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PubMed1989

Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos.

Using monoclonal antibodies, we followed the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53,000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gp188, a putative pore complex component, was completely redistributed through the surrounding cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies by other workers, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila. This 'spindle envelope', as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.

Animals↗
PubMed1990

Androgen-induced changes in nuclear pore number and in tight junctions in rat seminal vesicle epithelium.

The numbers of nuclear pore complexes and tight junctions were quantified in the seminal vesicle epithelial cells of castrated and castrated-plus-androgen-treated male rats, which received subcutaneous pellets of testosterone propionate (1 mg/kg body weight) for 1 week. Seminal vesicle weights were 0.284 +/- 0.02 g for castrated, 1.006 +/- 0.006 g for androgen-treated, and 0.918 +/- 0.04 g for untreated groups. Tissue samples were processed for light or electron microscopy and for freeze-fracture techniques. Nuclear areas were measured: controls were 279.34 +/- 8 microns 2; these increased significantly (P less than .001) in castrated-plus-androgen-treated rats (324.66 +/- 11 microns 2) and decreased (P less than .001) in castrated animals (173.14 +/- 6.3 microns 2). Nuclear pore density increased (P less than .001) in castrated-plus-androgen-stimulated rats (5.38 +/- 0.24 pores/microns 2) (control: 4.78 +/- 0.14 pores/microns 2), and decreased (P less than .001) in castrated rats (3.16 +/- 0.14 pores/microns 2). A significant (P less than .001) increase in numbers of tight junction strands that extended in the lateral cell membranes was detected in castrated-plus-androgen-treated rats vs. controls or castrated-only animals.

Androgens↗
PubMed2003

The nucleus of HeLa cells contains tubular structures for Ca2+ signaling with the involvement of mitochondria.

Calcium is an important messenger that controls many nuclear functions such as gene expression in mammalian cells but the regulation of nuclear Ca(2+) remains unclear. It has long been thought that Ca(2+) is translocated from the cytosol by a long distance to the nucleus through the nuclear pore complexes to activate or suppress gene transcription. However, this model is at best an incomplete one. With an aid of confocal and transmission electron microscopy, we demonstrated here that tubules, in a vertical or horizontal orientation, extended deep inside the nucleus of HeLa cells. These nuclear tubules (NTs) are double-membraned invaginations of the nuclear envelope and are usually associated with nucleolus. Also, membrane bound vesicles are found inside and inositol 1,4,5 trisphosphate (IP(3)) receptors are enriched in some but not all of these tubular structures. Interestingly, shuttling of mitochondria was observed in the NT and cytoplasm of the HeLa cells loaded with dihydro-rhod-2/AM. After stimulation with histamine that increases cytosolic [Ca(2+)] through IP(3) production, a slow rise of dihydro-rhod-2 fluorescence for the measurement of intra-mitochondrial Ca(2+) was observed in the area of NT indicating that Ca(2+) was sequestered by mitochondria inside the tubular invagination. Our work therefore suggests that the NTs and mitochondrial activities represent a specialized compartment and dynamic process involved in the regulation of Ca(2+) inside the cell nucleus.

Biological Transport↗
PubMed1986

Chemical factors that influence nucleocytoplasmic transport: a fluorescence photobleaching study.

The technique of fluorescence redistribution after photobleaching was used to measure the translocation rate of fluorescein-labeled dextrans across the nuclear pore complex in isolated rat liver nuclei. A transport assay system was established that could monitor the effect of biologically active molecules, e.g., ATP, GTP, cAMP on the translocation process. The results show that ATP, phosphoinositides, RNA, and insulin can enhance transport rates from 195 to 432%. It was further demonstrated that concanavalin A, but not wheat germ or soybean agglutinin, can block dextran transport completely. The effectors of dextran transport are similar to substances demonstrated to effect the efflux of RNA from isolated nuclei. A model for translocation through the nuclear pore is now presented that incorporates data from protein influx and RNA efflux experiments into a single pathway controlled by ATP.

Adenosine Triphosphate↗
PubMed1998

NTF2 mediates nuclear import of Ran.

Importin beta family transport receptors shuttle between the nucleus and the cytoplasm and mediate transport of macromolecules through nuclear pore complexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus complexed with RanGTP, and so should deplete RanGTP continuously from the nucleus. We describe here the development of an in vitro system to study how nuclear Ran is replenished. Nuclear import of Ran does not rely on simple diffusion as Ran's small size would permit, but instead is stimulated by soluble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocation of the NTF2-RanGDP complex to the nuclear side of the NPCs. A direct NTF2-RanGDP interaction is crucial for this process, since point mutations that disturb the RanGDP-NTF2 interaction also interfere with Ran import. The subsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrolysis. The release of Ran from NTF2 into the nucleus, and thus the directionality of Ran import, probably involves nucleotide exchange to generate RanGTP, for which NTF2 has no detectable affinity, followed by binding of the RanGTP to an importin beta family transport receptor.

Biological Transport↗
PubMedDate not supplied

TPEN, a Zn2+/Fe2+ chelator with low affinity for Ca2+, inhibits lamin assembly, destabilizes nuclear architecture and may independently protect nuclei from apoptosis in vitro.

We used Xenopus egg extracts to examine the effects of TPEN, a chelator with strong affinities for Zn2+, Fe2+, and Mn2+, on nuclear assembly in vitro. At concentrations above 1 mM, TPEN blocked the assembly of the nuclear lamina and produced nuclei that were profoundly sensitive to stress-induced balloon-like 'shedding' of nuclear membranes away from chromatin-associated membranes. TPEN-arrested nuclei were also defective for DNA replication, which could be explained as secondary to the lack of a lamina. Imaging of TPEN-arrested nuclei by field emission in-lens scanning electron microscopy (FEISEM) revealed clustered, structurally-perturbed nuclear pore complexes. TPEN-arrested nuclei were defective in the accumulation of fluorescent karyophilic proteins. All detectable effects caused by TPEN were downstream of the effects of BAPTA, a Ca2+/Zn2+ chelator that blocks pore complex assembly at two distinct early stages. Surprisingly, TPEN-arrested nuclei, but not control nuclei, remained active for replication in apoptotic extracts, as assayed by [32P]-dCTP incorporation into high molecular weight DNA, suggesting that TPEN blocks a metal-binding protein(s) required for nuclear destruction during programmed cell death.

Animals↗
PubMed2005

Pushing the envelope: microinjection of Minute virus of mice into Xenopus oocytes causes damage to the nuclear envelope.

Parvoviruses are small DNA viruses that replicate in the nucleus of their host cells. It has been largely assumed that parvoviruses enter the nucleus through the nuclear pore complex (NPC). However, the details of this mechanism remain undefined. To study this problem, the parvovirus Minute virus of mice (MVM) was microinjected into the cytoplasm of Xenopus oocytes and a transmission electron microscope was used to visualize the effect of the virus on the host cell. It was found that MVM caused damage to the nuclear envelope (NE) in a time- and concentration-dependent manner. Damage was predominantly to the outer nuclear membrane and was often near the NPCs. However, microinjection experiments in which the NPCs were blocked showed that NE damage induced by MVM was independent of the NPC. To address the question of whether this effect of MVM is specific to the NE, purified organelles were incubated with MVM. Visualization by electron microscopy revealed that MVM did not affect all intracellular membranes. These data represent a novel form of virus-induced damage to host cell nuclear structure and suggest that MVM is imported into the nucleus using a unique mechanism that is independent of the NPC, and involves disruption of the NE and import through the resulting breaks.

Animals↗
PubMed1998

Modification of Ran GTPase-activating protein by the small ubiquitin-related modifier SUMO-1 requires Ubc9, an E2-type ubiquitin-conjugating enzyme homologue.

Covalent modification of the Ran GTPase-activating protein RanGAP1 with the ubiquitin-related protein SUMO-1 promotes its association with Nup358, a component of the cytoplasmic fibrils emanating from the nuclear pore complex (1,2). In Xenopus egg extracts, Nup358 can be found in a complex with Ubc9 (3), a structural homologue of the E2-type ubiquitin-conjugating enzymes (UBCs). Here we show that a subset of the human homologue of Ubc9 (HsUbc9) colocalizes with RanGAP1 at the nuclear envelope. HsUbc9 forms thiolester conjugates with recombinant SUMO-1, but not with recombinant ubiquitin, indicating that it is functionally distinct from E2-type UBCs. Finally, HsUbc9 is required for the modification of RanGAP1 by SUMO-1. These results suggest that HsUbc9 is a component of a novel enzymatic cascade that modifies RanGAP1, and possibly other substrates, with SUMO-1.

Adenosine Triphosphate↗
PubMed2000

Phosphorylation of the nuclear transport machinery down-regulates nuclear protein import in vitro.

We have examined whether signal-mediated nucleocytoplasmic transport can be regulated by phosphorylation of the nuclear transport machinery. Using digitonin-permeabilized cell assays to measure nuclear import and export, we found that the phosphatase inhibitors okadaic acid and microcystin inhibit transport mediated by the import receptors importin beta and transportin, but not by the export receptor CRM1. Several lines of evidence, including the finding that transport inhibition is partially reversed by the broad specificity protein kinase inhibitor staurosporine, indicate that transport inhibition is due to elevated phosphorylation of a component of the nuclear transport machinery. The kinases and phosphatases involved in this regulation are present in the permeabilized cells. A phosphorylation-sensitive component of the nuclear transport machinery also is present in permeabilized cells and is most likely a component of the nuclear pore complex. Substrate binding by the importin alpha.beta complex and the association of the complex with the nucleoporins Nup358/RanBP2 and Nup153 are not affected by phosphatase inhibitors, suggesting that transport inhibition by protein phosphorylation does not involve these steps. These results suggest that cells have mechanisms to negatively regulate entire nuclear transport pathways, thus providing a means to globally control cellular activity through effects on nucleocytoplasmic trafficking.

Adenosine Triphosphate↗
PubMed1997

The activation domain of a hormone inducible HTLV-1 Rex protein determines colocalization with the nuclear pore.

Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Rex is an essential regulatory protein that acts at the posttranscriptional level to promote expression of unspliced and singly spliced genes of the virus. Rex functions have been attributed to at least three separate domains of the protein determining nuclear/nucleolar accumulation and RNA binding (overlapping), multimerization, and nuclear export of Rex-responsive RNA. The steady-state intracellular localization of functional Rex molecules is mainly nucleolar. Fusions of wild-type Rex and the ligand binding domain of human estrogen receptor (ER) produced conditional molecules (ERRex and ERalaRex), which remained cytoplasmic in the absence of hormone and in response to hormone colocalized with the nuclear pore complex (NPC). These molecules induced in a hormone-dependent manner the expression of a Rex reporter plasmid and of the HTLV-1 Env protein and fusion of Env expressing cells. In contrast, activation domain mutants (ERRex delta and ERRexGly) translocated from the cytoplasm and acquired a diffuse nuclear localization. These mutants did not associate with the NPC and failed to show any of the expected Rex functions. Rex functions were perturbed by inactivating the RNA binding domain (mutant ERM2) or the oligomerization domain (mutant ERM7). However, these two mutant fusion proteins exhibited a hormone-dependent NPC colocalization. These observations provide in vivo evidence that intranuclear translocation of intact Rex to the NPC is dependent exclusively on a functional activation domain and is not influenced by binding to the target RNA.

Binding Sites↗
PubMed1996

Protein import into the nucleus.

The transport of proteins from the cytoplasm into the nucleus is a multistep process. The nuclear localization sequence (NLS) of a transport substrate associates with the heterodimeric NLS-receptor which binds to a subset of proteins of the nuclear pore complex (NPC). Translocation through the NPC is energy-dependent and requires the small GTPase Ran. Proteins that interact with Ran in either the GDP-bound or the GTP-bound state coordinate transfer through the NPC. Lastly, the NLS-receptor/ substrate complex and Ran reach the nuclear side of the NPC where the complex disassembles.

Amino Acid Sequence↗
PubMed1995

Three classes of nuclear import signals bind to plant nuclei.

Three nuclear localization signals (NLS), including an unusual Mat alpha 2-like NLS from maize (Zea mays) R, were found to compete for binding to plant nuclei. In addition, the authentic yeast Mat alpha 2 NLS, which does not function in mammals, was shown to function in plants in vivo. Our results indicate that plants possess a site at the nuclear pore complex that recognizes the three known classes of NLSs.

Amino Acid Sequence↗
PubMed2004

Structure and function of HIV-1 auxiliary regulatory protein Vpr: novel clues to drug design.

Vpr is a 96-amino acid auxiliary regulatory protein that is packaged in the HIV-1 virion. It enhances the nuclear transport of the pre-integration complex, and regulates cell cycle, transcription and apoptosis. These biological activities suggest strongly that Vpr interacts with cellular biochemical pathways to regulate HIV-1 replication and pathogenesis. The karyophilic property of Vpr appears to be due to direct interaction of Vpr with nuclear transport factors and residents of the nuclear pore complex, whereas transcriptional effects of Vpr may be exerted through direct and indirect mechanisms. Cell cycle arrest at the G2/M checkpoint by Vpr is correlated with the hyperphosphorylation of Cdc2. The pro-apoptotic activity of Vpr is dependent on the subtype of the HIV-1 isolate, and may be dramatically enhanced by a single L64P mutation. Mitochondria- and caspase-dependent mechanisms appear to mediate Vpr-induced apoptosis. Recent evidence suggests that Vpr interacts with a cellular ubiquitination machinery and promotes degradation of Vpr mutants carrying the L64P mutation. Vpr interaction with the ubiquitination machinery may contribute to the regulation of the HIV-1 life cycle at various stages. NMR studies of Vpr have shown a Vpr monomer with three helical domains arranged in a twisted-U shape. However, Vpr most likely exists as a trimer in vivo. Structural/functional domains have been tentatively mapped for Vpr induction of apoptosis and for Vpr interaction with the ubiquitination machinery. Structural refinement of Vpr, specially by crystallography of the potential Vpr trimer, should help design therapeutic approaches to specifically target Vpr.

Amino Acid Sequence↗
PubMed2000

Nucleotides of the tRNA D-stem that play an important role in nuclear-tRNA export in Saccharomyces cerevisiae.

Nuclear export of tRNA in Saccharomyces cerevisiae involves Los1p and Arc1p. Los1p facilitates tRNA translocation across the nuclear pore complex whereas Arc1p plays a role in delivering some species of tRNA exiting the nucleus to their cognate aminoacyl-tRNA synthetases. Here, we show that mutations of C11 and G24 of the D-stem of the yeast tyrosine amber-suppressor tRNA have different effects on nuclear export of the tRNA. Changing G24 had no effect on export of the tRNA to the cytoplasm. In contrast, mutating C11 resulted in nuclear retention of the tRNA. Nuclear retention of the tRNA mutants was not due to lack of processing, since only the mature forms of the tRNA mutants were found. The fact that mutations of G24 did not affect export of the tRNA also indicates that the effect of mutating C11 is not due to gross alteration of the tertiary structure resulting from disruption of the C11/G24 base pair. Expression of Los1p and the mammalian tRNA export receptor exportin-t rescued nuclear export of the tRNA with changes at position 11. The export-defective mutations of the tRNA mutants were suppressed by introducing the complementary nucleotides at position 24. Taken together, these findings suggest that C11 is important for binding of the tRNA to the export receptor, and that this binding is influenced by the conformation of the base. Finally, the export-defective tRNA mutants described can be used as reporters to identify eukaryotic proteins involved in the nuclear-tRNA export process, and characterize the molecular interactions between known receptors and the tRNA substrate.

Base Sequence↗
PubMed2003

Importin-alpha mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain.

The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-alpha nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-alpha with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxy-terminal fragment of importin-alpha, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-alpha to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131-141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-alpha and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-alpha as well as the PI3-kinase-dependent phosphorylation of Sgk. Our results define a new role importin-alpha in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.

Active Transport, Cell Nucleus↗
PubMed1996

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.

Binding Sites↗
PubMed2004

Intracellular localization of histidyl-tRNA synthetase/Jo-1 antigen in T24 cells and some other cells.

Anti-Jo-1 antibody is characteristic of patients suffering from autoimmune-disease myositis. Since the antigen has been identified to be a histidyl-tRNA synthetase (HisRS), it is reasonable to suppose that it localizes mainly in the cytoplasm. However, contradictory results (localization in the nucleus, cytoplasm, or both) have been reported on this point. This study examined whether or not HisRS tagged with a green fluorescent protein (GFP) localizes, even if partially, in particular regions of the nucleus. The cDNA of human HisRS was ligated into either pEGFP-N1 or pEGFP-C1, and transfected into T24 cells. Transfectants expressed either HisRS-GFP or GFP-HisRS, both with the expected LDS-PAGE mobility. Observations with a confocal fluorescence microscope revealed that, in most cells, both GFP-tagged HisRSs were present solely in the cytoplasm. Occasional fluorescent spots seen in the nuclear region coincided with the immunofluorescent stain of the nuclear pore complex, indicating that they represent GFP-tagged HisRS in the cytoplasm that had invaginated deeply into the nucleus. Transient transfection into HeLa and L6 cells also resulted in the cytoplasmic localization of GFP-tagged HisRSs. These results indicate that HisRS would localize predominantly in the cytoplasm. The possible nuclear antigens other than HisRS that might be detected by anti-Jo-1 antisera are discussed.

Green Fluorescent Proteins↗
PubMed2000

Selective disruption of nuclear import by a functional mutant nuclear transport carrier.

p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-beta1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-beta1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.

Active Transport, Cell Nucleus↗