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PubMed1997

A domain distinct from nucleoplasmin's nuclear localization sequence influences its transport.

We constructed mutants of the prototypical, nuclear-accumulating protein nucleoplasmin and used them in both in vivo and in vitro nuclear transport assays to search for transport-influencing domains distinct from this protein's recognized nuclear localization sequence. We identified the polyglutamic acid tract on the amino flank of the nuclear localization sequence as being involved in two stages of nuclear transport. This poly-glu tract is required for the facilitated translocation of nucleoplasmin through the nuclear pore complex, and it also enhances the subsequent binding of nucleoplasmin within the nucleus.

Animals↗
PubMed2000

The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport.

Importin-beta is a nuclear transport factor which mediates the nuclear import of various nuclear proteins. The N-terminal 1-449 residue fragment of mouse importin-beta (impbeta449) possesses the ability to bidirectionally translocate through the nuclear pore complex (NPC), and to bind RanGTP. The structure of the uncomplexed form of impbeta449 has been solved at a 2.6 A resolution by X-ray crystallography. It consists of ten copies of the tandemly arrayed HEAT repeat and exhibits conformational flexibility which is involved in protein-protein interaction for nuclear transport. The overall conformation of the HEAT repeats shows that a twisted motion produces a significantly varied superhelical architecture from the previously reported structure of RanGTP-bound importin-beta. These conformational changes appear to be the sum of small conformational changes throughout the polypeptide. Such a flexibility, which resides in the stacked HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that impbeta449 has a structural similarity with another nuclear migrating protein, namely beta-catenin, which is composed of another type of helix-repeated structure of ARM repeat. Interestingly, the essential regions for NPC translocation for both importin-beta and beta-catenin are spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage.

Amino Acid Sequence↗
PubMed1998

How do animal DNA viruses get to the nucleus?

Genome and pre-genome replication in all animal DNA viruses except poxviruses occurs in the cell nucleus (Table 1). In order to reproduce, an infecting virion enters the cell and traverses through the cytoplasm toward the nucleus. Using the cell's own nuclear import machinery, the viral genome then enters the nucleus through the nuclear pore complex. Targeting of the infecting virion or viral genome to the multiplication site is therefore an essential process in productive viral infection as well as in latent infection and transformation. Yet little is known about how infecting genomes of animal DNA viruses reach the nucleus in order to reproduce. Moreover, this nuclear locus for viral multiplication is remarkable in that the sizes and composition of the infectious particles vary enormously. In this article, we discuss virion structure, life cycle to reproduce infectious particles, viral protein's nuclear import signal, and viral genome nuclear targeting.

Adenoviridae↗
PubMed2003

Passive transport of macromolecules through Xenopus laevis nuclear envelope.

Although nuclear pore complexes (NPC) are considered to be key structures in gene expression, little is known about their regulatory control. In order to explore the regulatory mechanism of passive transport of small macromolecules we examined the influence of different factors on the diffusional pathway of NPCs in isolated Xenopus laevis oocyte nuclei. Diffusion of fluorescence-labeled 10-kD dextran was measured across the nuclear envelope with confocal fluorescence microscopy. Surprisingly, the filling state of the perinuclear Ca(2+) store had no influence on passive transport of 10-kD dextran. Furthermore, nuclear envelope permeability was independent of cytoplasmic pH (pH range 8.3-6.3). In contrast, nuclear swelling, induced by omission of the endogenous cytosolic macromolecules, clearly increased nuclear permeability. An antibody against the glycoprotein gp62, located at the central channel entrance, reduced macromolecule diffusion. In addition, nuclei from transcriptionally active, early developmental stages (stage II) were less permeable compared to transcriptionally inactive, late-developmental-stage (stage VI) nuclei. In stage II nuclei, atomic force microscopy disclosed NPC central channels with plugs that most likely were ribonucleoproteins exiting the nucleus. In conclusion, the difference between macromolecule permeability and previous measurements of electrical resistance strongly indicates separate routes for macromolecules and ions across the nuclear envelope.

Adenosine Triphosphate↗
PubMed2026

ELYS associates with distinct DNA sequence environments during post-mitotic nuclear pore reassembly.

Nuclear pore complexes (NPCs) contribute to genome organization and cell identity, yet how post-mitotic NPC assembly is coordinated with chromatin architecture remains unclear. Here, we show that the nucleoporin ELYS preferentially associates with chromatin regions displaying distinct intrinsic DNA sequence features that are not explained by the repressive histone marks examined here. ELYS-bound regions are enriched for AT-rich sequences, whereas ELYS binding at super-enhancer-associated loci shift toward GC-rich sequence composition, revealing distinct sequence environments. These findings indicate that ELYS localization is associated with distinct intrinsic DNA sequence features and suggest a mechanism by which nuclear pore-associated architecture restores transcriptional programs after mitosis.

Journal Article↗
PubMed1996

A nuclear cap-binding complex binds Balbiani ring pre-mRNA cotranscriptionally and accompanies the ribonucleoprotein particle during nuclear export.

In vertebrates, a nuclear cap-binding complex (CBC) formed by two cap- binding proteins, CBP20 and CBP80, is involved in several steps of RNA metabolism, including pre-mRNA splicing and nuclear export of some RNA polymerase II-transcribed U snRNAs. The CBC is highly conserved, and antibodies against human CBP20 cross-react with the CBP20 counterpart in the dipteran Chironomus tentans. Using immunoelectron microscopy, the in situ association of CBP20 with a specific pre-mRNP particle, the Balbiani ring particle, has been analyzed at different stages of pre-mRNA synthesis, maturation, and nucleo-cytoplasmic transport. We demonstrate that CBP20 binds to the nascent pre-mRNA shortly after transcription initiation, stays in the RNP particles after splicing has been completed, and remains attached to the 5' domain during translocation of the RNP through the nuclear pore complex (NPC). The rapid association of CBP20 with nascent RNA transcripts in situ is consistent with the role of CBC in splicing, and the retention of CBC on the RNP during translocation through the NPC supports its proposed involvement in RNA export.

Animals↗
PubMed1992

Nopp140 shuttles on tracks between nucleolus and cytoplasm.

Nopp140 is a nucleolar phosphoprotein of 140 kd that we originally identified and purified as a nuclear localization signal (NLS)-binding protein. Molecular characterization revealed a 10-fold repeated motif of highly conserved acidic serine clusters that contain an abundance of phosphorylation consensus sites for casein kinase II (CK II). Indeed, Nopp140 is one of the most phosphorylated proteins in the cell, and NLS binding was dependent on phosphorylation. Nopp140 was shown to shuttle between the nucleolus and the cytoplasm. Shuttling is likely to proceed on tracks that were revealed by immunoelectron microscopy. These tracks extend from the dense fibrillar component of the nucleolus across the nucleoplasm to some nuclear pore complexes. We suggest that Nopp140 functions as a chaperone for import into and/or export from the nucleolus.

Amino Acid Sequence↗
PubMed2002

Nuclear import of factors involved in signaling is inhibited in C3H/10T1/2 cells treated with tetradecylthioacetic acid.

The non-beta-oxidisable tetradecylthioacetic acid (TTA) is incorporated into cellular membranes when C3H/10T1/2 cells are cultured in TTA-containing medium. We here demonstrate that this alteration in cellular membranes affect the nuclear translocation of proteins involved in signal transduction. Analysis of cellular fatty acid composition shows that TTA and TTA:1n-8 constitute approximately 40 mol% of total fatty acids in cellular/nuclear membranes. Activation of c-fos expression is significantly inhibited in TTA-treated cells but the enzymatic activation of mitogen activated protein kinase (ERK) is not affected. Immunofluorescence and confocal microscopy studies demonstrate that in mitogene-stimulated TTA-treated cells, the translocation of phosphorylated ERK1/2, protein kinase C alpha (PKC alpha), and PKC beta(1) from the cytoplasm into the nucleus is considerably decreased and delayed. Concomitant with a decreased nuclear import, ERK1/2 dephosphorylation is decreased in TTA-treated cells. There is no TTA-induced inhibition of nuclear import of proteins with a classical nuclear localization signal (NLS), as seen by in vitro nuclear import experiments of BSA fused to the NLS from SV40 large T, or in vivo studies of hnRNP A1 nuclear import. The expression levels of Importin alpha, Importin beta, Importin 7, and NTF2 are not altered in the TTA-treated cells. Taken together, our data indicate that TTA treatment causes changes in cellular fatty acid composition that negatively affect NLS-independent mechanisms of protein translocation through the nuclear pore complex.

Active Transport, Cell Nucleus↗
PubMed2005

Quantitative models of nuclear transport.

Nuclear pore complexes mediate the rapid trafficking of target macromolecules between the nucleus and the cytoplasm but exclude non-targets. Mathematical modeling helps to define the physical properties of a transport medium that can selectively enhance the permeation of some molecules but block others. Recent pioneering work has established a basis for quantitative modeling of nuclear translocation, and we expect this field to expand rapidly. A second area where modeling of nucleocytoplasmic transport has been prominently employed is in efforts to understand the regulatory networks by which signals pass between the nuclear and cytoplasmic compartments. Recent evidence suggests that the distinctive kinetics and spatial organization of nuclear transport processes can be used to efficiently propagate signals by new and unexpected pathways.

Active Transport, Cell Nucleus↗
PubMed2001

Germline and developmental roles of the nuclear transport factor importin alpha3 in C. elegans.

The importin alpha family of transport factors mediates the nuclear import of classical nuclear localization signal-containing proteins. In order to understand how multiple importin alpha proteins are regulated both in individual cells and in a whole organism, the three importin alpha (ima) genes of Caenorhabditis elegans have been identified and studied. All three IMAs are expressed in the germline; however, only IMA-3 is expressed in the soma. RNA interference (RNAi) experiments demonstrate that IMA-3 is required for the progression of meiotic prophase I during oocyte development. Loss of IMA-3 expression leads also to a disruption of the nuclear pore complex accompanied by the mis-localization of P granules. A range of defects occurring in ima-3(RNAi) F1 progeny further supports a role for IMA-3 during embryonic and larval development. The functional association of IMA-3 with distinct cellular events, its expression pattern and intracellular localization indicate that regulation of the nuclear transport machinery is involved in the control of developmental pathways.

Amino Acid Sequence↗
PubMed2005

[The relationship between nuclear-cytoplasmic transport and chromosome segregation].

Chromosomes/chromatids transport to cell division poles (chromosome segregation) and nuclear-cytoplasmic transport give way to each other during cell cycle. Disassembly and reassembly of the nuclear envelope during input or output mitosis are the critical time periods for changing one kind of transport system for the other one. The interest in the problem of relationships between nuclear-cytoplasmic transport and chromosome segregation came into view after revealing some factors involved in both transport systems. Among these factors are GTPase Ran, nuclear transport receptors and associated molecules, kinetochore proteins and proteins of nuclear pore complexes (nucleoprin, for example), spindle microtubules, microtubule motor proteins, and signal molecules. The role of all of these factors in the transport systems is not known. A decision of this problem will make it possible to determine mechanisms of both transport systems and to understand the character of evolutionary relationships between these systems.

Animals↗
PubMed1990

Nucleoplasmin: the archetypal molecular chaperone.

Nucleoplasmin was the first protein to be described as a molecular chaperone. Studies of nucleoplasmin have resulted in advances in two areas of cell biology. Firstly, the pathway of nucleosome assembly in Xenopus oocytes and eggs has been elucidated and is the only assembly pathway known in detail. Nucleosome assembly represents the major chaperoning function of nucleoplasmin. Secondly, nucleoplasmin has been used to elucidate the transport of proteins into the nucleus, revealing a selective entry mechanism for nuclear proteins, passage through the nuclear pore complex, and a two-step mechanism of transport. The properties and functions of nucleoplasmin are reviewed, together with other proteins which are related either structurally or functionally to nucleoplasmin.

Animals↗
PubMed1999

An unexpected link between the secretory path and the organization of the nucleus.

Yeast sec mutations define the machinery of vesicular traffic. Surprisingly, many of these mutations also inhibit ribosome biogenesis by reducing transcription of rRNA and genes encoding ribosomal proteins. We observe that these mutants reversibly inhibit protein import into the nucleus, with import cargo accumulating at the nucleoplasmic face of nuclear pore complexes, as when Ran-GTP cannot bind importins. They also rapidly and reversibly relocate multiple nucleolar and nucleoplasmic proteins to the cytoplasm. The import block and relocation are antagonized by overexpression of yeast Ran, Hog1p kinase, or Ssa/Hsp70 proteins or by inhibition of protein synthesis. These nucleocytoplasmic signaling events document an extraordinary plasticity of nuclear organization.

Biological Transport↗
PubMed1995

ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope.

Uptake and release of Ca2+ from isolated liver nuclei were studied with fluorescent probes. We show with the help of digital imaging and confocal microscopy that the Ca(2+)-sensitive fluorescent probe Fura 2 is concentrated in or around the nuclear envelope and that the distribution of Fura 2 fluorescence is similar to that of an endoplasmic reticulum marker. The previously demonstrated ATP-dependent uptake of Ca2+ into isolated nuclei and release of the accumulated Ca2+ by inositol 1,4,5-trisphosphate (IP3) are therefore due to transport of Ca2+ into and out of the nuclear envelope and not the nucleoplasm. Dextrans labeled with fluorescent Ca2+ indicators (calcium-Green 1 and Fura 2) are distributed uniformly in the nucleoplasm and can be used to show that changes in the external Ca2+ concentration produce rapid changes in the nucleoplasmic Ca2+ concentration. Nevertheless, IP3 and cyclic ADP-ribose evoke transient intranuclear Ca2+ elevations. The release from the Ca2+ stores in or around the nuclear envelope appears to be directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complexes.

Adenosine Diphosphate Ribose↗
PubMedDate not supplied

High-resolution field emission scanning electron microscope imaging of internal cell structures after Epon extraction from sections: a new approach to correlative ultrastructural and immunocytochemical studies.

The availability of high-resolution field emission scanning electron microscopes (FESEM) and the recent development of a less destructive method for extracting Epon from sections motivated us to investigate these techniques for the study of internal cell structures. We chose the nuclear pore complex (NPC) and insect striated muscle as test objects. Chemically fixed or rapidly cryoimmobilized samples were embedded in Epon 812. The Epon was extracted from 200- to 300-nm-thick sections with a modified potassium methoxide-crown ether complex. The samples were viewed with high-resolution FESEM at low voltages. In tangential sections of isolated nuclear envelopes from Xenopus oocytes the cytoplasmic and intranuclear components ("fishtraps") of NPCs appeared identical to what has been described from whole mounts. In cross sections, fishtraps are seen in side view, which is possible only with this technique. In longitudinal and cross sections of insect flight muscle the classical arrangement of myofilaments and cross-bridges is well preserved. This method now makes it possible to image internal cell structures from any desired angle by high-resolution FESEM. Immunolabeling studies on the rabbit psoas muscle demonstrated that antigenicity of alpha-actinin was retained in Epon-extracted sections. Immunogold labeling with antibodies against alpha-actinin conjugated to 3-nm gold beads was intense, highly specific, and restricted to the Z lines. This method can overcome the penetration problem of immunogold labeling, since any cell component can be positioned at the surface of the section. Obviously this approach can become a powerful new tool for many areas of structural cell biology.

Actinin↗
PubMed2003

The interplay of nuclear mRNP assembly, mRNA surveillance and export.

Fully processed mRNAs are exported to the cytoplasm where they direct protein synthesis. Export of mRNAs is mediated by a conserved heterodimeric transport receptor (known as NXF1-p15 in metazoa) that binds to mRNA cargoes either directly or indirectly by means of adaptor proteins. Upon binding, the receptor translocates the cargo across the central channel of the nuclear pore complex (NPC) by interacting directly with NPC proteins called nucleoporins. Our understanding of the molecular mechanisms by which the heterodimeric receptor is recruited to cellular mRNAs indicates that the adaptors, together with additional proteins, are loaded cotranscriptionally to nascent mRNAs to form large ribonucleoprotein complexes (mRNPs). This mRNP assembly process might be subject to quality control by a nuclear mRNP surveillance mechanism, so that aberrantly assembled mRNPs are degraded before the receptor delivers them to the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed1995

[The roles of two kinds of membrane vesicles in the formation of annulate lamellae and nuclear envelopes in a cell-free system from Xenopus egg extracts].

The eggs of Xenopus laevis were activated to complete their second meiosis and enter interphase by calcium ionophore A 23187. Then they were centrifuged at 10,000 g to get activated Xenopus egg extracts. Chromatin structure could form in such an extracts after exogenous Lambda DNA was added, and nuclear envelopes (NEs) could assemble around the chromatin. Furthermore, annulate lamellae (AL), a NE-like membrane structure, could form in chromatin-free regions simultaneously. After large amounts of observation and analysis at ultrastructural level, we propose that both AL and NEs assemble in the extracts by the fusion of membrane vesicles. There were two kinds of membrane vesicles in Xenopus egg extracts, small rough vesicles with diameters of 200 nm and large smooth vesicles. They had many differences in morphology, size and membrane structure. However, they both participate in the formation of AL and NEs, 200 nm membrane vesicles first fused each other to become parallel double membrane cisternae, nuclear pore complexes assembled in the double membranes in the mean time. The fusion of large smooth vesicles with the double membrane cirternae led to the growth of AL and the maturity of NEs. During the formation of NEs, large smooth vesicles were easily observed to link two neighboring double membrane fragments by fusing with them, so that integrated nuclear envelope could form. 200 nm small rough vesicles could attach to the surface of chromatin to start the assembly of nuclear envelopes, while others could fuse each other at chromatin-free regions and these fusion resulted in the formation of annulate lamellae.

Animals↗
PubMed2003

Virtual gating and nuclear transport: the hole picture.

The eukaryotic nucleus is surrounded by a protective nuclear envelope, which is perforated by trafficking machines termed nuclear pore complexes (NPCs). The NPCs are the sole mediators of exchange between the nucleus and the cytoplasm. Small molecules pass through the NPCs unchallenged; however, large macromolecules are excluded unless chaperoned across by transport factors. Here, we suggest a model, termed 'virtual gating', to explain the mechanism of this rapid and selective macromolecular trafficking.

Animals↗