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Results for “Nuclear Localization Signals”

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PubMed2007

Intracellular transport of hepatitis B virus.

For genome multiplication hepadnaviruses use the transcriptional machinery of the cell that is found within the nucleus. Thus the viral genome has to be transported through the cytoplasm and nuclear pore. The intracytosolic translocation is facilitated by the viral capsid that surrounds the genome and that interacts with cellular microtubules. The subsequent passage through the nuclear pore complexes (NPC) is mediated by the nuclear transport receptors importin alpha and beta. Importin alpha binds to the C-terminus of the capsid protein that comprises a nuclear localization signal (NLS). The exposure of the NLS is regulated and depends upon genome maturation and/or phosphorylation of the capsid protein. As for other karyophilic cargos using this pathway importin alpha interacts with importin beta that facilitates docking of the import complex to the NPC and the passage through the pore. Being a unique strategy, the import of the viral capsid is incomplete in that it becomes arrested inside the nuclear basket, which is a cage-like structure on the karyoplasmic face of the NPC. Presumably only this compartment provides the factors that are required for capsid disassembly and genome release that is restricted to those capsids comprising a mature viral DNA genome.

Active Transport, Cell Nucleus↗

PubMed1993

70-kDa heat-shock cognate protein colocalizes with karyophilic proteins into the nucleus during their transport in vitro.

Recently, we showed that antibodies against 70-kDa heat-shock cognate 66otein (hsc70) inhibit nuclear transport of karyophilic proteins in vivo. In this study, we examined the involvement of hsc70 in nuclear transport using a digitonin-permeabilized cell-free transport system. Depletion of the cytosolic extract required for nuclear transport of hsc70 by incubation with anti-hsc70 antibodies reduced the nuclear transport activity significantly, and addition of purified hsc70 to the depleted extract restored the transport activity. We examined the localization of hsc70 during nuclear transport in vitro by indirect immunofluorescence studies. Hsc70 accumulated in the nucleus when bovine serum albumin (BSA) conjugated to SV40 large T-antigen nuclear localization signal (NLS) peptides (T-BSA) or nucleoplasmin was added exogenously to the cytosolic extract, but not when BSA conjugated to transport-incompetent point-mutated NLS peptide was added. This karyophilic protein-dependent accumulation of hsc70 was dependent on the cytosolic extract, temperature, and ATP and was sensitive to wheat germ agglutinin. Addition of excess unlabeled T-BSA to the cytosolic extract competitively inhibited the nuclear accumulation of fluorescently labeled T-BSA or nucleoplasmin, but did not affect accumulation of hsc70 into the nucleus. These results show that hsc70 is required for nuclear transport and that it is colocalized with karyophilic proteins during their active import into the nucleus in vitro.

Adenosine Triphosphate↗

PubMed1996

Nuclear import in permeabilized protoplasts from higher plants has unique features.

The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.

Amino Acid Sequence↗

PubMed1998

Ataxin-3 is transported into the nucleus and associates with the nuclear matrix.

It has been reported that the ataxin-3 protein containing a polyglutamine sequence in the pathological range (61-84Q) is localized within the nucleus of neuronal cells, whereas ataxin-3 with a normal repeat length (12-37Q) is predominantly a cytoplasmic protein. In this study, the subcellular localization of the full-length ataxin-3 protein with a glutamine sequence in the normal range (Q3KQ22) was analysed in two mammalian cell lines. Using two affinity-purified polyclonal antibodies raised against the N- or C-terminal portion of ataxin-3, the protein was detected predominantly, but not exclusively, in the nucleus of COS-7 as well as neuroblastoma cells by immunofluorescence and confocal laser scanning microscopy (CLSM). The distribution of the protein in these cellular compartments was confirmed by biochemical subcellular fractionations. Furthermore, CLSM revealed that the ataxin-3 protein present in the nucleus of neuroblastoma cells is associated with the inner nuclear matrix. Our results taken together with the finding of a nuclear localization signal in ataxin-3 indicate that the ataxin-3 protein per se translocates to the nucleus and that an expanded glutamine repeat is not essential for this transport.

Animals↗

PubMed2005

HEXIM1 forms a transcriptionally abortive complex with glucocorticoid receptor without involving 7SK RNA and positive transcription elongation factor b.

The HEXIM1 protein has been shown to form a protein-RNA complex composed of 7SK small nuclear RNA and positive transcription elongation factor b (P-TEFb), which is composed of cyclin-dependent kinase 9 (CDK9) and cyclin T1, and to inhibit the kinase activity of CDK9, thereby suppressing RNA polymerase II-dependent transcriptional elongation. Here, we biochemically demonstrate that HEXIM1 forms a distinct complex with glucocorticoid receptor (GR) without RNA, CDK9, or cyclin T1. HEXIM1, through its arginine-rich nuclear localization signal, directly associates with the ligand-binding domain of GR. Introduction of HEXIM1 short interfering RNA and adenovirus-mediated exogenous expression of HEXIM1 positively and negatively modulated glucocorticoid-responsive gene activation, respectively. In the nucleus, HEXIM1 was shown to localize in a distinct compartment from that of the p160 coactivator transcriptional intermediary factor 2. Overexpression of HEXIM1 decreased ligand-dependent association between GR and transcriptional intermediary factor 2. Antisense-mediated disruption of 7SK blunted the negative effect of HEXIM1 on arylhydrocarbon receptor-dependent transcription but not on GR-mediated one, indicating that a class of transcription factors are direct targets of HEXIM1. These results indicate that HEXIM1 has dual roles in transcriptional regulation: inhibition of transcriptional elongation dependent on 7SK RNA and positive transcription elongation factor b and interference with the sequence-specific transcription factor GR via a direct protein-protein interaction. Moreover, the fact that the central nuclear localization signal of HEXIM1 is essential for both of these actions may argue the crosstalk of these functions.

Animals↗

PubMed2001

KIN241: a gene involved in cell morphogenesis in Paramecium tetraurelia reveals a novel protein family of cyclophilin-RNA interacting proteins (CRIPs) conserved from fission yeast to man.

In this study, we report cloning, by functional complementation of the KIN241 gene involved in Paramecium cell morphogenesis, cortical organization and nuclear reorganization. This gene is predicted to encode a protein of a novel type, comprising a cyclophilin-type, peptidyl-prolyl isomerase domain, an RNA recognition motif, followed by a region rich in glutamate and lysine (EK domain) and a C-terminal string of serines. As homologues of this protein are present in the genomes of Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, Arabidopsis thaliana and Homo sapiens, the Kin241p predicted sequence defines a new family of proteins that we propose to call 'CRIP', for cyclophilin-RNA interacting protein. We demonstrate that, in Paramecium, Kin241p is localized in the nucleus and that deletion of some nuclear localization signals (NLSs) decreases transport of the protein into the nucleus. No Kin241-1 protein is present in mutant cells, suggesting that the C-terminal serine-rich region is responsible for protein stability.

Amino Acid Motifs↗

PubMed1999

Cloning and characterization of ASH2L and Ash2l, human and mouse homologs of the Drosophila ash2 gene.

Drosophila ash2 belongs to the trithorax (trx) gene family. The ash2 product positively regulates expression of homeotic selector genes, and is implicated in early development and formation of various disc patterns in the fruit fly. Through large-scale sequencing of human genomic DNA coupled with in silico gene trapping, we identified a gene (ASH2L) on chromosome 8p11.2 whose predicted product was highly homologous to ash2, characterized it, and identified its mouse counterpart. The human ash2 cDNA is 2368 bp long, encoding 628 amino acids. The 16-exon gene spans more than 34 kb of genomic DNA between STS markers WI-9207 (centromere) and AFMA295ZD5 (telomere) on chromosome 8, with transcription oriented telomere to centromere. The ash2 genes are highly conserved among different species, including C. elegans and yeast. The presence of a conserved bipartite nuclear localization signal and a PHD finger motif in the human ash2 gene suggests that the gene product would function as a transcriptional regulator in humans, as its homologue does in Drosophila.

Amino Acid Sequence↗

PubMed2003

Regulation of PI4,5P2 synthesis by nuclear-cytoplasmic shuttling of the Mss4 lipid kinase.

The essential phospholipid PI4,5P(2) is generated by a well conserved PI4P 5-kinase, Mss4, in yeast. Balanced production and turnover of PI4,5P(2) is important for normal organization of the actin cytoskeleton and cell viability. Previous studies have shown that multiple PI phosphatases can regulate PI4,5P(2) levels. We report a new, unexpected regulatory mechanism for PI4,5P(2) homeostasis, directed by nuclear-cytoplasmic shuttling of the lipid kinase. We show that Mss4 is a phosphoprotein, which contains a functional nuclear localization signal (NLS) and can shuttle between the cytoplasm and the nucleus. Temperature-conditional mss4 cells that accumulate Mss4 protein in the nucleus exhibit reduced levels of PI4,5P(2), depolarization of the actin cytoskeleton and a block in Mss4 phosphorylation, suggesting an essential role for phosphorylated Mss4 at the plasma membrane. Through the isolation of gene dosage-dependent suppressors of mss4 mutants, we identified Bcp1, a protein enriched in the nucleus, which is required for Mss4 nuclear export and is related to the mammalian BRCA2-interacting protein BCCIP. Together, these studies suggest a new mechanism for lipid kinase regulation through regulated nuclear-cytoplasmic shuttling.

Actins↗

PubMed2000

Further characterization of the actin-related protein Act3p/Arp4 of S. cerevisiae through mutational analysis.

In the course of functional analysis of the yeast ARP4 (ACT3) gene, we further characterized the role of its protein product Arp4p in the cell. We report that although it is localized in the nucleus, Arp4p performs its function independently of binding to DNA directly. The roles of the core actin structure (the ATP-binding pocket) and a putative Nuclear Localization Signal (NLS) of Arp4p were tested by targeted mutagenesis. The results suggest that under normal conditions, the ATPase activity and the NLS are dispensable for the essential function of this protein in the cell. Furthermore, the use of reporter genes confirmed that Arp4p could be involved in some general mechanism of transcriptional regulation.

Actins↗

PubMed1996

Characterization of ES cells deficient for the p105 precursor (NF-kappa B1): role of p50 NLS.

Proteolytic processing of the p105 precursor (NF-kappa B1) generates the p50 subunit of NF-kappa B. To address the function of p105 precursor, we have generated embryonic stem (ES) cells which lack the C-terminal inhibiting ankyrin-containing domain of p105 (I kappa B gamma) but contain a functional p50 by homologous recombination. Two forms of p50 were found in ES cells lacking p105. While one was a p50 molecule with the expected size of 415 amino acids, the other was an isoform of 358 amino acids generated by alternative splicing. This isoform lacked the C-terminal 62 amino acids of p50 including the putative nuclear localization signal (NLS), but included five unrelated amino acids. This p50 molecule lacking the NLS behaved similar to wild type p50. Importantly, the p50 isoform-RelA complex interacted efficiently with I kappa B alpha, implying that the NLS of p50 is not essential for these functions. Also, p50 isoform dimers were present in the nucleus. Loss of p105 and the augment of total p50 produced an increase in the nuclear kappa B-binding activity of p50-RelA and p50 dimers. In wild type ES cells, a significant proportion of total p50 is associated with p105. Therefore, the mutation introduced in NF-kappa B1 eliminates both the processing to generate p50 and the trapping of p50 by p105. In contrast to p50, only a small fraction of RelA is associated with p105. These findings indicate that p105 plays a unique role in controlling p50 dimer activity.

Alternative Splicing↗

PubMed2004

A Theileria annulata DNA binding protein localized to the host cell nucleus alters the phenotype of a bovine macrophage cell line.

The apicomplexan parasite Theileria annulata is the only intracellular eukaryote that is known to induce the proliferation of mammalian cells. However, as the parasite undergoes stage differentiation, host cell proliferation is inhibited, and the leukocyte is eventually destroyed. We have isolated a parasite gene (SuAT1) encoding an AT hook DNA binding polypeptide that has a predicted signal peptide, PEST motifs, nuclear localization signals, and domains which indicate interaction with regulatory components of the higher eukaryotic cell cycle. The polypeptide is localized to the nuclei of macroschizont-infected cells and was detected at significant levels in cells that were undergoing parasite stage differentiation. Transfection of an uninfected transformed bovine macrophage cell line, BoMac, demonstrated that SuAT1 can modulate cellular morphology and alter the expression pattern of a cytoskeletal polypeptide in a manner similar to that found during the infection of leukocytes by the parasite. Our findings indicate that Theileria parasite molecules that are transported to the leukocyte nucleus have the potential to modulate the phenotype of infected cells.

AT-Hook Motifs↗

PubMed2000

Isolation of a cDNA representing the Fanconi anemia complementation group E gene.

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome with at least seven different complementation groups. Four FA genes (FANCA, FANCC, FANCF, and FANCG) have been identified, and two other FA genes (FANCD and FANCE) have been mapped. Here we report the identification, by complementation cloning, of the gene mutated in FA complementation group E (FANCE). FANCE has 10 exons and encodes a novel 536-amino acid protein with two potential nuclear localization signals.

Alternative Splicing↗

PubMed2005

Nuclear and mitochondrial localization signals overlap within bovine herpesvirus 1 tegument protein VP22.

VP22, a tegument protein of bovine herpesvirus 1, accumulates in the nucleus of infected and transiently transfected cells. Previous studies indicated a possible regulatory function of VP22 within nuclei, but how VP22 enters nuclei is unknown. Despite the abundance of basic residues within this protein, no classic nuclear localization signal (NLS) motif has been identified. To identify the signal directing nuclear accumulation, a series of truncations, internal deletions, and point mutations were constructed. Fluorescence microscopy of cells transfected with VP22 constructs indicated that a sequence of 103 residues is necessary and sufficient for nuclear localization. This NLS sequence is conformation-sensitive in contrast to a classical sequential NLS. Energy depletion assays and co-immunoprecipitation suggested that this NLS sequence also binds histone H4, resulting in nuclear retention of VP22. In addition, a mitochondrial targeting sequence was identified at the C-terminal 49 amino acids, which overlapped the sequence required for nuclear targeting. Our findings demonstrate the diversity of VP22 protein to localize within the cell and provide the opportunity for VP22 to direct cargo specifically to different subcellular compartments.

Animals↗

PubMed2003

Ste11p, a high-mobility-group box DNA-binding protein, undergoes pheromone- and nutrient-regulated nuclear-cytoplasmic shuttling.

The high-mobility-group (HMG) box is a conserved DNA-binding domain found in a family of transcription factors that regulate growth and development. One family member, Ste11p, directs sexual differentiation of Schizosaccharomyces pombe by binding specific DNA sequences upstream of genes required for mating and meiosis. Here, we show that Ste11p is a shuttling protein. In growing cells, Ste11p is present in low levels and is pancellular. Mating pheromones and nutrient limitation trigger nuclear accumulation and increased expression of the transcription factor. Several mechanisms likely control Ste11p localization. First, the 14-3-3 protein, Rad24p, binds phosphorylated Ste11p and inhibits its nuclear accumulation. Second, the HMG domain of Ste11p contains a basic cluster nuclear localization signal. Finally, treatment of cells with leptomycin B, an exportin inhibitor, results in the nuclear accumulation of Ste11p. A Ste11p deletion mutation, DeltaC54, mimics the effects of leptomycin B. The C54 region contains no identifiable nuclear export signal but instead is required for biological activity and to stimulate Ste11p target gene expression. These results provide evidence that both nuclear import and export mechanisms operate to regulate cellular localization of an HMG box protein. In addition, they establish a paradigm for the potential role of pheromone/hormone-like polypeptides in cellular localization of this important class of developmental regulators.

Active Transport, Cell Nucleus↗

PubMed2000

Nup2p, a yeast nucleoporin, functions in bidirectional transport of importin alpha.

Import of proteins containing a classical nuclear localization signal (NLS) into the nucleus is mediated by importin alpha and importin beta. Srp1p, the Saccharomyces cerevisiae homologue of importin alpha, returns from the nucleus in a complex with its export factor Cse1p and with Gsp1p (yeast Ran) in its GTP-bound state. We studied the role of the nucleoporin Nup2p in the transport cycle of Srp1p. Cells lacking NUP2 show a specific defect in both NLS import and Srp1p export, indicating that Nup2p is required for efficient bidirectional transport of Srp1p across the nuclear pore complex (NPC). Nup2p is located at the nuclear side of the central gated channel of the NPC and provides a binding site for Srp1p via its amino-terminal domain. We show that Nup2p effectively releases the NLS protein from importin alpha-importin and beta and strongly binds to the importin heterodimer via Srp1p. Kap95p (importin beta) is released from this complex by a direct interaction with Gsp1p-GTP. These data suggest that besides Gsp1p, which disassembles the NLS-importin alpha-importin beta complex upon binding to Kap95p in the nucleus, Nup2p can also dissociate the import complex by binding to Srp1p. We also show data indicating that Nup1p, a relative of Nup2p, plays a similar role in termination of NLS import. Cse1p and Gsp1p-GTP release Srp1p from Nup2p, which suggests that the Srp1p export complex can be formed directly at the NPC. The changed distribution of Cse1p at the NPC in nup2 mutants also supports a role for Nup2p in Srp1p export from the nucleus.

Active Transport, Cell Nucleus↗

PubMed2001

The N-terminal internal region of BLM is required for the formation of dots/rod-like structures which are associated with SUMO-1.

Bloom Syndrome (BS) is a human autosomal genetic disorder characterized by a predisposition to a variety of malignant tumors. The gene responsible for BS encodes a protein (BLM) consisting of 1417 amino acids with a nuclear localization signal in the C-terminal region, which is a member of the RecQ helicase family. We previously showed, using a yeast two-hybrid system, that BLM interacted with Ubc9, which is the conjugating enzyme of SUMO-1 (small ubiquitin-related modifier-1). In the present study, we exogenously expressed a green fluorescent protein-tagged Bloom syndrome protein, GFP-BLM, in human 293EBNA cells and found that it formed dots/rod-like structures associated with SUMO-1 in the nucleus. Deletion experiments indicated that the region from amino acids 238 to 586 of BLM is required for the formation of dots/rod-like structures associated with SUMO-1, and the DNA helicase domain, but not the helicase activity itself, slightly affected the formation and/or stability of these structures. Expression of a GFP-BLM which contained the 238-586 region, but lacked the C-terminal nuclear localization signal, resulted in localization to the cytoplasm without the formation of dots/rod-like structures and association with SUMO-1, indicating that these events occur only in the nucleus.

Adenosine Triphosphatases↗

PubMed1999

Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells.

Lipofection of nondividing cells is inefficient because much of the transfected DNA is retained in endosomes, and that which escapes to the cytoplasm enters the nucleus at low rates. To improve the final rate-limiting step of nuclear import, we conjugated a nonclassical nuclear localization signal (NLS) containing the M9 sequence of heterogeneous nuclear ribonucleoprotein (hnRNP) A1, to a cationic peptide scaffold derived from a scrambled sequence of the SV40 T-antigen consensus NLS (ScT). The ScT was added to improve DNA binding of the M9 sequence. Lipofection of confluent endothelium with plasmid complexed with the M9-ScT conjugate resulted in 83% transfection and a 63-fold increase in marker gene expression. The M9-ScT conjugate localized fluorescent plasmid into the nucleus of permeabilized cells, and addition of the nuclear pore blocker wheat germ agglutinin prevented nuclear import. This method of gene transfer may lead to viral- and lipid-free transfection of nondividing cells.

Animals↗

PubMed1998

A central region of Ku80 mediates interaction with Ku70 in vivo.

Ku, the DNA binding component of DNA-dependent protein kinase (DNA-PK), is a heterodimer composed of 70 and 86 kDa subunits, known as Ku70 and Ku80 respectively . Defects in DNA-PK subunits have been shown to result in a reduced capacity to repair DNA double-strand breaks. Assembly of the Ku heterodimer is required to obtain DNA end binding activity and association of the DNA-PK catalytic subunit. The regions of the Ku subunits responsible for heterodimerization have not been clearly defined in vivo . A previous study has suggested that the C-terminus of Ku80 is required for interaction with Ku70. Here we examine Ku subunit interaction using N- and C-terminal Ku80 deletions in a GAL4-based two-hybrid system and an independent mammalian in vivo system. Our two-hybrid study suggests that the central region of Ku80, not its C-terminus, is capable of mediating interaction with Ku70. To determine if this region mediates interaction with Ku70 in mammalian cells we transfected xrs-6 cells, which lack endogenous Ku80, with epitope-tagged Ku80 deletions carrying a nuclear localization signal. Immunoprecipitation from transfected cell extracts revealed that the central domain identified by the GAL4 two-hybrid studies stabilizes and co-immunoprecipitates with endogenous xrs-6 Ku70. The central interaction domain maps to the internally deleted regions of Ku80 in the mutant cell lines XR-V9B and XR-V15B. These findings indicate that the internally deleted Ku80 mutations carried in these cell lines are incapable of heterodimerization with Ku70.

Amino Acid Sequence↗