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PubMed1998

Transport of macromolecules between the nucleus and the cytoplasm.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor-substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor-cargo interaction and imparts directionality to the transport process. Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.

Biological Transport↗

PubMed2006

Hot spot formation in the nuclear envelope of oocytes in response to steroids.

A Glucocorticoid-sensitive cell rapidly responds to hormone stimulation with bidirectional exchange of specific macromolecules between cytosol and nucleus. Glucocorticoid-initiated macromolecules (GIMs) must overcome the nuclear envelope (NE) to enter or leave the nucleus. GIM translocation occurs through nuclear pore complexes (NPCs) that span the NE. We investigated the question whether transport of GIMs through NPCs occurs random or involves selected groups of NPCs (hot spots). Glucocorticoid receptors were expressed in Xenopus laevis oocytes and GIM transport was activated by triamcinolone acetonide, a potent synthetic glucocorticoid analogon. Glucocorticoid receptors associated with the NE and the chromatin were identified using western blot analysis and, at single molecule level, atomic force microscopy. Fluorescence-labeled dextran was used to describe passive NE permeability. We observed that after hormone injection (i) small GIMs, most likely GRs, localize within seconds on both sides of the NE. (ii) large GIMs, most likely ribonucleoproteins, localize within minutes on NPCs at the nucleoplasmic side (iii) both small and large GIMs accumulate on selected NPC clusters (iv) NE permeability transiently decreases when GIMs attach to NPCs. We conclude that GIM transport across the nuclear barrier does not randomly take place but is carried out by a selected population of NPCs.

Animals↗

PubMed2003

Role of the cytoskeleton in nuclear import.

The role of the cytoskeleton in regulating the intracellular localization of cellular organelles, viruses, and individual proteins has been the subject of much investigation in recent years. While regulated transport through the nuclear pore remains the primary determinant of nuclear localization, it has become clear that the nuclear localization of viruses and some cellular proteins is mediated by the ability of the cytoskeleton, usually microtubules, to direct their perinuclear accumulation in close proximity to the nuclear pore complex. We also discuss how the size of virions and the viscous nature of the cytoplasm would make it very unlikely or even impossible for viruses to achieve this localization by diffusion alone in the absence of active transport mechanisms. This review focuses on the known methods employed by different viruses and proteins to effect their perinuclear accumulation.

Active Transport, Cell Nucleus↗

PubMed2001

Delineation of mRNA export pathways by the use of cell-permeable peptides.

The transport of messenger RNAs (mRNAs) from the nucleus to the cytoplasm involves adapter proteins that bind the mRNA as well as receptor proteins that interact with the nuclear pore complex. We demonstrate the utility of cell-permeable peptides designed to interfere with interactions between potential adapter and receptor proteins to define the pathways accessed by particular mRNAs. We show that HuR, a protein implicated in the stabilization of short-lived mRNAs containing AU-rich elements (AREs), serves as an adapter for c-fos mRNA export through two pathways. One involves the HuR shuttling domain, HNS, which exhibits a heat shock-sensitive interaction with transportin 2 (Trn2); the other involves two protein ligands of HuR-pp32 and APRIL-which contain leucine-rich nuclear export signals (NES) recognized by the export receptor CRM1. Heterokaryon and in situ hybridization experiments reveal that the peptides selectively block the nucleocytoplasmic shuttling of their respective adapter proteins without perturbing the overall cellular distribution of polyadenylated mRNAs.

Amino Acid Sequence↗

PubMed2005

The Clr7 and Clr8 directionality factors and the Pcu4 cullin mediate heterochromatin formation in the fission yeast Schizosaccharomyces pombe.

Fission yeast heterochromatin is formed at centromeres, telomeres, and in the mating-type region where it mediates the transcriptional silencing of the mat2-P and mat3-M donor loci and the directionality of mating-type switching. We conducted a genetic screen for directionality mutants. This screen revealed the essential role of two previously uncharacterized factors, Clr7 and Clr8, in heterochromatin formation. Clr7 and Clr8 are required for localization of the Swi6 chromodomain protein and for histone H3 lysine 9 methylation, thereby influencing not only mating-type switching but also transcriptional silencing in all previously characterized heterochromatic regions, chromosome segregation, and meiotic recombination in the mating-type region. We present evidence for physical interactions between Clr7 and the mating-type region and between Clr7 and the S. pombe cullin Pcu4, indicating that a complex containing these proteins mediates an early step in heterochromatin formation and implying a role for ubiquitination at this early stage prior to the action of the Clr4 histone methyl-transferase. Like Clr7 and Clr8, Pcu4 is required for histone H3 lysine 9 methylation, and bidirectional centromeric transcripts that are normally processed into siRNA by the RNAi machinery in wild-type cells are easily detected in cells lacking Clr7, Clr8, or Pcu4. Another physical interaction, between the nucleoporin Nup189 and Clr8, suggests that Clr8 might be involved in tethering heterochromatic regions to the nuclear envelope by association with the nuclear-pore complex.

Blotting, Southern↗

PubMed1999

Subcellular distribution of survival motor neuron (SMN) protein: possible involvement in nucleocytoplasmic and dendritic transport.

Spinal muscular atrophy (SMA) is among the most common recessive autosomal diseases and is characterized by the loss of spinal motor neurons. A gene termed 'Survival of Motor Neurons' (SMN) has been identified as the SMA-determining gene. Recent work indicates the involvement of the SMN protein and its associated protein SIP1 in spliceosomal snRNP biogenesis. However, the function of SMN remains unknown. Here, we have studied the subcellular localization of SMN in the rat spinal cord and more generally in the central nervous system (CNS), by light fluorescence and electron microscopy. SMN immunoreactivity (IR) was found in the different regions of the spinal cord but also in various regions of the CNS such as the brainstem, cerebellum, thalamus, cortex and hippocampus. In most neurons, we observed a speckled labelling of the cytoplasm and a discontinuous staining of the nuclear envelope. For some neurons (e.g. brainstem nuclei, dentate gyrus, cortex: layer V) and, in particular in motoneurons, SMN-IR was also present as prominent nuclear dot-like-structures. In these nuclear dots, SMN colocalized with SIP1 and with fibrillarin, a marker of coiled bodies. Ultrastructural studies in the anterior horn of the spinal cord confirmed the presence of SMN in the coiled bodies and also revealed the protein at the external side of nuclear pores complexes, in association with polyribosomes, and in dendrites, associated with microtubules. These localizations suggest that, in addition to its involvement in the spliceosome biogenesis, the SMN protein could also play a part in nucleocytoplasmic and dendritic transport.

Animals↗

PubMed1995

Nup358, a cytoplasmically exposed nucleoporin with peptide repeats, Ran-GTP binding sites, zinc fingers, a cyclophilin A homologous domain, and a leucine-rich region.

The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell lambda expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.

Amino Acid Isomerases↗

PubMed1997

Yeast acetyl-CoA carboxylase is associated with the cytoplasmic surface of the endoplasmic reticulum.

Acetyl-CoA carboxylase (ACC1) catalyzes the first and rate limiting step of de novo fatty acid synthesis. Defects in Acc1p were recently correlated with an altered structure/function of the nuclear envelope in yeast. The subcellular distribution of the enzyme was determined in wild-type and mutant cells by cell fractionation and confocal immunofluorescence microscopy. Even though fatty acid synthesis is generally considered to be a cytosolic reaction, we found that Acc1p cofractionated with nuclei and the ER (endoplasmic reticulum) marker BiP/Kar2p. Membrane-bound Acc1p was susceptible to proteinase K digestion and was solubilized by mild salt treatment indicating that it is loosely associated with the cytosolic surface of the nuclear ER membrane. Consistent with these observations, immunofluorescence analysis revealed that Acc1p was distributed in a gradient within the cytoplasm that had its highest concentration around the ER. Possible association of Acc1p with the nuclear pore complexes (NPCs) was investigated in strains that display NPC clustering. Results of these experiments suggest that Acc1p localization is independent of NPC distribution. We propose that association of Acc1p with the cytoplasmic surface of the ER membrane is physiologically relevant to "channel" the enzymatic product of Acc1p, malonyl-CoA, to a putative ER-localized fatty acid chain elongase complex.

Acetyl-CoA Carboxylase↗

PubMedDate not supplied

The use of field emission in-lens scanning electron microscopy to study the steps of assembly of the nuclear envelope in vitro.

At mitosis the nuclear envelope (NE) is disassembled to allow chromosome separation. In telophase it is reassembled as the chromosomes decondense. Cell-free extracts of Xenopus eggs have been used extensively to study assembly of the NE and the nuclear pore complexes (NPCs), providing several models for the steps involved. The NE is a surface structure which in cell-free extracts is easily exposed. It is appropriate, therefore, to use a surface imaging technique to study NE dynamics. Field emission in-lens scanning electron microscopy (FEISEM) provides the opportunity to image surfaces, directly, and to visualise details of structures such as the NPC. Here we show the feasibility and value of FEISEM to study the steps of NE formation. Nuclei have been assembled in vitro and fixed at different time points during assembly, followed by conductive staining, platinum coating, and visualisation by FEISEM. Changes on the nuclear surface with time are shown. Details of the surface of chromatin and the cytoplasmic face of NPC structure are demonstrated without the need to isolate the structures from the nucleus.

Animals↗

PubMed1995

Isolation and characterization of nuclear envelopes from the yeast Saccharomyces.

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.

Antibodies, Monoclonal↗

PubMed1999

Active nuclear import and export is independent of lumenal Ca2+ stores in intact mammalian cells.

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (approximately 10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)-tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N', N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

Calcium↗

PubMed2001

U2AF participates in the binding of TAP (NXF1) to mRNA.

TAP/NXF1 is a conserved mRNA export receptor serving as a link between messenger ribonucleoproteins (mRNPs) and the nuclear pore complex. The mechanism by which TAP recognizes its export substrate is unclear. We show here that TAP is added to spliced mRNP in human cells. We identified a distinct region of TAP that targets it to mRNP. Using yeast two-hybrid screens and in vitro binding studies, we found that this region coincides with a direct binding site for U2AF35, the small subunit of the splicing factor U2AF. This interaction is evolutionarily conserved across metazoa, indicating its significance. We further found in human cells that the exogenously expressed large U2AF subunit, U2AF65, accumulates in spliced mRNP, leading to the recruitment of U2AF35 and TAP. Similarly to TAP, U2AF65 stimulated directly the nuclear export and expression of an mRNA that is otherwise retained in the nucleus. Together with our finding that U2AF is continuously exported from the nucleus, these data suggest that U2AF participates in nuclear export, by facilitating TAP's addition to its mRNA substrates.

Binding Sites↗

PubMed2005

The genome of HSV-1 translocates through the nuclear pore as a condensed rod-like structure.

Incoming herpes simplex virus type-1 (HSV-1) capsids are known to dock to the nuclear pore complex (NPC) and release their genome. It has remained elusive, however, how the huge viral DNA translocates through the comparatively small NPC channel. In the present study, the interaction of HSV-1 with NPCs was analyzed by atomic force microscopy. In addition to capsids, smaller subviral structures--most with a diameter of 35-40 nm and a length of 130-160 nm--were visualized at the cytoplasmic side of the NPC. These components differed from capsids in their adhesion and stiffness properties, and were the sole subviral structures translocated through dilated NPCs towards the nucleus. It is presumed that they are the HSV-1 genome, and that a change in NPC conformation allows translocation of this genome as a densely packaged, rodlike structure.

Animals↗

PubMed2004

RXRalpha acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.

Retinoid X receptor (RXR) plays a crucial role in the cross talk between retinoid receptors and other hormone receptors including the orphan receptor TR3, forming different heterodimers that transduce diverse steroid/thyroid hormone signaling. Here we show that RXRalpha exhibits nucleocytoplasmic shuttling in MGC80-3 gastric cancer cells and that RXRalpha shuttling is energy-dependent through a nuclear pore complex (NPC)-mediated pathway for its import and an intact DNA binding domain-mediated pathway for its export. In the presence of its ligand 9-cis retinoic acid, RXRalpha was almost exclusively located in the cytoplasm. More importantly, we also show that RXRalpha acts as a carrier to assist translocation of TR3, which plays an important role in apoptosis. Both RXRalpha and TR3 colocalized in the nucleus; however, upon stimulation by 9-cis retinoic acid they cotranslocated to the cytoplasm and then localized in the mitochondria. TR3 export depends on RXRalpha, as in living cells GFP-TR3 alone did not result in export from the nucleus even in the presence of 9-cis retinoic acid, whereas GFP-TR3 cotransfected with RXRalpha was exported out of the nucleus in response to 9-cis retinoic acid. Moreover, specific reduction of RXRalpha levels caused by anti-sense RXRalpha abolished TR3 nuclear export. In contrast, specific knockdown of TR3 by antisense-TR3 or TR3-siRNA did not affect RXRalpha shuttling. These results indicate that RXRalpha is responsible for TR3 nucleocytoplasmic translocation, which is facilitated by the RXRalpha ligand 9-cis retinoic acid. In addition, mitochondrial TR3, but not RXRalpha, was critical for apoptosis, as TR3 mutants that were distributed in the mitochondria induced apoptosis in the presence or absence of 9-cis retinoic acid. These data reveal a novel aspect of RXRalpha function, in which it acts as a carrier for nucleocytoplasmic translocation of orphan receptors.

3T3 Cells↗

PubMed1998

The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.

Amino Acid Sequence↗

PubMed2006

Correlation of initial autoantibody profile and clinical outcome in primary biliary cirrhosis.

Although there have been significant advances in understanding the clinical and biochemical features of primary biliary cirrhosis (PBC), there is still a paucity of data on the usefulness of biomarkers as prognostic indicators. This is particularly important at the time of initial diagnosis. Indeed, the widespread use of antimitochondrial antibody testing has led to an earlier diagnosis of asymptomatic PBC and it is difficult to predict which patients will experience a benign versus a rapidly progressive course. To address this issue, we examined a unique population of 127 newly diagnosed patients with PBC during a 15-year period of observation that began in January 1990. Sera from these patients were analyzed for antimitochondrial, antinuclear, and anti-smooth muscle antibodies, and immunoblotting was performed for nuclear pore complex (NPC). The patients were then followed up longitudinally using biochemical liver function tests. No patient was under any medical therapy for PBC at the time of the initial sera collection. Data were analyzed based not only on the clinical features, but also the Mayo score and specific outcome measures, including time to death, need for liver transplantation, and complication free survival. Among patients with early disease, bilirubin increased to >2 mg/dL in the anti-NPC(+) patients (26% vs. 5%, P = .019). Anti-NPC antibodies remained stable or slightly increased over the period of observation. In condusion, anti-NPC identifies patients likely to experience an unfavorable clinical course and more rapid disease progression.

Adult↗

PubMed2001

Anti-nuclear envelope antibodies: Clinical associations.

OBJECTIVES: Characterization of the clinical associations and clinical implications of antibodies reacting with antigens of the nuclear envelope. METHODS: Description of an illustrative case and a MEDLINE search-assisted literature review of relevant cases. RESULTS: With indirect immunofluorescence, autoantibodies directed against various antigens of the nuclear envelope stain the nucleus in a ring-like (rim) pattern. Autoantibodies against 5 antigenic components of the nuclear envelope have been described: anti-gp210, p62, lamina, lamina-associated polypeptides, and lamin B receptor. Antibodies to antigens of the nuclear pore complex, such as gp210 and p62, are highly specific (> 95%) for primary biliary cirrhosis and may aid in the serologic diagnosis of this condition, especially in cases in which antimitochondrial antibodies are not detectable. In contrast, antilamin antibodies are not disease-specific but seem to be associated with lupus anticoagulant or anticardiolipin antibodies, antiphospholipid syndrome, thrombocytopenia, autoimmune liver diseases, and arthralgia. High-titered antilamin antibodies help to define a subset of lupus patients with antiphospholipid antibodies who are at a lower risk of developing thrombotic events. In addition, preliminary data suggest that the presence of antilamin antibodies may be helpful in the diagnosis of chronic fatigue syndrome. CONCLUSIONS: Each of the antibodies reacting with nuclear membrane antigens has its own spectrum of disease associations. RELEVANCE: Determination of anti-nuclear envelope antibody pattern by indirect immunofluorescence, with subsequent determination of the specific antibody, carries important diagnostic and prognostic implications in various autoimmune conditions.

Antibodies, Antinuclear↗

PubMed1997

Dynamic and equilibrium studies on the interaction of Ran with its effector, RanBP1.

Ran, a small nuclear GTP-binding protein, is one of the most abundant Ras-related proteins in eucaryotic cells. Ran is essential for nucleo-cytoplasmatic transport and is primarily localized in the nucleus and at the nuclear pore complex. Here, we characterize the kinetics and equilibrium of the interaction between Ran and RanBP1 by two independent biophysical approaches: fluorescence spectroscopy using analogues of guanine nucleotides and surface plasmon resonance in the BIAcore system. Both approaches result in kinetic and equilibrium data which are in good agreement with each other. Affinities of RanBP1 for Ran in the GTP-bound state were in the nanomolar range, while Ran.GDP bound RanBP1 with a dissociation constant around 10 microM. Interestingly, the difference in affinity of RanBP1 for Ran.GDP was mostly due to a dramatic increase of the dissociation rate constant. Mutant Ran protein lacking the last five amino acids of the C-terminus (RanDeltaC) is unable to facilitate nuclear import in vitro and does not bind to RanBP1. Here, we show that RanBP1 binds RanDeltaC.mGppNHp with KD values around 10 microM, as is the case for its association with full-length Ran.GDP. The loss of affinity of RanBP1 for the triphosphate form of RanDeltaC was a result of both a decrease of the association rate and a moderately increased dissociation of the RanDeltaC.RanBP1 complex. Circular dichroism spectra indicate significant changes in the secondary structure of either Ran.GppNHp, RanBP1, or both proteins upon forming a stable complex with each other.

Biological Transport↗