plasmodium spp. Anti-sera against the protein showed a low level of anti-parasitic activity. Immunization with recombinant PfKbeta fragments was only partially protective against a heterologous challenge infection in mice. Our results show that the parasite releases a highly immunogenic, cytoplasmic protein into the host which may not contribute to the development of protective immunity.

Animals↗
PubMed2000

A novel karyopherin-beta homolog is developmentally and hormonally regulated in fetal lung.

To investigate molecular mechanisms of lung organogenesis, we used representational difference analysis to search for glucocorticoid-inducible genes in developing lung in a fetal rat model. Messenger RNA prepared from fetal and adult rat lung was used to prepare "representative amplicons." Adult-lung complementary DNA (cDNA) amplicons were used as "driver" in successive rounds of subtractive hybridization/amplification to isolate target fetal lung-specific cDNAs. A single clone, which was conserved and had near-perfect homology to eight human/rodent expressed sequence tags, was used as template for 5' and 3' rapid amplification of cDNA ends and SPICE (system for polymerase chain reaction amplification of cDNA ends) reactions to obtain the 3.6-kb cDNA, LGL2 (Genbank, AF 110195) encoding a deduced polypeptide (lgl2) of 963 amino acids. Northern analysis confirmed that LGL2 is differentially expressed in fetal lung (maximal during the pseudoglandular stage, gestational Days 14 to 16), induced by glucocorticoid, and enriched in epithelium relative to the mesenchyme. LGL2 was also detected in human fetal lung at gestational Week 16 as well as in human and rat fetal brain, heart, intestine, and kidney. We mapped LGL2 to chromosome 1p33-34.2. Comparison with sequences in the genome database identified lgl2 as a member of the karyopherin-beta family of nuclear import proteins, with greatest homology to transportin SR. Maximal expression of LGL2 in the pseudoglandular stage of development is coordinate with that of key transcription factors that regulate prominent signal transduction pathways in fetal lung organogenesis. We propose a role for lgl2 in nuclear import of transcription factors that regulate signal transduction during fetal lung development.

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↗
PubMed2000

The nucleoporin Nup98 is a site for GDP/GTP exchange on ran and termination of karyopherin beta 2-mediated nuclear import.

Karyopherin beta2 (Kapbeta2, transportin) binds the M9 sequence of human ribonucleoprotein A1 and mediates its nuclear import. Here we show a role for the nucleoporin Nup98 in the disassembly of Kapbeta2 import complexes at the nuclear side of the nuclear pore complex (NPC). Kapbeta2 bound to a region at the N terminus of Nup98 that contains an M9-like sequence. The human ribonucleoprotein A1 M9 sequence competed with Nup98 for binding to Kapbeta2, indicating that Nup98 can dissociate Kapbeta2 from its substrate. Binding of Kapbeta2 to Nup98 was inhibited by Ran loaded with guanylyl imidophosphate, suggesting that RanGTP dissociates Kapbeta2 from Nup98. RanGTP is produced from RanGDP through nucleotide exchange mediated by RanGEF (RCC1). Immunoelectron microscopy and nucleotide exchange assays revealed functional RanGEF on both sides of the NPC. On the nuclear side, the localization of RanGEF coincided with that of Nup98. RanGEF bound to Nup98 at a region adjacent to the Kapbeta2-binding site. These findings suggest a model where 1) import substrate is released from Kapbeta2 at the nucleoplasmic side of the NPC by competition with the Nup98 M9-like site, 2) Nup98-bound RanGEF catalyzes the formation of RanGTP, and 3) RanGTP dissociates Kapbeta2 from Nup98 allowing repeated cycles of import.

Amino Acid Sequence↗
PubMed2002

Karyopherin beta 2B participates in mRNA export from the nucleus.

Transport of macromolecules between the cell nucleus and cytoplasm occurs through the nuclear pores and is mediated by soluble carriers known as karyopherins (Kaps), transportins, importins, or exportins. We report that Kap beta2B (transportin-2) forms complexes with the mRNA export factor TAP in the presence of RanGTP, as shown by coimmunoprecipitation from HeLa cells. The interaction strictly depends on the presence of RanGTP. In digitonin-permeabilized cells, Kap beta2B mediates TAP-GFP export from the nuclei in the presence of RanGTP. A TAP mutant that does not coimmunoprecipitate with Kap beta2B is also not exported by Kap beta2B. In the permeabilized cells assay, TAP is also exported independently of Kap beta2B by direct interaction with nucleoporins, in agreement with previous reports. The export rate is, however, significantly lower than the Kap beta2B-mediated pathway. Both Kap beta2B and TAP are present and enriched in the poly(A)(+) RNA complexes isolated from HeLa cell nuclear lysates. Poly(A)(+) RNA strongly accumulates in the nuclei of HeLa cells treated with Kap beta2B short interfering RNA, indicating that Kap beta2B is involved in the export of at least a large proportion of the mRNA species. The export of beta-actin and GAPDH mRNA is also inhibited, whereas 28S RNA is not affected. The data support the conclusion that Kap beta2B participates directly in the export of a large proportion of cellular mRNAs, and TAP connects Kap beta2B to the mRNAs to be exported.

Active Transport, Cell Nucleus↗
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↗
PubMed2006

Classical nuclear localization signals: definition, function, and interaction with importin alpha.

The best understood system for the transport of macromolecules between the cytoplasm and the nucleus is the classical nuclear import pathway. In this pathway, a protein containing a classical basic nuclear localization signal (NLS) is imported by a heterodimeric import receptor consisting of the beta-karyopherin importin beta, which mediates interactions with the nuclear pore complex, and the adaptor protein importin alpha, which directly binds the classical NLS. Here we review recent studies that have advanced our understanding of this pathway and also take a bioinformatics approach to analyze the likely prevalence of this system in vivo. Finally, we describe how a predicted NLS within a protein of interest can be confirmed experimentally to be functionally important.

Active Transport, Cell Nucleus↗
PubMed1996

Ran binding domains promote the interaction of Ran with p97/beta-karyopherin, linking the docking and translocation steps of nuclear import.

Nuclear protein import is accomplished by two sequential events; docking at the nuclear pore complex followed by ATP-dependent translocation across the nuclear envelope. Docking of nuclear targeted proteins requires a 56-kDa nuclear localization signal receptor (alpha-karyopherin, importin-alpha, SRP1 alpha) and a 97-kDa protein (beta-karyopherin, importin-beta). Components necessary for translocation include the Ran/TC4 GTPase and NTF2/B-2. The functions of these factors at a molecular level remain unclear. We have now found that a complex of Ran, in the GTP-bound state, with either the Ran binding protein, RanBP1, or an isolated Ran binding domain binds with high affinity and specificity to beta-karyopherin to form a ternary complex. We find that a C-terminal truncation mutant of Ran, delta-DE Ran, also binds to beta-karyopherin and that delta-DE Ran can associate with a cytosolic, multiprotein complex that contains beta-karyopherin and another delta-DE Ran binding protein of 115/120 kDa. These data suggest a physical link between docking and translocation mediated by a Ran GTPase-Ran binding protein complex.

Amino Acid Sequence↗