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At least 127 records · Page 7Linked to original sources
PubMed2005

Dissection of a novel nuclear localization signal in open reading frame 29 of varicella-zoster virus.

Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin alpha- and beta-dependent mechanism. These data are further supported by the demonstration that a glutathione S-transferase-karyopherin alpha fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin alpha. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.

Cell Line↗
PubMed2004

The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation.

To genetically transform plants, Agrobacterium exports its transferred DNA (T-DNA) and several virulence (Vir) proteins into the host cell. Among these proteins, VirE3 is the only one whose biological function is completely unknown. Here, we demonstrate that VirE3 is transferred from Agrobacterium to the plant cell and then imported into its nucleus via the karyopherin alpha-dependent pathway. In addition to binding plant karyopherin alpha, VirE3 interacts with VirE2, a major bacterial protein that directly associates with the T-DNA and facilitates its nuclear import. The VirE2 nuclear import in turn is mediated by a plant protein, VIP1. Our data indicate that VirE3 can mimic this VIP1 function, acting as an 'adapter' molecule between VirE2 and karyopherin alpha and 'piggy-backing' VirE2 into the host cell nucleus. As VIP1 is not an abundant protein, representing one of the limiting factors for transformation, Agrobacterium may have evolved to produce and export to the host cells its own virulence protein that at least partially complements the cellular VIP1 function necessary for the T-DNA nuclear import and subsequent expression within the infected cell.

Bacterial Proteins↗
PubMed2003

Conformational variability of nucleo-cytoplasmic transport factors.

The transport of macromolecules between the nucleus and cytoplasm of eukaryotic cells is largely mediated by a single family of transport factors, the karyopherin or importin beta-like family. Structural and biochemical evidence suggests conformational flexibility of these modular HEAT-repeat proteins is crucial for their regulation. Here we use small angle x-ray scattering to assess the extent of conformational variation within a set of nuclear import and export factors. The study reveals that importin beta, transportin, and the exportin Xpo-t share a similar S-like superhelical conformation in their unbound state. There are no obvious differences in the overall structures that might generally distinguish nuclear export from nuclear import mediators. Two other members of the family, the exportins Cse1 and Xpo1, possess a significantly more globular conformation, indicating that the extended S-like architecture is not a hallmark of all karyopherins. Binding of RanGTP/cargo to importin beta, transportin, and Xpo-t triggers distinct conformational responses, suggesting that even closely related karyopherins employ different mechanisms of conformational regulation and that cargo and nuclear pore-interacting surfaces of the different receptors may be unique.

Karyopherins↗
PubMed2005

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein.

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.

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↗
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↗
PubMed2002

Age-associated reduction of nuclear protein import in human fibroblasts.

Age-dependent decreases in the protein concentrations of the nucleocytoplasmic transport factors karyopherin alpha2, CAS, and RanBP1 were found by comparing fibroblast cultures obtained from young, mature, and old human donors. Karyopherin beta1 levels do not change with age and present very little variation among donors. The decrease in the concentration of transport factors is accompanied by a reduction in the protein import rate in fibroblasts from old donors, as detected by a change in the intracellular localization of a test transport substrate that shuttles between the cytoplasm and the nucleus. Measurements of concentrations of the same import factors in organs and tissues of old mice revealed a decrease of CAS in kidney, lung, and spleen. The import reduction in old age is expected to lead to impaired activity of proteins whose functions depend on timely import into the nuclei.

Active Transport, Cell Nucleus↗
PubMed2003

Nuclear entry of high-risk human papillomavirus type 16 E6 oncoprotein occurs via several pathways.

The E6 oncoprotein of high-risk human papillomavirus type 16 (HPV16) interacts with several nuclear transcription factors and coactivators in addition to cytoplasmic proteins, suggesting that nuclear import of HPV16 E6 plays a role in the cellular transformation process. In this study we have investigated the nuclear import pathways of HPV16 E6 in digitonin-permeabilized HeLa cells. We found that HPV16 E6 interacted with the karyopherin (Kap) alpha2 adapter and could enter the nucleus via a classical Kap alpha2beta1-mediated pathway. Interestingly, HPV16 E6 also interacted, via its basic nuclear localization signal (NLS) located at the C terminus, with both Kap beta1 and Kap beta2 import receptors. Binding of RanGTP to these Kap betas inhibited their interaction with HPV16 E6 NLS. In agreement with these binding data, nuclear import of the HPV16 E6 oncoprotein in digitonin-permeabilized HeLa cells could be mediated by either Kap beta1 or Kap beta2. Nuclear import via these pathways required RanGDP and was independent of GTP hydrolysis by Ran. Significantly, an E6(R124G) mutant had reduced nuclear import activity, and the E6 deletion mutant lacking (121)KKQR(124) was not imported into the nucleus. The data reveal that the HPV16 E6 oncoprotein interacts via its C-terminal NLS with several karyopherins and exploits these interactions to enter the nucleus of host cells via multiple pathways.

Active Transport, Cell Nucleus↗
PubMed1999

Phosphorylation-dependent binding of hepatitis B virus core particles to the nuclear pore complex.

Although many viruses replicate in the nucleus, little is known about the processes involved in the nuclear import of viral genomes. We show here that in vitro generated core particles of human hepatitis B virus bind to nuclear pore complexes (NPCs) in digitonin-permeabilized mammalian cells. This only occurred if the cores contained phosphorylated core proteins. Binding was inhibited by wheat germ agglutinin, by antinuclear pore complex antibodies, and by peptides corresponding either to classical nuclear localization signals (NLS) or to COOH-terminal sequences of the core protein. Binding was dependent on the nuclear transport factors importins (karyopherins) alpha and beta. The results suggested that phosphorylation induces exposure of NLS in the COOH-terminal portion of the core protein that allows core binding to the NPCs by the importin- (karyopherin-) mediated pathway. Thus, phosphorylation of the core protein emerged as an important step in the viral replication cycle necessary for transport of the viral genome to the nucleus.

Biological Transport↗
PubMed2003

Nuclear import of zinc binuclear cluster proteins proceeds through multiple, overlapping transport pathways.

In Aspergillus nidulans, the high transcriptional level of the ethanol utilization pathway genes (alc) is regulated by the specific activator AlcR. Here we have analyzed the mechanism of the nuclear import of AlcR, as well as that of other proteins belonging to the Zn(2)Cys(6) binuclear cluster family. The nuclear localization signal of AlcR maps within the N-terminal 75 amino acid residues and overlaps with its DNA-binding domain. It consists of five clusters rich in basic residues. Four of them are necessary and sufficient for nuclear targeting. The first two basic regions are crucial for both nuclear localization and recognition of AlcR-specific DNA targets. This nuclear localization signal (NLS) motif is recognized by the nuclear transport machinery of Saccharomyces cerevisiae and requires both Ran/Gsp1p activity and specific transport receptors. AlcR can be imported into nuclei via multiple transport pathways mediated by a distinct set of karyopherins composed of Kap104p, Sxm1p, and Nmd5p transport receptors. The two former karyopherins interact with the NLS of AlcR directly. Other Zn binuclear cluster proteins from S. cerevisiae, such as Gal4p and Pdr3p, also appear to be transported to the nuclei in a nonclassical, importin-alpha-independent manner and can share common importin beta receptors.

Active Transport, Cell Nucleus↗
PubMed2001

Identification of nuclear-import and cell-cycle regulatory proteins that bind to prothymosin alpha.

Prothymosin alpha (ProT alpha) is a nuclear protein that is widely distributed in mammalian tissues, and is thought to play a role in cell proliferation. In an attempt to shed light on this role, affinity chromatography on ProT alpha-Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes (NC37 cells) that interact with ProT alpha in vitro, and thus may interact with ProT alpha in vivo. Immunoblotting techniques were used to screen the ProT alpha-binding fractions for histones and other proteins involved in nuclear transport and cell-cycle control. The most abundant ProT alpha-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear-transport proteins, karyopherin beta1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; while tranportin, CAS, and Ran BPI were not detected. Of the cell-cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at very low concentrations; while cyclin D1, cyclin D3, Cip1, and Kip1 were not detected. These results suggest (i) that ProT alpha is transported into the nucleus by the karyopherin beta1-Rch-1 complex, and (ii) that ProT alpha may interact in the nucleus with proteins involved in DNA metabolism and cell-cycle control.

Active Transport, Cell Nucleus↗
PubMed2005

Nucleocytoplasmic distribution of opioid growth factor and its receptor in tongue epithelium.

The subcellular distributions of the opioid growth factor (OGF), [Met(5)]-enkephalin, and opioid growth factor receptor (OGFr) in the epithelium of the rat tongue were determined in order to reveal structure-function relationships. Laser scanning confocal microscopic analysis showed that both OGF and OGFr were colocalized in the paranuclear cytoplasm and in the nuclei of keratinocytes in the stratum basale. Using immunoelectron microscopy and postembedding techniques, double labeling experiments disclosed that complexes of OGF-OGFr were colocalized on the outer nuclear envelope, in the paranuclear cytoplasm, perpendicular to the nuclear envelope in a putative nuclear pore complex, and in the nucleus adjacent to heterochromatin. Anti-OGF IgG alone was detected in the cytoplasm, and anti-OGFr IgG alone was associated with the outer nuclear envelope. Study of chronic treatment with the opioid antagonist, naltrexone (NTX), which blocks opioid-receptor binding, revealed the presence of OGFr immunoreactivity alone in the cytoplasm and the nucleus; some OGF-OGFr complexes were also observed. Colocalization of OGFr and karyopherin (importin) beta was recorded in the cytoplasm and nucleus. These results in tongue epithelium are the first to suggest that OGFr resides on the outer nuclear envelope, where OGF interacts with OGFr; that the OGF-OGFr complex translocates between cytoplasm and nucleus at the nuclear pore; and that the nuclear localization signal of OGFr interacts with karyopherin beta for nuclear transport. These novel data also indicate that signal transduction for cell proliferation appears to involve an OGF-OGFr complex that interfaces with chromatin in the nucleus. Moreover, the unique finding that OGFr was found in the cytoplasm and nucleus in NTX-treated specimens may suggest that NTX-OGFr complexes have the same pathway as OGF-OGFr.

Animals↗
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↗
PubMed2026

Signaling into the nucleus through the importin 7 pathway.

Importin 7 (IPO7) is a nuclear transport receptor of the β-karyopherin family that mediates the translocation of a broad spectrum of macromolecules, commonly referred to as cargoes. Discovered nearly three decades ago, IPO7 was initially identified as an import receptor for constitutive cellular cargoes, including histone H1 and ribosomal proteins, and was shown to function synergistically and partially redundantly with canonical receptors such as importin β1 and karyopherin β2. Over the past 15 years, however, accumulating evidence has established IPO7 as an important mediator of signal-dependent nuclear trafficking in response to extracellular stimuli, including cytokines, growth factors, and cellular stress. Thus, IPO7 has emerged as a versatile nuclear transport receptor that couples extracellular signaling to dynamic changes in nuclear composition and gene expression. Mechanistically, many IPO7 cargoes lack classical nuclear localization signals and instead contain noncanonical targeting motifs that directly engage IPO7. In several cases, phosphorylation-dependent conformational changes expose these motifs, promoting IPO7 binding and translocation through the nuclear pore complex. The expanding repertoire of IPO7 cargoes, including ERK, SMAD3, EGR1, GLI1, the glucocorticoid receptor, HIF-1α, YAP1, and RUNX2, highlights its prominent role at the interface of signaling and transcriptional control. Consistent with these functions, dysregulation of IPO7-mediated transport has been implicated in cancer, hypoxia, and other pathological states. Beyond cellular signaling, IPO7 also contributes to the nuclear trafficking of viral genomes and proteins. Here, we review the molecular mechanisms of IPO7-dependent nuclear import, emerging principles of cargo recognition, and pathways that regulate IPO7 activity during cellular signaling.

NLS↗
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↗
PubMed2004

Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization.

The association of genes with the nuclear pore complex (NPC) and nuclear transport factors has been implicated in transcriptional regulation. We therefore examined the association of components of the nuclear transport machinery including karyopherins, nucleoporins, and the Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome. We find that most nucleoporins and karyopherins preferentially associate with a subset of highly transcribed genes and with genes that possess Rap1 binding sites whereas the RanGEF preferentially associates with transcriptionally inactive genes. Consistent with coupling of transcription to the nuclear pore, we show that transcriptional activation of the GAL genes results in their association with nuclear pore proteins, relocation to the nuclear periphery, and loss of RanGEF association. Taken together, these results indicate that the organization of the genome is coupled via transcriptional state to the nuclear transport machinery.

Active Transport, Cell Nucleus↗
PubMed1998

Viral protein R regulates docking of the HIV-1 preintegration complex to the nuclear pore complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells depends critically on import of the viral preintegration complex into the nucleus. Recent evidence suggests that viral protein R (Vpr) plays a key regulatory role in this process by binding to karyopherin alpha, a cellular receptor for nuclear localization signals, and increasing its affinity for the nuclear localization signals. An in vitro binding assay was used to investigate the role of Vpr in docking of the HIV-1 preintegration complex (PIC) to the nuclear pore complex. Mutant HIV-1 PICs that lack Vpr were impaired in the ability to dock to isolated nuclei and recombinant nucleoporins. Although Vpr by itself associated with nucleoporins, the docking of Vpr+ PICs was dependent on karyopherin beta and was blocked by antibodies to beta. Vpr stabilized docking by preventing nucleoporin-stimulated dissociation of the import complex. These results suggest a biochemical mechanism for Vpr function in transport of the HIV-1 genome across the nuclear pore complex.

Cell Line↗
PubMed1999

GTP hydrolysis links initiation and termination of nuclear import on the nucleoporin nup358.

Binding of GTP-bound Ran (RanGTP) to karyopherin beta1 (Kapbeta1) releases import cargo into the nucleus. Using an ultrastructural, biochemical, and functional approach, we have studied the mechanism by which Kapbeta1.RanGTP is recycled at the nuclear pore complex for repeated rounds of import. In vitro, Kapbeta1 bound to the RanBP1-homologous (RBH) domains of Nup358 in the presence of either RanGTP or RanGDP, forming trimeric complexes. The Kapbeta1.RanGTP. RBH complex resisted dissociation by RanBP1 and GTP hydrolysis by Ran GTPase activating protein 1. Ran-dependent binding of gold-conjugated Kapbeta1 to the cytoplasmic fibers of the nuclear pore complex in digitonin-permeabilized cells and RanBP1 competition confirmed the in vitro binding data. Interaction of karyopherin alpha and a classical nuclear localization sequence peptide with the Kapbeta1.RanGTP.RBH complex stimulated GTP hydrolysis by Ran GTPase activating protein 1 both in vitro and in permeabilized cells. This GTP hydrolysis was required for reinitiation of import of a nuclear localization sequence-bearing substrate in permeabilized cells. These data suggest that GTP hydrolysis on the RBH domains of Nup358 couples the termination of one cycle of nuclear import with the initiation of the next.

Binding Sites↗