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PubMed2000

Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localization signals by karyopherin alpha.

BACKGROUND: Karyopherin alpha (importin alpha) is an adaptor molecule that recognizes proteins containing nuclear localization signals (NLSs). The prototypical NLS that is able to bind to karyopherin alpha is that of the SV40 T antigen, and consists of a short positively charged sequence motif. Distinct classes of NLSs (monopartite and bipartite) have been identified that are only partly conserved with respect to one another but are nevertheless recognized by the same receptor. RESULTS: We report the crystal structures of two peptide complexes of yeast karyopherin alpha (Kapalpha): one with a human c-myc NLS peptide, determined at 2.1 A resolution, and one with a Xenopus nucleoplasmin NLS peptide, determined at 2.4 A resolution. Analysis of these structures reveals the determinants of specificity for the binding of a relatively hydrophobic monopartite NLS and of a bipartite NLS peptide. The peptides bind Kapalpha in its extended surface groove, which presents a modular array of tandem binding pockets for amino acid residues. CONCLUSIONS: Monopartite and bipartite NLSs bind to a different number of amino acid binding pockets and make different interactions within them. The relatively hydrophobic monopartite c-myc NLS binds extensively at a few binding pockets in a similar manner to that of the SV40 T antigen NLS. In contrast, the bipartite nucleoplasmin NLS engages the whole array of pockets with individually more limited but overall more abundant interactions, which include the NLS two basic clusters and the backbone of its non-conserved linker region. Versatility in the specific recognition of NLSs relies on the modular.

Amino Acid Sequence↗

PubMed2006

Karyopherin-mediated import of integral inner nuclear membrane proteins.

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-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 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.

Amino Acid Motifs↗

PubMed2004

The karyopherin Msn5/Kap142 requires Nup82 for nuclear export and performs a function distinct from translocation in RPA protein import.

Protein transport between the nucleus and cytoplasm requires interactions between nuclear pore complex proteins (nucleoporins) and soluble nuclear transport factors (karyopherins, importins, and exportins). Exactly how these interactions contribute to the nucleocytoplasmic transport of substrates remains unclear. Using a synthetic lethal screen with the nucleoporin NUP1, we have identified a conditional allele of NUP82, encoding an essential nuclear pore complex protein in Saccharomyces cerevisiae. This nup82-3 allele also exhibits synthetic genetic interactions with mutants of the karyopherin MSN5. nup82-3 mutants accumulate the Msn5 export substrate Pho4 within the nucleus at non-permissive temperatures. The nuclear import of the RPA complex subunit Rfa2 is impaired in nup82-3 and in mutants of the karyopherin KAP95, but is not affected by the loss of MSN5. Interestingly, deletion of MSN5 results in retention of Rfa2-GFP within the nucleus under conditions in which it normally diffuses out. These data provide evidence that Nup82 is important for Msn5-mediated nuclear protein export and Kap95-mediated protein import. In addition, Msn5 may play a role independent of import in the localization of Rfa2.

Active Transport, Cell Nucleus↗

PubMed2003

Cleavage stage porcine embryos may have differing developmental requirements for karyopherins alpha2 and alpha3.

Numerous cellular proteins are able to localize to the nucleus due to the fact that they possess a nuclear localization signal (NLS) in their amino acid sequence. Nuclear localization sequences recognized by the importin alpha/beta heterodimer are found in cellular proteins capable of performing many diverse functions, ranging from chromatin remodeling to cell cycle regulation. Evidence has been presented that suggests individual importin alpha homologues are present at varying levels in different adult tissues. Other data have shown that specific subsets of NLSs found in different cellular proteins are recognized by individual importin alpha homologues with varying affinities. This evidence led us to hypothesize that due to the specific cargoes they carry, the mammalian embryo has different developmental requirements for individual importin alpha homologues. The results of the studies presented here indicate that importin alpha/beta-mediated import occurs throughout early cleavage in the porcine embryo, as determined by a reporter protein microinjection assay, and that multiple importin alpha homologues are present throughout early cleavage, as determined by immunocytochemical analysis. An RNA interference approach was used in an attempt to determine the developmental requirements for specific importin alpha homologues during early cleavage in the porcine embryo. Results from this study showed that fertilized porcine embryos injected with double stranded RNA (dsRNA) corresponding to the importin alpha homologue karyopherin alpha3 had significantly fewer nuclei following four days of culture than did embryos injected with dsRNA for another importin alpha homologue, karyopherin alpha2, or two control groups. This is the first report indicating that mammalian embryos may have differential developmental requirements for specific nuclear trafficking pathways.

Animals↗

PubMed2003

Nuclear import of yeast Gcn4p requires karyopherins Srp1p and Kap95p.

The yeast transcription factor Gcn4p contains two stretches of amino acid residues, NLS1 and NLS2, which are independently able to relocate the cytoplasmic protein chorismate mutase into the nucleus. Only NLS2 is conserved among fungi. A truncated version of CPCA (the counterpart of Gcn4p in Aspergillus nidulans), which lacks the conserved NLS, accumulates in the cytoplasm instead of the nucleus. Nuclear uptake mediated by the NLS1 of Gcn4p is impaired by defects in genes for several different karyopherins, whereas NLS2-dependent nuclear import specifically requires the alpha-importin Srp1p and the beta-importin Kap95p. Yeast strains that are defective in either of these two karyopherins are unable to respond to amino acid starvation. We have thus identified Gcn4p as a substrate for the Srp1p/Kap95p transport complex. Our data suggest that NLS2 is the essential and specific nuclear transport signal; NLS1 may play only an unspecific or accessory role.

Active Transport, Cell Nucleus↗

PubMed2005

Nuclear import of TFIIB is mediated by Kap114p, a karyopherin with multiple cargo-binding domains.

Nuclear import and export is mediated by an evolutionarily conserved family of soluble transport factors, the karyopherins (referred to as importins and exportins). The yeast karyopherin Kap114p has previously been shown to import histones H2A and H2B, Nap1p, and a component of the preinitiation complex (PIC), TBP. Using a proteomic approach, we have identified several potentially new cargoes for Kap114p. These cargoes include another PIC component, the general transcription factor IIB or Sua7p, which interacted directly with Kap114p. Consistent with its role as a Sua7p import factor, deletion of KAP114 led to specific mislocalization of Sua7p to the cytoplasm. An interaction between Sua7p and TBP was also detected in cytosol, raising the possibility that both Sua7p and TBP can be coimported by Kap114p. We have also shown that Kap114p possesses multiple overlapping binding sites for its partners, Sua7p, Nap1p, and H2A and H2B, as well as RanGTP and nucleoporins. In addition, we have assembled an in vitro complex containing Sua7p, Nap1p, and histones H2A and H2B, suggesting that this Kap may import several proteins simultaneously. The import of more than one cargo at a time would increase the efficiency of each import cycle and may allow the regulation of coimported cargoes.

Active Transport, Cell Nucleus↗

PubMed2003

Karyopherin-mediated nuclear import of the homing endonuclease VMA1-derived endonuclease is required for self-propagation of the coding region.

VMA1-derived endonuclease (VDE), a site-specific endonuclease in Saccharomyces cerevisiae, enters the nucleus to generate a double-strand break in the VDE-negative allelic locus, mediating the self-propagating gene conversion called homing. Although VDE is excluded from the nucleus in mitotic cells, it relocalizes at premeiosis, becoming localized in both the nucleus and the cytoplasm in meiosis. The nuclear localization of VDE is induced by inactivation of TOR kinases, which constitute central regulators of cell differentiation in S. cerevisiae, and by nutrient depletion. A functional genomic approach revealed that at least two karyopherins, Srp1p and Kap142p, are required for the nuclear localization pattern. Genetic and physical interactions between Srp1p and VDE imply direct involvement of karyopherin-mediated nuclear transport in this process. Inactivation of TOR signaling or acquisition of an extra nuclear localization signal in the VDE coding region leads to artificial nuclear localization of VDE and thereby induces homing even during mitosis. These results serve as evidence that VDE utilizes the host systems of nutrient signal transduction and nucleocytoplasmic transport to ensure the propagation of its coding region.

Active Transport, Cell Nucleus↗

PubMed2006

Karyopherin flexibility in nucleocytoplasmic transport.

Recent structural work on nuclear transport factors of the importin-beta superfamily of karyopherins has shown that these proteins are superhelices of HEAT repeats that are able to assume different conformations in different functional states. The inherent flexibility of these helicoids facilitates the accommodation of different binding partners by an induced-fit type of mechanism. Moreover, the energy stored by distorting these molecules may partially balance binding energies to enable assembly and disassembly of their complexes with relatively small energy changes. Flexibility appears to be an intrinsic feature of such superhelices and might be functionally important not only for karyopherins and nuclear transport, but also for HEAT repeat proteins from other biological systems.

Active Transport, Cell Nucleus↗

PubMed2000

A simple, solid-phase binding assay for the nuclear import receptor karyopherin alpha. Part 1: direct binding.

The nuclear import receptor karyopherin alpha recognizes nuclear localization signals (NLSs), peptides that direct the transport of proteins into the nucleus. A simple, colorimetric assay has been developed to facilitate the identification and comparison of karyopherin ligands by direct and competitive binding using NLSs immobilized on the solid phase (TentaGel resin).

Alkaline Phosphatase↗

PubMed2003

Unconventional tethering of Ulp1 to the transport channel of the nuclear pore complex by karyopherins.

The ubiquitin-like protein SUMO-1 (small ubiquitin-related modifier 1) is covalently attached to substrate proteins by ligases and cleaved by isopeptidases. Yeast has two SUMO-1-deconjugating enzymes, Ulp1 and Ulp2, which are located at nuclear pores and in the nucleoplasm, respectively. Here we show that the catalytic C-domain of Ulp1 must be excluded from the nucleoplasm for cell viability. This is achieved by the noncatalytic N-domain, which tethers Ulp1 to the nuclear pores. The bulk of cellular Ulp1 is not associated with nucleoporins but instead associates with three karyopherins (Pse1, Kap95 and Kap60), in a complex that is not dissociated by RanGTP in vitro. The Ulp1 N-domain has two distinct binding sites for Pse1 and Kap95/Kap60, both of which are required for anchoring to the nuclear pore complex. We propose that Ulp1 is tethered to the nuclear pores by a Ran-insensitive interaction with karyopherins associated with nucleoporins. This location could allow Ulp1 to remove SUMO-1 from sumoylated cargo proteins during their passage through the nuclear pore channel.

Active Transport, Cell Nucleus↗

PubMed2003

Domains of Gln3p interacting with karyopherins, Ure2p, and the target of rapamycin protein.

Gln3p is a GATA-type transcription factor responsive to the quality of nitrogen and carbon. In preferred nitrogen such as glutamine, Gln3p is phosphorylated and sequestered in the cytoplasm in a manner that is dependent on the target of rapamycin (TOR) protein and Ure2p. In nonpreferred nitrogen or nitrogen starvation, Gln3p is dephosphorylated and imported into the nucleus via karyopherin alpha/Srp1p. Upon reintroduction of preferred nitrogen, Gln3p is exported from the nucleus by Crm1p/Xpo1p. Although recent work has provided insights into Gln3p, a more detailed understanding is needed to elucidate the mechanism of its localization and function. In this study, we show that Gln3p contains canonical nuclear localization signal and nuclear export signal sequences necessary for its localization and interaction with its relevant karyopherins. In addition, we identify an N-terminal domain of Gln3p interacting with Ure2p and a C-terminal region for binding to TOR. Finally, we find a lysine/arginine-rich domain essential for the rapamycin-sensitive function, but dispensable for its localization. Our results reveal key domains of Gln3p important for its function and regulation.

Binding Sites↗

PubMed2003

Molecular basis for the rapid dissociation of nuclear localization signals from karyopherin alpha in the nucleoplasm.

The yeast karyopherin heterodimer Kap60p.Kap95p facilitates nuclear import of proteins bearing a classic nuclear localization signal (NLS). The alpha subunit Kap60p binds to the NLS of cargo molecules in the cytoplasm, forming stable complexes that must ultimately dissociate in the nucleoplasm. Although Kap60p can release NLSs on its own using an autoinhibitory sequence (AIS) motif that can occupy the NLS binding site, that mechanism is too slow to support rapid nuclear import. We previously showed that the nuclear basket nucleoporin Nup2p and the exportin complex Cse1p.Gsp1p.GTP function as karyopherin release factors (KaRFs) because they can accelerate the rate of dissociation of NLSs from Kap60p. Here we dissect the molecular mechanics of their KaRF activity. We show that Cse1p accelerates dissociation of Kap60p.NLS-cargo complexes and Kap60p.Nup2p complexes by increasing the affinity of Kap60p for its AIS motif. In contrast, Nup2p uses a conserved sequence motif (VMXXRKIA) coupled to an AIS-like motif to accelerate dissociation of Kap60p.NLS complexes in a vectorial reaction mechanism. Mutation of either motif in Nup2p leads to a loss of KaRF activity and to the accumulation of Kap60p.NLS-cargo complexes in the nucleoplasm of yeast. We discuss a model whereby Nup2p, Cse1p, and Gsp1p cooperate to establish directionality in the movement of Kap60p and NLS-cargos across the nuclear pore complex.

Active Transport, Cell Nucleus↗

PubMed2001

The karyopherin Kap142p/Msn5p mediates nuclear import and nuclear export of different cargo proteins.

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap-substrate complexes, the endogenous RPA-Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482-486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284-2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231-1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501-512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.

4-Nitroquinoline-1-oxide↗

PubMed2001

Nuclear localization of the tyrosine kinase Itk and interaction of its SH3 domain with karyopherin alpha (Rch1alpha).

We report a physical and functional association between the Tec-family tyrosine kinase Itk (Emt/Tsk) and the nuclear import chaperone karyopherin alpha (Rch1alpha) in human T cells. The Itk-SH3 domain and the Rch1alpha proline-rich (PR) motif were crucial for the Itk/Rch1alpha constitutive interaction as demonstrated by directed mutagenesis of the Rch1alpha PR motif (proline 242 to alanine, P242A). TCR-CD3 stimulation of Jurkat T cells resulted in increased Itk/Rch1alpha complex formation, recruitment of karyopherin beta to the protein complex and Rch1alpha tyrosine phosphorylation. Analysis of in vitro kinase reactions with a panel of recombinant glutathione-S-transferase (GST) fusion tyrosine kinases (Itk, Lck, ZAP-70 and Jak3) revealed that only GST-Itk efficiently phosphorylated a recombinant GST-Rch1alpha fusion. We observed constitutive nuclear localization of Itk that was up-regulated following either TCR-CD3 stimulation or over-expression of wild-type Rch1alpha in T cells. Further, nuclear localization of Itk and TCR-CD3-mediated IL-2 production were significantly down-regulated following expression of the Rch1alpha-P242A mutant, implicating a role for Rch1alpha in the nuclear translocation of Itk.

Active Transport, Cell Nucleus↗

PubMed1996

Role of the karyopherin pathway in human immunodeficiency virus type 1 nuclear import.

The interaction of the human immunodeficiency virus type 1 (HIV-1) nucleoprotein complex with the cell nuclear import machinery is necessary for viral replication in macrophages and for the establishment of infection in quiescent T lymphocytes. The karyophilic properties of two viral proteins, matrix (MA) and Vpr, are keys to this process. Here, we show that an early step of HIV-1 nuclear import is the recognition of the MA nuclear localization signal (NLS) by Rch1, a member of the karyopherin-alpha family. Furthermore, we demonstrate that an N-terminally truncated form of Rch1 which binds MA but fails to localize to the nucleus efficiently blocks MA- but not Vpr-mediated HIV-1 nuclear import. Correspondingly, NLS peptide inhibits the nuclear migration of MA but not that of Vpr and prevents the infection of terminally differentiated macrophages by vpr-defective virus but not wild-type virus. These results are consistent with a model in which Rch1 or another member of the karyopherin-alpha family, through the recognition of the MA NLS, participates in docking the HIV-1 nucleoprotein complex at the nuclear pore. In addition, our data suggest that Vpr governs HIV-1 nuclear import through a distinct pathway.

Amino Acid Sequence↗

PubMed2004

Paired-type homeodomain transcription factors are imported into the nucleus by karyopherin 13.

We report that the paired homeodomain transcription factor Pax6 is imported into the nucleus by the Karyopherin beta family member Karyopherin 13 (Kap13). Pax6 was identified as a potential cargo for Kap13 by a yeast two-hybrid screen. Direct binding of Pax6 to Kap13 was subsequently confirmed by in vitro assays with recombinant proteins, and binding in vivo was shown by coimmunoprecipitation. Ran-dependent import of Pax6 by Kap13 was shown to occur by using a digitonin-permeabilized cells assay. Kap13 binds to Pax6 via a nuclear localization sequence (NLS), which is located within a segment of 80 amino acid residues that includes the homeodomain. Kap13 showed reduced binding to Pax6 when either region located at each end of the homeodomain (208 to 214 and 261 to 267) was deleted. The paired-type homeodomain transcription factor family includes more than 20 members. All members contain a region similar to the NLS found in Pax6 and are therefore likely to be imported by Kap13. We confirmed this hypothesis for Pax3 and Crx, which bind to and are imported by Kap13.

Amino Acid Sequence↗

PubMed2001

[Dynamics of the interaction of Stat1 transcription factor with the internalized EGF receptor and karyopherins alpha in response to stimulation of cells with epidermal growth factor].

The present paper deals with two aspects of EGF-induced signal transduction via transcriptional factor STAT1. Utilizing vesicle fractionation in Percoll density gradient followed by co-immunoprecipitation, we observed the association of STAT1 with EGF receptor internalized in early endosomes. Co-immunoprecipitation studies with antikaryopherin alpha antibody showed the binding of activated STAT1 to nuclear import factors--karyopherins alpha. Both complexes demonstrate similar dynamics upon EGF treatment: they are formed at the early times, cannot be detected within 15 min, and reappear in 20 min or later. The complex of STAT1 and karyopherin alpha is localized in the cytoskeletal fraction.

Animals↗

PubMed2001

The dynamics of karyopherin-mediated nuclear transport.

The regulated exchange of proteins and nucleic acids between the nucleus and cytoplasm demands a complex interplay between nuclear pore complexes (NPCs), which provide conduits in the nuclear envelope, and mobile transport receptors (or karyopherins, also known as importins/exportins) that bind and mediate the translocation of cargoes through the NPCs. Biochemical characterization of individual karyopherins has led to the identification of many of their cargoes and to the elucidation of the mechanisms by which they mediate transport. Likewise, the characterization of numerous NPC-associated components, in combination with structural studies of NPCs, have begun to address the possible mechanisms that drive nucleocytoplasmic transport, and the role that different nucleoporins play in the transport process. Some recent studies indicate that several NPC-associated factors, previously thought to be stable components of the NPC, dynamically interact with both nuclear and cytoplasmic aspects of the NPC. The mobility of these components challenges our conventional view of the NPC as the stationary phase of transport. These components and their potiential roles in nucleo-cytoplasmic transport are discussed.

Active Transport, Cell Nucleus↗