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PubMed1995

Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins.

The molecular dynamics of nuclear protein import were examined in a solution binding assay by testing for interactions between a protein containing a nuclear localization signal (NLS), the transport factors karyopherin alpha, karyopherin beta, and Ran, and FXFG or GLFG repeat regions of nucleoporins. We found that karyopherins alpha and beta cooperate to bind FXFG but not GLFG repeat regions. Binding of the NLS protein to karyopherin alpha was enhanced by karyopherin beta. Two novel reactions were discovered. First, incubation of a karyopherin heterodimer-NLS protein complex with an FXFG repeat region stimulated the dissociation of the NLS protein from the karyopherin heterodimer. Second, incubation of the karyopherin heterodimer with RanGTP (or with a Ran mutant that cannot hydrolyze GTP) led to the dissociation of karyopherin alpha from beta and to an association of Ran with karyopherin beta; RanGDP had no effect. We propose that movement of NLS proteins across the nuclear pore complex is a stochastic process that operates via repeated association-dissociation reactions.

Amino Acid Sequence↗
PubMed2006

Actively transcribed GAL genes can be physically linked to the nuclear pore by the SAGA chromatin modifying complex.

Recent work has demonstrated that some actively transcribed genes closely associate with nuclear pore complexes (NPC) at the nuclear periphery. The Saccharomyces cerevisiae Mlp1 and Mlp2 proteins are components of the inner nuclear basket of the nuclear pore that mediate interactions with these active genes. To investigate the physical link between the NPC and active loci, we identified proteins that interact with the carboxyl-terminal globular domain of Mlp1 by tandem affinity purification coupled with mass spectrometry. This analysis led to the identification of several components of the Spt-Ada-Gcn5-acetyltransferase (SAGA) histone acetyltransferase complex, Gcn5, Ada2, and Spt7. We utilized co-immunoprecipitation and in vitro binding assays to confirm the interaction between the Mlp proteins and SAGA components. Chromatin immunoprecipitation experiments revealed that Mlp1 and SAGA components associate with the same region of the GAL promoters. Critically, this Mlp-promoter interaction depends on the integrity of the SAGA complex. These results identify a physical association between SAGA and the NPC, and support previous results that relied upon visualization of GAL loci at the nuclear periphery by microscopy (Cabal, G. G. Genovesio, A., Rodriguez-Navarro, S., Zimmer, C., Gadal, O., Lesne, A., Buc, H., Feuerbach-Fournier, F., Olivo-Marin, J.-C., Hurt, E. C., and Nehrbass, U. (2006) Nature 441, 770-773). We propose that a physical interaction between nuclear pore components and the SAGA complex can link the actively transcribed GAL genes to the nuclear pore.

Binding Sites↗
PubMed2001

Nucleocytoplasmic transport enters the atomic age.

Nucleocytoplasmic transport occurs through nuclear pore complexes (NPCs) and is mediated by saturable transport receptors that shuttle between the nucleus and cytoplasm. Our understanding of the molecular interactions underlying this process has improved dramatically as a result of the elucidation of the crystal structures of several nuclear transport factors either alone or in a complex with other components of the nuclear transport machinery. Furthermore, a conserved family of proteins, which is distinct from the well characterized family of importin beta-like nuclear export receptors, is implicated in the export of messenger RNA to the cytoplasm.

Active Transport, Cell Nucleus↗
PubMed2006

Probing nucleocytoplasmic transport by two-photon activation of PA-GFP.

Two-photon activation of photoactivatable green fluorescent protein (PA-GFP) provides a unique tool for probing cellular transport processes, because activation is strictly limited to the sub-femtoliter optical volume of the two-photon spot. We demonstrate two-photon activation of PA-GFP immobilized in a gel and freely diffusing within cells and recover a quadratic power dependence. Illumination at 820 nm allows simultaneous activation and fluorescence monitoring by two-photon excitation. Alternatively, we activate PA-GFP using two-photon excitation and monitor the fluorescence of the photoconverted product with one-photon excitation. We probe nucleocytoplasmic transport through the nuclear pore complex of COS-1 cells, by observing the time-dependent fluorescence at various locations within the cell after two-photon activation of PA-GFP in the nucleus and in the cytoplasm. Two-photon activation of a tandem construct of two PA-GFPs showed a markedly slower rate of crossing through the nuclear pore. Analysis based on a restricted diffusion model yields a nuclear pore radius of 4.5 nm, which is in good agreement with previously reported values. This application demonstrates the attractive features of two-photon photoactivation over traditional techniques, such as photobleaching, for studying transport processes in cells.

Active Transport, Cell Nucleus↗
PubMed2005

The SUMO pathway is essential for nuclear integrity and chromosome segregation in mice.

Covalent modification by SUMO regulates a wide range of cellular processes, including transcription, cell cycle, and chromatin dynamics. To address the biological function of the SUMO pathway in mammals, we generated mice deficient for the SUMO E2-conjugating enzyme Ubc9. Ubc9-deficient embryos die at the early postimplantation stage. In culture, Ubc9 mutant blastocysts are viable, but fail to expand after 2 days and show apoptosis of the inner cell mass. Loss of Ubc9 leads to major chromosome condensation and segregation defects. Ubc9-deficient cells also show severe defects in nuclear organization, including nuclear envelope dysmorphy and disruption of nucleoli and PML nuclear bodies. Moreover, RanGAP1 fails to accumulate at the nuclear pore complex in mutant cells that show a collapse in Ran distribution. Together, these findings reveal a major role for Ubc9, and, by implication, for the SUMO pathway, in nuclear architecture and function, chromosome segregation, and embryonic viability in mammals.

Animals↗
PubMed1985

The organizational fate of intermediate filament networks in two epithelial cell types during mitosis.

Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.

Animals↗
PubMed1987

Epidermal growth factor and insulin stimulate nuclear pore-mediated macromolecular transport in isolated rat liver nuclei.

Fluorescence photobleaching was used to measure the effect of epidermal growth factor (EGF), insulin, and glucagon on the nuclear transport of fluorescent-labeled dextrans across the nuclear pore complex. EGF and insulin were found to stimulate transport approximately 200%, while boiling these polypeptide growth factors greatly diminished this enhancement activity. Glucagon demonstrated no enhancement effect. The nuclear transport enhancement effects were observed at EGF and insulin concentrations that elicit the various physiological responses, e.g., nanomolar range.

Animals↗
PubMed2006

The nuclear GTPase Gsp1p can affect proper telomeric function through the Sir4 protein in Saccharomyces cerevisiae.

The small Ras-like GTPase Ran/Gsp1p is a highly conserved nuclear protein required for the nucleocytoplasmic trafficking of macromolecules. Recent findings suggest that the Ran/Gsp1p pathway may have additional roles in several aspects of nuclear structure and function, including spindle assembly, nuclear envelope formation, nuclear pore complex assembly and RNA processing. Here, we provide evidence that Gsp1p can regulate telomeric function in Saccharomyces cerevisiae. We show that overexpression of PRP20, encoding the Gsp1p GDP/GTP nuclear exchange factor, specifically weakens telomeric silencing without detectably affecting nucleocytoplasmic transport. In addition to this silencing defect, we show that Rap1p and Sir3p delocalize from their normal telomeric foci. Interestingly, Gsp1p was found to interact genetically and physically with the telomeric component Sir4p. Taken together, these results suggest that the GSP1 pathway could regulate proper telomeric function in yeast through Sir4p.

Chromatin Immunoprecipitation↗
PubMed2002

REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export.

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon-exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, we show that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, we conclude that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. Our results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1.

Active Transport, Cell Nucleus↗
PubMed2003

The Chironomus tentans translation initiation factor eIF4H is present in the nucleus but does not bind to mRNA until the mRNA reaches the cytoplasmic perinuclear region.

In the cell nucleus, precursors to mRNA, pre-mRNAs, associate with a large number of proteins and are processed to mRNA-protein complexes, mRNPs. The mRNPs are then exported to the cytoplasm and the mRNAs are translated into proteins. The mRNAs containing in-frame premature stop codons are recognized and degraded in the nonsense-mediated mRNA decay process. This mRNA surveillence may also occur in the nucleus and presumably involves components of the translation machinery. Several translation factors have been detected in the nucleus, but their functional relationship to the dynamic protein composition of pre-mRNPs and mRNPs in the nucleus is still unclear. Here, we have identified and characterized the translation initiation factor eIF4H in the dipteran Chironomus tentans. In the cytoplasm, Ct-eIF4H is associated with poly(A+) RNA in polysomes. We show that a minor fraction of Ct-eIF4H enters the nucleus. This fraction is independent on the level of transcription. CteIF4H could not be detected in gene-specific pre-mRNPs or mRNPs, nor in bulk mRNPs in the nucleus. Our immunoelectron microscopy data suggest that Ct-eIF4H associates with mRNP in the cytoplasmic perinuclear region, immediately as the mRNP exits from the nuclear pore complex.

Amino Acid Sequence↗
PubMed2005

Null mutants of Drosophila B-type lamin Dm(0) show aberrant tissue differentiation rather than obvious nuclear shape distortion or specific defects during cell proliferation.

To elucidate the function of metazoan B-type lamins during development, new null mutations of the Drosophila B-type lamin gene, lamDm(0), were analyzed in parallel with the misg(sz18) mutation, a lamDm(0) allele reported previously. Although in all these mutants, lamin Dm(0) protein was undetectable in neuroblasts and imaginal disc cells from the second instar larval stage onward, cells continued to proliferate. In contrast to the embryonic lethality of another Drosophila lamDm(0) allele, lam(PM15), reported previously, lethality did not occur until late pupal stages. Chromosomal structure and the overall nuclear shape remained normal even at these late pupal stages, although obviously abnormal nuclear pore complex distribution was observed concomitant with the loss of lamin Dm(0) protein. Compensating expression of lamin C was not induced in the absence of lamin Dm(0). Thus, no lamin-containing nuclear structures were found in proliferating larval neuroblasts. We did find that developmental abnormalities appeared in specific organs during the late pupal stage, preceding lethality. Surprisingly, coordinated size increase (hypertrophy) of the ventriculus was observed accompanied by cell division and muscle layer formation. Hypertrophy of the ventriculus correlated with a decrease in ecdysteroid hormone receptor B1 (EcRB1) protein, and furthermore could be suppressed by a heat-inducible EcRB1 transgene. In contrast, both gonadal and CNS tissues exhibited underdevelopment.

Animals↗
PubMed2006

Studying nuclear disassembly in vitro using Xenopus egg extract.

Xenopus egg extract provides an extremely powerful approach in the study of cell cycle regulated aspects of nuclear form and function. Each egg contains enough membrane and protein components to support multiple rounds of cell division. Remarkably, incubation of egg extract with DNA in the presence of an energy regeneration system is sufficient to induce formation of a nuclear envelope around DNA. In addition, these in vitro nuclei contain functional nuclear pore complexes, which form de novo and are capable of supporting nucleocytoplasmic transport. Mitotic entry can be induced by the addition of recombinant cyclin to an interphase extract. This initiates signaling that leads to disassembly of the nuclei. Thus, this cell-free system can be used to decipher events involved in mitotic remodeling of the nuclear envelope such as changes in nuclear pore permeability, dispersal of membrane, and disassembly of the lamina. Both general mechanisms and individual players required for orchestrating these events can be identified via biochemical manipulation of the egg extract. Here, we describe a procedure for the assembly and disassembly of in vitro nuclei, including the production of Xenopus egg extract and sperm chromatin DNA.

Animals↗
PubMed2006

The Nup107-160 nucleoporin complex is required for correct bipolar spindle assembly.

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

Animals↗
PubMed1993

Most nuclear proteins are imported by a single pathway.

Using kinetic competition analysis, we show that the import of most Xenopus oocyte nuclear proteins is directed by a single family of nuclear localization signals (NLS). The transport of microinjected [35S]methionine-labeled nuclear proteins into oocyte nuclei was challenged with saturating concentrations of SV40 large T-antigen NLS peptide-BSA (P(Lys)-BSA) and was assessed by SDS-acrylamide gel electrophoresis and fluorography. P(Lys)-BSA was effective at competing the import of virtually all larger polypeptides but had a less marked effect on the mediated import of many smaller polypeptides. Wheat germ agglutinin, an inhibitor of nuclear pore complex function, enhanced the competitive effects of P(Lys)-BSA on the import of both large and small polypeptides. These data indicate that most 35S-labeled Xenopus oocyte nuclear proteins, including nucleoplasmin, contain NLSs that are functionally related to the T-antigen NLS and probably employ the same cytosolic NLS receptor(s).

Amino Acid Sequence↗
PubMed1998

Characterization of mouse ubiquitin-like SMT3A and SMT3B cDNAs and gene/pseudogenes.

Mouse SMT3A and SMT3B cDNAs encoding ubiquitin-like proteins of 110 and 95 amino acids, respectively, were isolated and sequenced. The sequence of the first 92 amino acids (ending with the conserved Gly-Gly) of mouse SMT3A exhibited two differences at amino acid no. 38 and 76 in comparison with that of human SMT3A. The C-terminal 18 amino acid sequence of mouse SMT3A was completely different from the C-terminal 11 amino acid sequence of human SMT3A. Mouse and human SMT3B were identical for a sequence of 95 amino acids. Mouse SMT3A genomic DNAs were amplified by polymerase-chain-reaction and sequenced. The nucleotide sequence of a PCR-amplified SMT3A genomic DNA fragment was found to be identical to that of SMT3A cDNA, indicating the absence of intron(s) in its protein coding region. Another genomic DNA fragment of 1,531 nucleotides, containing 7% differences from that of cDNA, is unable to encode a functional protein, and thus, it is a SMT3A processed pseudogene. Three mouse SMT3B processed pseudogenes were cloned and sequenced. The genuine mouse SMT3B gene has not yet been isolated. Mouse SMT3A transcript of 1.8 kb was predominantly expressed in most tissues, while SMT3B transcript of 1.0 kb was abundantly present in all tissues analyzed. A family of ubiquitin-like proteins was recently discovered. One distinguishing feature of ubiquitin and ubiquitin-like proteins is the capacity to conjugate with other proteins post-translationally. The ubiquitin-like proteins are cleaved endoproteolytically after a diglycine sequence, corresponding to the C-terminal Gly75-Gly76 of ubiquitin. The cleavage activates the molecule for conjugation. The yeast SMT3 gene was originally identified as a suppressor of mutations in MIF2 gene, which encodes an essential protein binding to the A+T-rich CDEII region of centromere DNA (1). Studies using temperature-sensitive mutants showed that the loss of yeast Mif2 protein function results in chromosome missegregation, mitotic delay, and aberrant microtubule morphologies (2). The yeast Mif2 protein shares at least two regions of similarity with mammalian centromere protein CENP-C, an integral component of active kinetochores (3, 4). Human SMT3A cDNA was identified from the genome sequencing project of chromosome 21 (5). We have cloned human SMT3B (formerly designated as HSMT3) cDNA (6). Human SMT3C protein was independently isolated by several groups and denoted as SUMO-1 (7), GMP1 (8), PICI (9), UBL1 (10), sentrin (11). SUMO-1/GMP1 was found to be covalently linked to the Ran GTPase-activating protein RanGAP1, and attachment of SUMO-1 targets the otherwise cytosolic RanGAP1 to the nuclear pore complex. The modified form of RanGAP1 also appeared to associate with the mitotic spindle apparatus during mitosis (7, 8). PIC1 was shown to interact with the PML component of nuclear multiprotein complex that is disrupted in acute promyelocytic leukemia (9). UBL1 was found to associate with human RAD51/RAD52 proteins involved in DNA recombination and DNA double-strand break repair (10). Sentrin was shown to interact with Fas/APO-1 or the TNF receptor 1 death domain, and the overexpression of sentrin provided protection against both anti-Fas/APO-1 and TNF-induced cell death (11). Here we report the characterization of mouse SMT3A and SMT3B cDNAs, gene/pseudogenes, and mRNA expression.

Amino Acid Sequence↗
PubMed1977

RNA transport in isolated myeloma nuclei. Transport from membrane-denuded nuclei.

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.

Adenosine Triphosphate↗
PubMed2004

Transient permeability leak of nuclear envelope induced by aldosterone.

The mineralocorticoid hormone aldosterone controls fluid and electrolyte transport in target cells of the kidney and the cardiovascular system. Classic genomic aldosterone action involves the activation of cytosolic mineralocorticoid receptors and translocation into the cell nucleus where specific transcription processes are initiated. A key barrier of the intracellular signalling pathway is the nuclear envelope, which physically separates the nucleoplasm from the cytoplasm. It was shown recently that aldosterone changes ion conductivity of the nuclear envelope mediated by nuclear pore complexes. The latter are supramolecular nanomachines responsible for import and export of inorganic ions and macromolecules. The aim of the present study was to test whether aldosterone changes the macromolecule permeability of the nuclear envelope. Aldosterone-responsive Xenopus laevis oocytes were used as a model system. We isolated the cell nuclei at defined times after hormone injection. By means of confocal fluorescence microscopy and fluorescence-labelled dextrans we evaluated passive macromolecule import and export in isolated nuclei. 10 minutes after aldosterone injection nuclear envelope permeability of 10 kD dextran was found sharply increased. At the same time cell nuclei were found swollen by about 28%. Changes in nuclear volume and nuclear envelope permeability lasted 5 to 15 minutes and could be inhibited by the mineralocorticoid receptor blocker spironolactone. We conclude that aldosterone transiently changes the barrier function of the nuclear envelope. This short-lasting permeability change signals the start of a sustained transcription process that follows in response to steroids.

Active Transport, Cell Nucleus↗
PubMed1990

Active transport of proteins into the nucleus.

Nuclear proteins are actively and posttranslationally transported across the nuclear envelope. This transport is a highly selective process that can be divided into two steps, receptor-binding followed by translocation through the nuclear envelope. Receptor-binding is mediated by nuclear localization signals that have been identified in many nuclear proteins. Translocation is energy-dependent and occurs through the nuclear pore complex.

Amino Acid Sequence↗