Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Nuclear Pore Complex”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 235 records · Page 13Linked to original sources
PubMed1998

Molecular segments of protein Tpr that confer nuclear targeting and association with the nuclear pore complex.

Tpr is a 267-kDa protein of unknown function recently identified as a constitutive component of the nuclear pore complex (NPC2)-attached intranuclear filaments. Secondary structure predictions suggest that the protein is divided into a large, coiled-coil forming aminoterminal domain and a shorter, highly acidic carboxyterminal domain. To identify which of Tpr's molecular segments determine its specific intranuclear localization, we have constructed expression vectors encoding various Tpr deletion mutants as well as chimeric combinations of Tpr sequences with the soluble cytoplasmic protein pyruvate kinase. Following transfection of cultured mammalian cells, we have identified a short region within Tpr's carboxyterminal domain that is essential and sufficient to mediate nuclear import of Tpr and which can also confer nuclear accumulation of pyruvate kinase. Tpr deletion mutants that contain this nuclear targeting segment, but lack the aminoterminal domain, appear evenly dispersed throughout the nucleus without any noticeable association to the NPC. In contrast, the aminoterminal domain lacking the carboxyterminal region remains located within the cytoplasm, forming aggregate-like structures not associated with the nuclear envelope. However, when tagged to Tpr's short nuclear targeting segment or to the nuclear localization signal of the SV40 large T protein, the aminoterminal domain is imported into the nucleus, where it then associates with the NPC. This association is mediated by shorter molecular segments within the aminoterminal domain which contain clusters of heptad repeats, whereas other regions are dispensable. This assignment of different topogenetic properties to distinct molecular segments of Tpr will now allow the design of future experiments to study the protein's structural properties further and determine its actual function.

Amino Acid Sequence↗
PubMed1989

Protein import through the nuclear pore complex is a multistep process.

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.

Acetylglucosamine↗
PubMed2003

The Ran GTPase cycle is required for yeast nuclear pore complex assembly.

Here, we report the first evidence that the Ran GTPase cycle is required for nuclear pore complex (NPC) assembly. Using a genetic approach, factors required for NPC assembly were identified in Saccharomyces cerevisiae. Four mutant complementation groups were characterized that correspond to respective mutations in genes encoding Ran (gsp1), and essential Ran regulatory factors Ran GTPase-activating protein (rna1), Ran guanine nucleotide exchange factor (prp20), and the RanGDP import factor (ntf2). All the mutants showed temperature-dependent mislocalization of green fluorescence protein (GFP)-tagged nucleoporins (nups) and the pore-membrane protein Pom152. A decrease in GFP fluorescence associated with the nuclear envelope was observed along with an increase in the diffuse, cytoplasmic signal with GFP foci. The defects did not affect the stability of existing NPCs, and nup mislocalization was dependent on de novo protein synthesis and continued cell growth. Electron microscopy analysis revealed striking membrane perturbations and the accumulation of vesicles in arrested mutants. Using both biochemical fractionation and immunoelectron microscopy methods, these vesicles were shown to contain nups. We propose a model wherein a Ran-mediated vesicular fusion step is required for NPC assembly into intact nuclear envelopes.

Alleles↗
PubMed2006

NDC1: a crucial membrane-integral nucleoporin of metazoan nuclear pore complexes.

POM121 and gp210 were, until this point, the only known membrane-integral nucleoporins (Nups) of vertebrates and, thus, the only candidate anchors for nuclear pore complexes (NPCs) within the nuclear membrane. In an accompanying study (Stavru et al.), we provided evidence that NPCs can exist independently of POM121 and gp210, and we predicted that vertebrate NPCs contain additional membrane-integral constituents. We identify such an additional membrane protein in the NPCs of mammals, frogs, insects, and nematodes as the orthologue to yeast Ndc1p/Cut11p. Human NDC1 (hNDC1) likely possesses six transmembrane segments, and it is located at the nuclear pore wall. Depletion of hNDC1 from human HeLa cells interferes with the assembly of phenylalanine-glycine repeat Nups into NPCs. The loss of NDC1 function in Caenorhabditis elegans also causes severe NPC defects and very high larval and embryonic mortality. However, it is not ultimately lethal. Instead, homozygous NDC1-deficient worms can be propagated. This indicates that none of the membrane-integral Nups is universally essential for NPC assembly, and suggests that NPC biogenesis is an extremely fault-tolerant process.

Amino Acid Sequence↗
PubMed1990

Structure and function of the nuclear pore complex: new perspectives.

The double membrane of the nuclear envelope is a formidable barrier separating the nucleus and cytoplasm of eukaryotic cells. However, movement of specific macromolecules across the nuclear envelope is critical for embryonic development, cell growth and differentiation. Transfer of molecules between the nucleus and cytoplasm occurs through the aqueous channel formed by the nuclear pore complex (NPC). Although small molecules may simply diffuse across the NPC, transport of large proteins and RNA requires specific transport signals and is energy dependent. A family of pore glycoproteins modified by O-linked N-acetylglucosamine moieties are essential for transport through the NPC. Recent evidence suggests that the regulation of nuclear transport may also involve the interaction of RNA and nuclear proteins with specific binding proteins that recognize these transport signals. Are these nuclear pore glycoproteins and signal binding proteins the 'gatekeepers' that control access to the genetic material? Recent evidence obtained from a combination of biochemical and genetic approaches suggests--perhaps.

Animals↗
PubMed1994

Architecture of the nuclear pore complex and its involvement in nucleocytoplasmic transport.

Recent structural analyses of the nuclear pore complex (NPC) have described in some detail the numerous sub-domains which make up this supramolecular assembly. Three dimensional image analysis of detergent-extracted NPCs reveals that the NPC framework is made up of spoke units, each containing four major domains, arranged with 822 symmetry. As shown by freeze-drying/metal shadowing techniques, attached to this framework are several peripheral components including particles and fibers on the cytoplasmic face and a cage-like structure on the nucleoplasmic face. While it is known that traffic between cytoplasm and nucleus occurs exclusively through the nuclear pore, the specifics of NPC involvement in such traffic remain unknown at present. Proteins destined for the nucleus contain nuclear localization sequences (NLSs). Several proteins have been identified which bind to these NLSs and may act to direct these proteins to the NPC, either releasing them prior to or remaining attached during translocation through the NPC. These NLS-binding proteins have been localized to the cytoplasm, nucleus, nuclear envelope and nucleolus, suggesting some of these proteins transverse the NPC bound or unbound to NLS-containing proteins.

Animals↗
PubMed1999

Cell cycle dependent localization of the telomeric PARP, tankyrase, to nuclear pore complexes and centrosomes.

Tankyrase is a human poly(ADP-ribose) polymerase that was initially identified through its interaction with the telomeric protein TRF1, a negative regulator of telomere length. In vitro poly(ADP-ribosyl)ation by tankyrase inhibits TRF1 binding to telomeric DNA suggesting a role for tankyrase in telomere function. We previously demonstrated that tankyrase co-localizes with TRF1 at the ends of human chromosomes in metaphase. Here we show that tankyrase localizes to additional subcellular sites in a cell cycle dependent manner. In interphase, tankyrase co-localized with TRF1 to telomeres, but in addition was found to reside at nuclear pore complexes, as evidenced by indirect immunofluorescence, subcellular fractionation and immunoelectron microscopy. At mitosis, concomitant with nuclear envelope breakdown and nuclear pore complex disassembly, tankyrase was found to relocate around the pericentriolar matrix of mitotic centrosomes. This complex staining pattern along with the observation that tankyrase did not contain a nuclear localization signal suggested that its telomeric localization might be regulated, perhaps by TRF1. Indeed, localization of exogenously-expressed tankyrase to telomeres was dependent upon co-transfection with TRF1. These data indicate that the subcellular localization of tankyrase can be regulated by both the cell cycle and TRF1.

Cell Cycle↗
PubMed2001

Import of adenovirus DNA involves the nuclear pore complex receptor CAN/Nup214 and histone H1.

Adenovirus type 2 (Ad2) imports its DNA genome through the nuclear pore complex (NPC) of cells in interphase for viral production. Here we identify the NPC-filament protein CAN/Nup214 as a docking site for incoming Ad2 capsids. Binding to CAN is independent of cytosolic factors. Capsids disassemble at NPCs to free their DNA for import. This process requires binding of nuclear histone H1 to the stably docked capsids and involves H1-import factors, restricting this irreversible process to the proximity of the nucleus. Our results provide a molecular mechanism for disassembly of Ad2 and reveal an unexpected function of histone H1 in virus-mediated DNA import.

Active Transport, Cell Nucleus↗
PubMed2000

The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm.

Compositional analysis of nuclear pore complexes (NPCs) is nearing completion, and efforts are now focused on understanding how these protein machines work. Recent analysis of soluble transport factor interactions with NPC proteins reveals distinct and overlapping pathways for movement between the nucleus and cytoplasm. New fluorescence- and microscopy-based strategies have been used to monitor the pathway of NPC assembly and to reveal the dynamics of the NPC during transport.

Animals↗
PubMed1998

Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export.

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments. Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emergence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.

Animals↗
PubMed1996

Yeast N1e3p/Nup170p is required for normal stoichiometry of FG nucleoporins within the nuclear pore complex.

The FG nucleoporins are a conserved family of proteins, some of which bind to the nuclear localization sequence receptor, karyopherin. Distinct members of this family are found in each region of the nuclear pore complex (NPC), spanning from the cytoplasmically disposed filaments to the distal end of the nuclear basket. Movement of karyopherin from one FG nucleoporin to the next may be required for translocation of substrates across the NPC. So far, nothing is known about how the FG nucleoporins are localized within the NPC. To identify proteins that interact functionally with one member of this family, the Saccharomyces cerevisiae protein Nup1p, we previously identified 16 complementation groups containing mutants that are lethal in the absence of NUP1 These mutants were referred to as nle (Nup-lethal) mutants. Mutants in the nle3/nlel7 complementation group are lethal in combination with amino-terminal nup1 truncation mutants, which we have previously shown to be defective for localization to the NPC. Here we show that NLE3 (which is allelic to NUP170) encodes a protein with similarity to the mammalian nucleoporin Nup155. We show that Nle3p coprecipitates with glutathione S-transferase fusions containing the amino-terminal domain of Nup1p. Furthermore, a deletion of Nle3p leads to changes in the stoichiometry of several of the XFXFG nucleoporins, including the loss of Nup1p and Nup2p. These results suggest that Nle3p plays a role in localizing specific FG nucleoporins within the NPC. The broad spectrum of synthetic phenotypes observed with the nle3delta mutant provides support for this model. We also identify a redundant yeast homolog that can partially substitute for Nle3p and show that together these proteins are required for viability.

Amino Acid Sequence↗
PubMed1995

Autoantibodies from patients with primary biliary cirrhosis preferentially react with the amino-terminal domain of nuclear pore complex glycoprotein gp210.

Patients with primary biliary cirrhosis frequently develop autoantibodies directed to gp210, a major glycoprotein of the nuclear pore complex. This protein contains a large glycosylated cisternal domain, a single transmembrane segment, and a short cytoplasmic tail. It has been previously shown that autoantibodies from primary biliary cirrhosis patients exclusively react with the cytoplasmic tail. We demonstrate that autoantibodies against gp210 recognize at least two different epitopes. 4 out of 12 anti-gp210 positive sera reacted with the fragment consisting of the cytoplasmic tail, and 8 sera targeted a novel epitope located within the large glycosylated lumenal domain. Moreover, our data prove that carbohydrate moieties are an essential part of this novel epitope. We propose, therefore, that future screening assays should be performed with antigens possessing both epitopes to detect all sera with anti-gp210 specificity.

Antibody Specificity↗
PubMed2004

The FG-repeat asymmetry of the nuclear pore complex is dispensable for bulk nucleocytoplasmic transport in vivo.

Nucleocytoplasmic transport occurs through gigantic proteinaceous channels called nuclear pore complexes (NPCs). Translocation through the NPC is exquisitely selective and is mediated by interactions between soluble transport carriers and insoluble NPC proteins that contain phenylalanine-glycine (FG) repeats. Although most FG nucleoporins (Nups) are organized symmetrically about the planar axis of the nuclear envelope, very few localize exclusively to one side of the NPC. We constructed Saccharomyces cerevisiae mutants with asymmetric FG repeats either deleted or swapped to generate NPCs with inverted FG asymmetry. The mutant Nups localize properly within the NPC and exhibit exchanged binding specificity for the export factor Xpo1. Surprisingly, we were unable to detect any defects in the Kap95, Kap121, Xpo1, or mRNA transport pathways in cells expressing the mutant FG Nups. These findings suggest that the biased distribution of FG repeats is not required for major nucleocytoplasmic trafficking events across the NPC.

Active Transport, Cell Nucleus↗
PubMed1997

RanGTP targets p97 to RanBP2, a filamentous protein localized at the cytoplasmic periphery of the nuclear pore complex.

RanBP2, a protein containing FG repeat motifs and four binding sites for the guanosine triphosphatase Ran, is localized at the cytoplasmic periphery of the nuclear pore complex (NPC) and is believed to play a critical role in nuclear protein import. We purified RanBP2 from rat liver nuclear envelopes and examined its structural and biochemical properties. Electron microscopy showed that RanBP2 forms a flexible filamentous molecule with a length of approximately 36 nm, suggesting that it comprises a major portion of the cytoplasmic fibrils implicated in initial binding of import substrates to the NPC. Using in vitro assays, we characterized the ability of RanBP2 to bind p97, a cytosolic factor implicated in the association of the nuclear localization signal receptor with the NPC. We found that RanGTP promotes the binding of p97 to RanBP2, whereas it inhibits the binding of p97 to other FG repeat nucleoporins. These data suggest that RanGTP acts to specifically target p97 to RanBP2, where p97 may support the binding of an nuclear localization signal receptor/substrate complex to RanBP2 in an early step of nuclear import.

Animals↗
PubMed2006

Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells.

So far, POM121 and gp210 are the only known anchoring sites of vertebrate nuclear pore complexes (NPCs) within the lipid bilayer of the nuclear envelope (NE) and, thus, are excellent candidates for initiating the NPC assembly process. Indeed, we demonstrate that POM121 can recruit several nucleoporins, such as Nup62 or Nup358, to ectopic assembly sites. It thus appears to act as a nucleation site for the assembly of NPC substructures. Nonetheless, we observed functional NPCs and intact NEs in severely POM121-depleted cells. Double knockdowns of gp210 and POM121 in HeLa cells, as well as depletion of POM121 from human fibroblasts, which do not express gp210, further suggest that NPCs can assemble or at least persist in a POM121- and gp210-free form. This points to extensive redundancies in protein-protein interactions within NPCs and suggests that vertebrate NPCs contain additional membrane-integral nucleoporins for anchorage within the lipid bilayer of the NE. In Stavru et al., we describe such an additional transmembrane nucleoporin as the metazoan orthologue of yeast Ndc1p.

Fibroblasts↗
PubMed2004

Actin- and protein-4.1-containing filaments link nuclear pore complexes to subnuclear organelles in Xenopus oocyte nuclei.

We imaged the interiors of relatively intact Xenopus oocyte nuclei by field emission scanning electron microscopy (feSEM) and visualized a network of filaments that attach to nuclear pore complexes and extend throughout the nucleus. Within the nucleus, these 'pore-linked filaments' (PLFs) were embedded into spherical structures 100 nm to approximately 5 microm in diameter. A subset of spheres was identified as Cajal bodies by immuno-gold labeling; the rest were inferred to be nucleoli and snurposomes both of which are abundant in Xenopus oocyte nuclei. Most PLFs were independent of chromatin. The thickness of a typical PLF was 40 nm (range, approximately 12-100 nm), including the 4 nm chromium coat. PLFs located inside the nucleus merged, bundled and forked, suggesting architectural adaptability. The PLF network collapsed upon treatment with latrunculin A, which depolymerizes actin filaments. Jasplakinolide, which stabilizes actin filaments, produced PLFs with more open substructure including individual filaments with evenly-spaced rows of radially projecting short filaments. Immuno-gold labeling of untreated oocyte nuclei showed that actin and protein 4.1 each localized on PLFs. Protein 4.1-gold epitopes were spaced at approximately 120 nm intervals along filaments, and were often paired ( approximately 70 nm apart) at filament junctions. We suggest that protein 4.1 and actin contribute to the structure of a network of heterogeneous filaments that link nuclear pore complexes to subnuclear organelles, and discuss possible functions for PLFs in nuclear assembly and intranuclear traffic.

Actins↗
PubMed1987

Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway.

Using a monoclonal antibody (mAb 414), we previously identified a protein of 62 kDa (p62) that was localized to the nuclear pore complex by immunoelectron microscopy. We also showed that p62 binds specifically to wheat germ agglutinin. Therefore, we proposed that this nuclear pore complex protein might be a member of a recently characterized family of glycoproteins that are labeled by in vitro galactosylation of rat liver nuclei and contain O-linked monosaccharidic GlcNAc residues. In support of this, we now show that incubation with N-acetylglucosaminidase reduces the molecular mass of p62 by approximately 3 kDa because of the removal of terminal GlcNAc residues. Moreover, p62 can be galactosylated in vitro by using UDP-[3H]galactose and galactosyltransferase. We also show that most of the GlcNAc residues are added within 5 min of synthesis, when p62 is soluble and cytosolic. Thus, the addition of GlcNAc is carried out in the cytoplasm and is clearly distinct from the N- and O-linked glycosylation pathways of the endoplasmic reticulum and Golgi complex. Using another mAb with a broad specificity for nuclear GlcNAc-containing proteins, we show by immunofluorescence and protein blotting of subnuclear fractions that some of these proteins are in the interior of the nucleus, and others are most likely located in the pore complex.

Animals↗
PubMed2001

Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos.

The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional 'spindle envelope'. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.

Animals↗