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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↗
PubMed2003

NLS bioconjugates for targeting therapeutic genes to the nucleus.

One of the major steps limiting non-viral gene transfer efficiency is the entry of plasmid DNA from the cytoplasm into the nucleus of transfected cells. Trafficking of nuclear proteins from the cytoplasm into the nucleus through nuclear pore complexes is mediated by the presence of nuclear localization sequences (NLS) on proteins. Viral DNA and RNA also require interaction with cellular machinery for efficient nuclear import. In this article, we review the various strategies used to provide plasmid DNA with nuclear localization sequences, and discuss the possibility of developing efficient gene delivery systems based on these strategies.

Active Transport, Cell Nucleus↗
PubMed2001

Mrnp41 (Rae 1p) associates with microtubules in HeLa cells and in neurons.

Mrnp41 (hRae1p) is an evolutionarily highly conserved protein, which is a potential component of mRNP particles and plays a role in nuclear mRNA export. The protein is mainly localized at the nuclear pore complex, but is also associated with distinct nuclear domains and with a meshwork of numerous small particles in the cytoplasm (Kraemer and Blobel (1997): Proc. Natl. Acad. Sci. USA 91, 1519-1523). We show that the cytoplasmic pattern of mrnp41 is sensitive to treatment with the microtubule (MT)-depolymerizing drug nocodazole which causes disappearance of mrnp41 from the cell periphery and concentration around the nucleus. By immunofluorescence we demonstrate that mrnp41 colocalizes with MT in HeLa cells and displays an MT-like distribution in cultured neurons. Association of mrnp41 with MT is further demonstrated by copurification with MT from pig brain throughout several steps of polymerization and depolymerization. Separation of MT-associated proteins (MAPs) by phosphocellulose (PC) chromatography showed copurification of mrnp41 with MAPs. These data show an association of mrnp41 with MT and, moreover, demonstrate that an intact MT system is necessary for dispersion of mrnp41-containing particles to the cellular periphery. The essential role of mrnp41 in spindle pole separation and cell cycle progression may also be related to its ability to bind to MTs.

Animals↗
PubMed2005

Phosphorylation of RanGAP1 stabilizes its interaction with Ran and RanBP1.

Ran is a nuclear Ras-like GTPase that is required for various nuclear events including the bi-directional transport of proteins and ribonucleoproteins through the nuclear pore complex, spindle formation, and reassembly of the nuclear envelope. One of the key regulators of Ran is RanGAP1, a Ran specific GTPase activating protein. The question of whether a mechanism exists for controlling nucleocytoplasmic transport through the regulation of RanGAP1 activity continues to be debated. Here we show that RanGAP1 is phosphorylated in vivo and in vitro. Serine-358 (358S) was identified as the major phosphorylation site, by MALDI-TOF-MS spectrometry. Site directed mutagenesis at this position abolished the phosphorylation. Experiments using purified recombinant kinase and specific inhibitors such as DRB and apigenin strongly suggest that casein kinase II (CK2) is the responsible kinase. Although the phosphorylation of 358S of RanGAP1 did not significantly alter its GAP activity, the phosphorylated wild type RanGAP1, but not a mutant harboring a mutation at the phosphorylation site 358S, efficiently formed a stable ternary complex with Ran and RanBP1 in vivo, suggesting that the 358S phosphorylation of RanGAP1 affects the Ran system.

Amino Acid Sequence↗
PubMedDate not supplied

Ultrastructural, cytochemical, and immunocytochemical study of nuclei and cytoskeleton of thyroid papillary carcinoma cells.

One of the most constant and typical histological markers of thyroid papillary carcinoma is the presence of multilobated and ground-glass nuclei. With the intention of extending and characterizing the ultrastructural comprehension of this feature, thyroid papillary carcinoma cells and normal follicular cells were studied using chromatin and ribonucleoprotein preferential staining techniques, as well as immunolabeling with antibodies against DNA, lamins, vimentin, and desmin. Carcinoma cells showed scant compact chromatin arranged in small masses. A special type of nuclear bodies with DNA immunoreactive fibrils was found in these cells. Some nucleoli were surrounded by a double fibrillar layer limiting a perinucleolar space occupied by RNP fibrogranular structures that differed from those of normal nuclei. Perichromatin granules were scarce compared to normal follicular cells. Immunoelectron microscopic studies of lamins showed diminished immunoreactivity in the nuclei of carcinoma cells. A perinuclear distribution of desmin and vimentin filament was found in tumor cells. The cytoplasm of normal follicular cells showed scarce immunoreactivity to vimentin and no immunolabeling for desmin. Both types of filaments attach to the nuclear pore complex and to other regions of the nuclear envelope. Contacts between labeled filaments and desmosomes or hemidesmosomes were frequent. The results show that in papillary thyroid carcinoma cells, changes in the distribution of chromatin and ribonucleoproteins, either alone or in conjunction with scarce lamin immunolabeling and perinuclear vimentin and desmin filamentous rings, may be responsible for the characteristic ground glass and multilobated nuclei.

Carcinoma, Papillary↗
PubMed2002

[Nuclear pores: from yeast to higher eukaryotes].

In eukaryotes, bidirectional transport of macro-molecules between the cytoplasm and the nucleus occurs through elaborate supramolecular structures embedded in the nuclear envelope, the nuclear pore complexes (NPCs), whose overall architecture has been evolutionary conserved from yeast to vertebrates. In recent years, fast progress in characterizing the NPCs components (the nucleoporins or Nups) has been made in the yeast S. cerevisiae, and to a lesser extent in vertebrates. In addition, despite the low homology between most yeast and vertebrate nucleoporins, their organization and their topological mapping within the NPC substructures have been broadly conserved during evolution.

Animals↗
PubMed1996

HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs.

BACKGROUND: The structural proteins of human immunodeficiency virus type 1 (HIV-1) are encoded by intron-containing mRNAs that normally are retained in the nucleus. A viral regulatory protein, Rev, specifically induces the accumulation of these transcripts in the cytoplasm. Rev is an RNA-binding protein that also contains an 'effector' domain. The Rev effector domain has recently been shown to function as an autonomous nuclear export signal (NES) that, when fused to a foreign protein, will cause its rapid nuclear export. We and others have recently reported the cloning of a human protein (hRIP/Rab), that specifically interacts with the effector domain of Rev. RESULTS: Here we show that the NESs contained within two cellular proteins, PKI and I kappa B, which are not involved in RNA metabolism, also interact with hRIP. Fusion of these cellular sequences to the Rev RNA-binding domain reconstitutes a functional Rev protein. In addition to hRIP, these NESs also bind to several nuclear pore complex (NPC). We show that this protein export pathway is highly conserved by demonstrating that mammalian NESs also function in yeast. CONCLUSIONS: Our results indicate that the HIV-1 Rev protein evolved to take advantage of a cellular protein export pathway in order to allow the nucleocytoplasmic transport of unspliced viral RNA. Our data suggest a model in which the export substrate is translocated through the NPC by sequential interactions with different nucleoporins. Finally, our experiment suggests a mechanism by which I kappa B can downregulate nuclear NF kappa B activity by causing its rapid export from the nucleus.

Binding Sites↗
PubMed2003

The SONB(NUP98) nucleoporin interacts with the NIMA kinase in Aspergillus nidulans.

The Aspergillus nidulans NIMA kinase is essential for mitotic entry. At restrictive temperature, temperature-sensitive nimA alleles arrest in G2, before accumulation of NIMA in the nucleus. We performed a screen for extragenic suppressors of the nimA1 allele and isolated two cold-sensitive son (suppressor of nimA1) mutants. The sonA1 mutant encoded a nucleoporin that is a homolog of yeast Gle2/Rae1. We have now cloned SONB, a second nucleoporin genetically interacting with NIMA. sonB is essential and encodes a homolog of the human NUP98/NUP96 precursor. Similar to NUP98/NUP96, SONB(NUP98/NUP96) is autoproteolytically cleaved to generate SONB(NUP98) and SONB(NUP96). SONB(NUP98) localizes to the nuclear pore complex and contains a GLEBS domain (Gle2 binding sequence) that binds SONA(GLE2). A point mutation within the GLEBS domain of SONB1(NUP98) suppresses the temperature sensitivity of the nimA1 allele and compromises the physical interaction between SONA(GLE2) and SONB1(NUP98). The sonB1 mutation also causes sensitivity to hydroxyurea. We isolated the histone H2A-H2B gene pair as a copy-number suppressor of sonB1 cold sensitivity and hydroxyurea sensitivity. The data suggest that the nucleoporins SONA(GLE2) and SONB(NUP98) and the NIMA kinase interact and regulate nuclear accumulation of mitotic regulators to help promote mitosis.

Amino Acid Sequence↗
PubMed2001

Perturbation of SUMOlation enzyme Ubc9 by distinct domain within nucleoporin RanBP2/Nup358.

Ubc9, a conjugation enzyme for the ubiquitin-related modifier SUMO, is present predominantly in the nucleus and at the nuclear pore complex. The functional significance of its subcellular compartmentalization, however, remains to be elucidated. Here, we define a Pro-Glu-Asp-Ser-Thr-rich element containing 129 amino acid residues, designated IR1+2, on the human nucleoporin RanBP2/Nup358, which binds directly to Ubc9 with high affinity both in vitro and in vivo. When IR1+2 tagged with green fluorescence protein at its amino terminus (GFP-IR1+2) was transfected into COS-7 cells, we found that approximately 90% of the nuclear Ubc9 was sequestered in the cytoplasm. We also observed that both SUMO-1 and SUMO-2/3 were mislocalized, and promyelocytic leukemia protein PML formed an enlarged aggregate in the nucleus. Moreover, the homologous recombination protein Rad51 mislocalized to the cytoplasm, and Rad51 foci, a hallmark of functional association of Rad51 with damaged DNA, did not form efficiently even in the presence of a DNA strand breaker. These findings emphasize that the IR1+2 domain is a useful tool for manipulating the nuclear localization of Ubc9 and perturbing the subcellular localization of SUMOs and/or SUMOlated proteins, and they emphasize the important role of nuclear Ubc9 in the Rad51-mediated homologous recombination pathway, possibly by modulating intracellular trafficking of Rad51.

Active Transport, Cell Nucleus↗
PubMed1999

A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export.

In order to identify additional factors required for nuclear export of messenger RNA, a genetic screen was conducted with a yeast mutant deficient in a factor Gle1p, which associates with the nuclear pore complex (NPC). The three genes identified encode phospholipase C and two potential inositol polyphosphate kinases. Together, these constitute a signaling pathway from phosphatidylinositol 4, 5-bisphosphate to inositol hexakisphosphate (IP6). The common downstream effects of mutations in each component were deficiencies in IP6 synthesis and messenger RNA export, indicating a role for IP6 in GLE1 function and messenger RNA export.

Biological Transport↗
PubMed2005

The integral membrane protein Pom34p functionally links nucleoporin subcomplexes.

Here we have examined the function of Pom34p, a novel membrane protein in Saccharomyces cerevisiae, localized to nuclear pore complexes (NPCs). Membrane topology analysis revealed that Pom34p is a double-pass transmembrane protein with both the amino (N) and carboxy (C) termini positioned on the cytosolic/pore face. The network of genetic interactions between POM34 and genes encoding other nucleoporins was established and showed specific links between Pom34p function and Nup170p, Nup188p, Nup59p, Gle2p, Nup159p, and Nup82p. The transmembrane domains of Pom34p in addition to either the N- or C-terminal region were necessary for its function in different double mutants. We further characterized the pom34deltaN nup188delta mutant and found it to be perturbed in both NPC structure and function. Mislocalization of a subset of nucleoporins harboring phenylalanine-glycine repeats was observed, and nuclear import capacity for the Kap104p and Kap121p pathways was inhibited. In contrast, the pom34delta pom152delta double mutant was viable at all temperatures and showed no such defects. Interestingly, POM152 overexpression suppressed the synthetic lethality of pom34delta nup170delta and pom34delta nup59delta mutants. We speculate that multiple integral membrane proteins, either within the nuclear pore domain or in the nuclear envelope, execute coordinated roles in NPC structure and function.

Cytosol↗
PubMed1998

Involvement of p62 nucleoporin in angiotensin II-induced nuclear translocation of STAT3 in brain neurons.

Chronic stimulation of brain neurons by angiotensin II (Ang II) results in a increase in norepinephrine (NE) uptake. This involves stimulation of transcription of NE transporter and tyrosine hydroxylase genes and is associated with translocation of signaling molecules and transcription factors from the cytoplasmic compartment into the neuronal nucleus (). We report here that the phosphorylation of p62, a glycoprotein nucleoporin of the nuclear pore complex (NPC), by MAP kinase is involved in this process. Ang II caused a time-dependent translocation of signal transducers and activators of transcription (STAT3) from the cytoplasmic compartment into the nucleus. This translocation was attenuated by pretreatment with antisense oligonucleotide (AON) to MAP kinase. Ang II also stimulated phosphorylation of p62, and a maximal phosphorylation of 12-fold was observed with 100 nM Ang II. This stimulation was blocked by losartan, an AT1 receptor subtype-specific antagonist. The conclusion that MAP kinase is involved in Ang II-induced phosphorylation of p62 and nuclear translocation of STAT3 is supported by the following. (1) p62 phosphorylation was blocked by a peptide that competes with p62 as a MAP kinase substrate both in vitro and in vivo; (2) AON to MAP kinase attenuated Ang II stimulation of p62 phosphorylation; and (3) in addition, it also blocked nuclear translocation of STAT3. Intracellular loading of the peptide containing MAP kinase substrate consensus of the p62 reduced Ang II stimulation of p62 phosphorylation and nuclear translocation of STAT3 in both in vivo and in vitro experiments. These observations suggest that Ang II-induced phosphorylation of p62 may accelerate the activity of the NPC, which would result in an increase in the nuclear transport of transcription factors and signaling molecules. This will stimulate transcriptional processes associated with Ang II regulation of NE neuromodulation.

Angiotensin II↗
PubMed2006

Mutated nup62 causes autosomal recessive infantile bilateral striatal necrosis.

OBJECTIVE: The objective of this study was to identify the gene causing autosomal recessive infantile bilateral striatal necrosis. METHODS: We have mapped the disease gene in the candidate region to approximately 230kb on 19q13.33 in 8 interrelated families including a total of 12 patients and 39 unaffected individuals. RESULTS: Sequencing of the nup62 gene showed a missense mutation causing a change from glutamine to proline (Q391P) in all the patients, producing a substitution from a polar, hydrophilic residue to a nonpolar, neutral residue. All the other 12 candidate genes were sequenced, and no pathogenic sequence changes were found. Comparisons of p62 protein sequences from diverse species indicate that glutamine at position 391 is highly conserved. Five prenatal diagnoses were performed in three at-risk families. INTERPRETATION: This is the second example of a nuclear pore complex protein causing mendelian disease in humans (the first one is triple A syndrome). Our findings suggest that p62 has a cell type-specific role and is important in the degeneration of the basal ganglia in humans.

Adult↗
PubMed1976

Arrangement of chromosome ends and axial core formation during early meiotic prophase in the male grasshopper Brachystola magna by 3D, E.M. reconstruction.

Evidence is presented that chromosome ends are attached to the nuclear envelope prior to the formation of axial cores during early meiotic prophase in the grasshopper Brachystola magna. The attachment sites of distal and proximal chromosome ends are clustered in a small region of the inner nuclear envelope resulting in a classical bouquet arrangement of the chromosomes. Proximal ends are tightly clustered due to the presence of chromocenters. Distal chromosome ends are more widely scattered throughout the base of the bouquet.--Axial core formation can be initiated at chromosome ends or at internal chromosome sites. However, there is a preference for axial cores to form in distal chromosome regions rather than proximal regions during early meiotic prophase.--Virtually all of the nuclear pore complexes are located in the general vicinity of the chromosome attachment sites but each specific attachment site is surrounded by a small area of nuclear envelope which is devoid of pore complexes.

Animals↗
PubMed2006

A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis.

Nuclear-cytoplasmic partitioning and traffic between cytoplasmic and nuclear compartments are fundamental processes in eukaryotic cells. Nuclear pore complexes mediate transport of proteins, RNAs and ribonucleoprotein particles in and out of the nucleus. Here we present positional cloning of a plant nucleoporin gene, Nup133, essential for a symbiotic signal transduction pathway shared by Rhizobium bacteria and mycorrhizal fungi. Mutation of Nup133 results in a temperature sensitive nodulation deficient phenotype and absence of mycorrhizal colonization. Root nodules developing with reduced frequency at permissive temperatures are ineffective and electron microscopy show that Rhizobium bacteria are not released from infection threads. Measurement of ion fluxes using a calcium-sensitive dye show that Nup133 is required for the Ca2+ spiking normally detectable within minutes after application of purified rhizobial Nod-factor signal molecules to root hairs. Localization of NUP133 in the nuclear envelope of root cells and root hair cells shown with enhanced yellow fluorescent protein fusion proteins suggests a novel role for NUP133 nucleoporins in a rapid nuclear-cytoplasmic communication after host-plant recognition of symbiotic microbes. Our results identify a component of an intriguing signal process requiring interaction at the cell plasma membrane and at intracellular nuclear and plastid organelle-membranes to induce a second messenger.

Alleles↗
PubMed2005

Parkin ubiquitinates and promotes the degradation of RanBP2.

Parkinson disease (PD) is a common neurodegenerative disorder, which involves the deterioration of dopaminergic neurons in the pars compacta of the substantia nigra. The etiology of PD is still unknown, but recent identification of mutations in familial cases of PD has advanced the understanding of the molecular mechanisms of this neurological disease. Mutations in the parkin gene, which encodes for ubiquitin-protein ligase (E3), have been implicated in autosomal recessive juvenile Parkinsonism, an early onset and common familial form of PD. Here we reported that Parkin selectively binds to RanBP2, which is localized in the cytoplasmic filament of the nuclear pore complex and belongs to the small ubiquitin-related modifier E3 ligase family. We also demonstrated that RanBP2 becomes a target for Parkin E3 ubiquitin-ligase and is processed via Parkin-mediated ubiquitination and subsequent proteasomal degradation. Furthermore, Parkin controls the intracellular levels of sumoylated HDAC4, as a result of the ubiquitination and degradation of RanBP2. Our findings suggested that the intracellular levels of RanBP2 and its functional activity may be modulated by Parkin-mediated ubiquitination and proteasomal pathways.

Amino Acid Motifs↗
PubMed2004

The role of NUP98 gene fusions in hematologic malignancy.

Chromosomal aberrations occur with great frequency and some specificity in leukemia and other hematologic malignancies. The most common outcome of these rearrangements is the formation of a fusion gene, comprising portions of 2 genes normally present in the cell. These fusion proteins are presumed to be oncogenic; in many cases, animal models have proven them to be oncogenic. One of the most promiscuous fusion partner genes is the newly identified NUP98 gene, located on chromosome 11p15.5, which to date has been observed fused to 15 different fusion partners. NUP98 encodes a 98 kD protein that is an important component of the nuclear pore complex, which mediates nucleo-cytoplasmic transport of protein and RNA. The fusion partners of NUP98 form 2 distinct groups: homeobox genes and non-homeobox genes. All NUP98 fusions join the N-terminal GLFG repeats of NUP98 to the C-terminal portion of the partner gene, which, in the case of the homeobox gene partners, includes the homeodomain. Clinical findings are reviewed here, along with the findings of several in vivo and in vitro models have been employed to investigate the mechanisms by which NUP98 fusion genes contribute to the pathogenesis of leukemia.

Acute Disease↗
PubMed2006

Changes in nucleoporin domain topology in response to chemical effectors.

Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca2+ and 2 mM Mg2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.

Animals↗