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PubMed1995

The essential yeast nucleoporin NUP159 is located on the cytoplasmic side of the nuclear pore complex and serves in karyopherin-mediated binding of transport substrate.

We have identified a new yeast nucleoporin of 159 kDa that we term NUP159. Immunofluorescence microscopy with a monospecific monoclonal antibody against NUP159 gave the punctate nuclear rim staining characteristic of nucleoporins. Immunogold electron microscopy with isolated yeast NEs yielded decoration of only the cytoplasmic side of the nuclear pore complex. The gene encoding NUP159 is essential, and, like some other nucleoporins, NUP159 contains a coiled-coil domain as well as a domain of repeated motifs. Five segments of NUP159, covering its entire length, were expressed in Escherichia coli. The repeat motif-containing segment was found to bind a nuclear transport substrate in the presence of vertebrate cytosolic extract containing nuclear transport factors. This segment also bound 35S-labeled mammalian karyopherin beta, one such transport factor that mediates the docking of substrates to the nuclear pore complex. These data establish a direct biochemical link between the repeat motif domain of a yeast nucleoporin, transport factors, (specifically karyopherin beta), and nuclear transport substrates. Its cytoplasmic aspect implies a role for NUP159 in nuclear import.

Amino Acid Sequence↗

PubMed1999

Domains of Crm1 involved in the formation of the Crm1, RanGTP, and leucine-rich nuclear export sequences trimeric complex.

Nuclear export of proteins containing a leucine-rich nuclear export sequence (NES) is mediated by a specific NES receptor known as Crm1. This protein, which is related to the karyopherin beta family, interacts directly with NES in a RanGTP-dependent manner. To characterize the domains of Crm1 involved in formation of the trimeric Crm1-NES-RanGTP complex, N- and C-terminal deletion mutants of Crm1 were generated and their ability to bind NES and RanGTP in vitro was analyzed. Our results indicate that two regions of Crm1 are required for the formation of the trimeric Crm1-NES-RanGTP complex, the N-terminal domain of Crm1 and the central domain of the receptor, starting after residue 160 with an essential region between 566 and 720. The N-terminal domain is homologous to the RanGTP-binding domain of karyopherin beta and therefore is likely involved in the interaction with RanGTP. Consequently, the central domain likely corresponds to the NES-binding site of Crm1.

Amino Acid Sequence↗

PubMed2000

Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF).

Epsin (Eps15 interactor) is a cytosolic protein involved in clathrin-mediated endocytosis via its direct interactions with clathrin, the clathrin adaptor AP-2, and Eps15. The NH(2)-terminal portion of epsin contains a phylogenetically conserved module of unknown function, known as the ENTH domain (epsin NH(2)-terminal homology domain). We have now solved the crystal structure of rat epsin 1 ENTH domain to 1.8 A resolution. This domain is structurally similar to armadillo and Heat repeats of beta-catenin and karyopherin-beta, respectively. We have also identified and characterized the interaction of epsin 1, via the ENTH domain, with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). Leptomycin B, an antifungal antibiotic, which inhibits the Crm1- dependent nuclear export pathway, induces an accumulation of epsin 1 in the nucleus. These findings suggest that epsin 1 may function in a signaling pathway connecting the endocytic machinery to the regulation of nuclear function.

Adaptor Proteins, Vesicular Transport↗

PubMed1997

Evolutionary specialization of the nuclear targeting apparatus.

The alpha- and beta-karyopherins (Kaps), also called importins, mediate the nuclear transport of proteins. All alpha-Kaps contain a central domain composed of eight approximately 40 amino acid, tandemly arranged, armadillo-like (Arm) repeats. The number and order of these repeats have not changed since the common origin of fungi, plants, and mammals. Phylogenetic analysis suggests that the various alpha-Kaps fall into two groups, alpha1 and alpha2. Whereas animals encode both types, the yeast genome encodes only an alpha1-Kap. The beta-Kaps are characterized by 14-15 tandemly arranged HEAT motifs. We show that the Arm repeats of alpha-Kaps and the HEAT motifs of beta-Kaps are similar, suggesting that the alpha-Kaps and beta-Kaps (and for that matter, all Arm and HEAT repeat-containing proteins) are members of the same protein superfamily. Phylogenetic analysis indicates that there are at least three major groups of beta-Kaps, consistent with their proposed cargo specificities. We present a model in which an alpha-independent beta-Kap progenitor gave rise to the alpha-dependent beta-Kaps and the alpha-Kaps.

Amino Acid Sequence↗

PubMed2003

Intersection of the Kap123p-mediated nuclear import and ribosome export pathways.

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.

Active Transport, Cell Nucleus↗

PubMed2006

The l2 minor capsid protein of low-risk human papillomavirus type 11 interacts with host nuclear import receptors and viral DNA.

Analysis of the interactions of low-risk human papillomavirus type 11 (HPV11) L2 with karyopherin beta (Kap beta) nuclear import receptors revealed that L2 interacted with Kap beta 1, Kap beta 2, and Kap beta 3 and formed a complex with the Kap alpha 2 beta 1 heterodimer. HPV11 L2 contains two nuclear localization signals (NLSs)-in the N terminus and the C terminus-that could mediate its nuclear import via a classical pathway. Each NLS was functional in vivo, and deletion of both of them abolished L2 nuclear localization. Both NLSs interacted with the viral DNA. Thus, HPV11 L2 can interact with several karyopherins and the viral DNA and may enter the nucleus via multiple pathways.

Active Transport, Cell Nucleus↗

PubMed1998

Regulation of NF-kappa B, AP-1, NFAT, and STAT1 nuclear import in T lymphocytes by noninvasive delivery of peptide carrying the nuclear localization sequence of NF-kappa B p50.

Activation of T lymphocytes by Ags or cytokines results in translocation of the transcription factors NF-kappa B, AP-1, NFAT, and STAT from the cytoplasm into the nucleus. The first step in the nuclear import process is recognition of a nuclear localization sequence (NLS) within the karyophilic protein by a cytoplasmic receptor such as the importin (karyopherin)-alpha subunit. The NLSs of NF-kappa B, AP-1, and NFAT differ and the NLS of STAT1 has not yet been identified. Herein we demonstrate that the inducible nuclear import of NF-kappa B, AP-1, NFAT, and STAT1 in Jurkat T lymphocytes is significantly inhibited by a cell-permeable peptide carrying the NLS of the NF-kappa B p50 subunit. NLS peptide-mediated disruption of the nuclear import of these transcription factors results in inhibition of I kappa B alpha and IL-2 gene expression, processes dependent on NF-kappa B or the combination of NF-kappa B, AP-1, and NFAT. Further, we show that inhibitory NLS peptide interacts in vitro with a cytoplasmic NLS receptor complex comprised of the Rch1/importin (karyopherin)-beta heterodimer expressed in Jurkat T cells. Taken together, these data indicate that the inducible nuclear import of NF-kappa B, AP-1, NFAT, and STAT1 in Jurkat T cells can be regulated by NLS peptide delivered noninvasively to the cytoplasm of Jurkat T cells to target members of the importin (karyopherin)-alpha beta NLS receptor complex.

Amino Acid Sequence↗

PubMed1999

Kap104p-mediated nuclear import. Nuclear localization signals in mRNA-binding proteins and the role of Ran and Rna.

Kap104p is a Saccharomyces cerevisiae nuclear import receptor for two essential mRNA-binding proteins, Nab2p and Nab4p/Hrp1p. We demonstrate direct binding of Kap104p to each of these substrates. We have defined the nuclear localization signals in both Nab2p and Nab4p/Hrp1p by Kap104p binding in vitro and KAP104-dependent nuclear import in vivo. The nuclear localization signals map to similar arginine/glycine-rich RNA-binding domains in both proteins and are thus termed rg-nuclear localization signals to distinguish them from classical nuclear localization signals. We also demonstrate that Kap104p, like other known beta-karyopherins (or importins), interacts directly with the small GTPase Ran/Gsp1. However, unlike other known import factors, Ran binding is not sufficient to mediate release of substrates from Kap104p; efficient Ran-GTP-mediated substrate release requires RNA. Also, addition of Kap104p to Nab2p and Nab4p/Hrp1p prebound to single-stranded DNA-cellulose stimulated release of both proteins from the resin. We suggest a simple cycle in which Nab2p and Nab4p/Hrp1p, upon import, are released in the nucleus at sites of transcription by the concerted action of Ran-GTP and binding to newly synthesized mRNA. The resulting ribonucleoprotein complexes are exported to the cytoplasm, where Kap104p rebinds to Nab2p and Nab4p/Hrp1p, contributing to their release from mRNA.

Amino Acid Sequence↗

PubMed1997

A distinct nuclear import pathway used by ribosomal proteins.

Protein transport into the nucleus is governed by the interaction of soluble transport factors with their import substrates and nuclear pore complexes. Here, we identify a major distinct nuclear import pathway, mediated by a previously uncharacterized yeast beta karyopherin Kap123p. The predominant substrates for this pathway are ribosomal proteins, which must be imported into the nucleus prior to assembly into pre-ribosomes. Kap123p binds directly to its transport substrates, repeat motif-containing nucleoporins, and Ran-GTP. We show that the related protein Pse1p is also a karyopherin and can functionally substitute for Kap123p; both are capable of specifically directing a ribosomal nuclear localization signal reporter to the nucleus in vivo.

Amino Acid Sequence↗

PubMed2005

Zinc finger domain of Snail functions as a nuclear localization signal for importin beta-mediated nuclear import pathway.

Snail, a DNA-binding zinc finger protein, functions as a transcriptional repressor for genes including E-cadherin during development and the acquisition of tumor cell invasiveness. Human Snail is a 264-amino acid nuclear protein with an amino-terminal basic amino acid-rich domain (SNAG domain) and a carboxyl-terminal DNA-binding domain (zinc finger domain). A series of fusion proteins composed of green fluorescent protein (GFP) and portions of the Snail protein were generated, and their subcellular localization was examined. Fusion of the four zinc fingers to GFP led to the targeting of GFP to the nucleus, demonstrating that the zinc finger domain is sufficient for nuclear localization. Using an in vitro transport system, the nuclear import of Snail was reconstituted by importin (karyopherin) beta in the presence of Ran and NTF2. We further demonstrated that Snail binds directly to importin beta in a zinc finger domain-dependent manner. These results indicate that zinc finger domain of Snail functions as a nuclear localization signal and Snail can be transported into the nucleus in an importin beta-mediated manner.

Animals↗

PubMed2002

A defect of Kap104 alleviates the requirement of mitotic exit network gene functions in Saccharomyces cerevisiae.

A subgroup of the karyopherin beta (also called importin beta) protein that includes budding yeast Kap104 and human transportin/karyopherin beta2 is reported to function as a receptor for the transport of mRNA-binding proteins into the nucleus. We identified KAP104 as a responsible gene for a suppressor mutation of cdc15-2. We found that the kap104-E604K mutation suppressed the temperature-sensitive growth of cdc15-2 cells by promoting the exit from mitosis and suppressed the temperature sensitivity of various mitotic-exit mutations. The cytokinesis defect of these mitotic-exit mutants was not suppressed by kap104-E604K. Furthermore, the kap104-E604K mutation delays entry into DNA synthesis even at a permissive temperature. In cdc15-2 kap104-E604K cells, SWI5 and SIC1, but not CDH1, became essential at a high temperature, suggesting that the kap104-E604K mutation promotes mitotic exit via the Swi5-Sic1 pathway. Interestingly, SPO12, which is involved in the release of Cdc14 from the nucleolus during early anaphase, also became essential in cdc15-2 kap104-E604K cells at a high temperature. The kap104-E604K mutation caused a partial delocalization of Cdc14 from the nucleolus during interphase. This delocalization of Cdc14 was suppressed by the deletion of SPO12. These results suggest that a mutation in Kap104 stimulates exit from mitosis through the activation of Cdc14 and implies a novel role for Kap104 in cell-cycle progression in budding yeast.

Base Sequence↗

PubMed2004

Importin alpha: a multipurpose nuclear-transport receptor.

The importin alpha/beta heterodimer targets hundreds of proteins to the nuclear-pore complex (NPC) and facilitates their translocation across the nuclear envelope. Importin alpha binds to classical nuclear localization signal (cNLS)-containing proteins and links them to importin beta, the karyopherin that ferries the ternary complex through the NPC. A second karyopherin, the exportin CAS, recycles importin alpha back to the cytoplasm. In this article, we discuss control mechanisms that importin alpha exerts over the assembly and disassembly of the ternary complex and we describe how new groups of importin alpha genes arose during the evolution of metazoan animals to function in development and differentiation. We also describe activities of importin alpha that seem to be distinct from its housekeeping functions in nuclear transport.

Active Transport, Cell Nucleus↗

PubMed1998

Transport routes through the nuclear pore complex.

The nuclear pore complex can be considered to be the stationary phase of bidirectional traffic between the nucleus and the cytoplasm. The mobile phase consists of karyopherins, transport substrates, and the small GTPase Ran and its modulators. Recently, the family of karyopherins was expanded with the recognition of numerous open reading frames with limited homology to karyopherin beta 1. In several cases, the specific substrates transported by the new karyopherins have been identified, allowing the characterization of new pathways into and out of the nucleus. However, the mechanisms of transport, particularly the role of Ran, remain poorly understood.

Animals↗

PubMed1998

Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein.

The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin alpha/importin alpha, which acts as the NLS receptor, and karyopherin beta1/importin beta, which binds karyopherin alpha and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin beta1, termed karyopherin beta2 or transportin, and does not require a karyopherin alpha-like adapter protein. A yeast homolog of karyopherin beta2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin beta1, but not the Kap104p homolog karyopherin beta2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin alpha. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

Amino Acid Sequence↗

PubMed2000

Mobility within the nucleus and neighboring cytosol is a key feature of prothymosin-alpha.

Prothymosin alpha is a small, unfolded, negatively charged, poorly antigenic mammalian protein with a potent nuclear localization signal. Although it is apparently essential for growth, its precise function is unknown. We examined the location and behavior of the protein bearing different epitope tags using in situ immunolocalization in COS-1 and NIH3T3 cells. Tagged prothymosin alpha appeared to be punctate and widely dispersed throughout the nucleus, with the exception of the nucleolus. A tiny cytoplasmic component, which persisted in the presence of cycloheximide and actinomycin D during interphase, became pronounced immediately before, during, and after mitosis. When nuclear uptake was abrogated, small tagged prothymosin alpha molecules, but not prothymosin alpha fused to beta-galactosidase, accumulated significantly in the cytoplasm. Tagged prothymosin alpha shared domains with mobile proteins such as Ran, transportin, and karyopherin beta, which also traverse the nuclear membrane, and co-localized with active RNA polymerase II. Mild digitonin treatment resulted in nuclei devoid of prothymosin alpha. The data do not support tight binding to any nuclear component. Therefore, we propose that prothymosin alpha is a highly diffusible bolus of salt and infer that it facilitates movement of charged molecules in highly charged environments within and near the nucleus.

3T3 Cells↗

PubMed2004

Karyopherins: from nuclear-transport mediators to nuclear-function regulators.

The karyopherin beta (or importin beta) family comprises soluble transport factors that mediate the movement of proteins and RNAs between the nucleus and cytoplasm. Recent studies have extended the role of karyopherins to regulating assembly of the nuclear pore complex (NPC), assembly of the nuclear envelope, mitosis and replication. New data also address how karyopherins specifically recognize and transport many distinct cargoes and traverse the NPC. These data raise the possibility that, although there might be a universal mechanism for nuclear transport, specific interactions between karyopherins and components of the NPC might function to regulate differentially the ability of the different karyopherins to cross the NPC.

Active Transport, Cell Nucleus↗

PubMed2004

Monitoring the expression profiles of doxorubicin-resistant K562 human leukemia cells by serial analysis of gene expression.

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis.

Delta-5 Fatty Acid Desaturase↗

PubMed2001

Proteomic analysis of nucleoporin interacting proteins.

The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport. Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways. Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts. We find that many proteins bind to FG Nups in highly reproducible patterns. Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins. The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins. Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p. In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer. When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding. The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex.

Cell Nucleus↗