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PubMed1997

C-terminal truncations of the yeast nucleoporin Nup145p produce a rapid temperature-conditional mRNA export defect and alterations to nuclear structure.

A screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective in nucleocytoplasmic trafficking of poly(A)+ RNA has identified an allele of the NUP145 gene, which encodes an essential nucleoporin. NUP145 was previously identified by using a genetic synthetic lethal screen (E. Fabre, W. C. Boelens, C. Wimmer, I. W. Mattaj, and E. C. Hurt, Cell 78:275-289, 1994) and by using a monoclonal antibody which recognizes the GLFG family of vertebrate and yeast nucleoporins (S. R. Wente and G. Blobel, J. Cell Biol. 125:955-969, 1994). Cells carrying the new allele, nup145-10, grew at 23 and 30 degrees C but were unable to grow at 37 degrees C. Many cells displayed a modest accumulation of poly(A)+ RNA under permissive growth conditions, and all cells showed dramatic and rapid nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. The mutant allele contains a nonsense codon which truncates the 1,317-amino-acid protein to 698 amino acids. This prompted us to examine the role of the carboxyl half of Nup145p. Several additional alleles that encode C-terminally truncated proteins or proteins containing internal deletions of portions of the carboxyl half of Nup145p were constructed. Analysis of these mutants indicates that some sequences between amino acids 698 and 1095 are essential for RNA export and for growth at 37 degrees C. In these strains, nuclear accumulation of poly(A)+ RNA and fragmentation of the nucleolus occurred rapidly following a shift to 37 degrees C. Constitutive defects in nuclear pore complex distribution and nuclear structure were also seen in these strains. Although cells lacking Nup145p grew extremely slowly at 23 degrees C and did not grow at 30 degrees C, efficient growth at 23 or 30 degrees C occurred as long as cells produced either the amino 58% or the carboxyl 53% of Nup145p. Strains carrying alleles of NUP145 lacking up to 200 amino acids from the carboxy terminus were viable at 37 degrees C but displayed nucleolar fragmentation and some nuclear accumulation of poly(A)+ RNA following a shift to 37 degrees C. Surprisingly, these strains grew efficiently at 37 degrees C in spite of a reduction in the level of synthesis of rRNAs to approximately 25% of the wild-type level.

Biological Transport↗
PubMed2024

MX2 forms nucleoporin-comprising cytoplasmic biomolecular condensates that lure viral capsids.

Human myxovirus resistance 2 (MX2) can restrict HIV-1 and herpesviruses at a post-entry step through a process requiring an interaction between MX2 and the viral capsids. The involvement of other host cell factors, however, remains poorly understood. Here, we mapped the proximity interactome of MX2, revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and anti-herpes simplex virus 1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region and MX2 dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates, preventing nuclear entry of their viral genomes. Thus, MX2 forms cytoplasmic condensates that likely act as nuclear pore decoys, trapping capsids and inducing premature viral genome release to interfere with nuclear targeting of HIV-1 and HSV-1.

Humans↗
PubMed1998

Karyopherins and kissing cousins.

In eukaryotic cells, a regulated flux of molecules between the cytoplasm and the nucleus maintains two very different environments while allowing the controlled exchange of macromolecules necessary for their individual functions. Molecules entering or leaving the nucleus use nuclear localization signals or nuclear export signals to pass through selective channels in the nuclear envelope formed by nuclear pore complexes. The recognition of signal-bearing cargo, its interaction with the nuclear pore complex and its translocation through the pore complex are mediated by soluble transport factors. Recently, the list of potential transport factors has grown rapidly, suggesting a previously unanticipated level of complexity for nuclear transport.

Animals↗
PubMed2006

Karyopherin-mediated import of integral inner nuclear membrane proteins.

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) remains a notable exception. Here we show that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-alpha and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-alpha, karyopherin-beta1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. We also provide evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.

Amino Acid Motifs↗
PubMed1997

A role for the divergent actin gene, ACT2, in nuclear pore structure and function.

We have identified a temperature-sensitive allele of the yeast divergent actin gene ACT2, act2-1, which displays defects in nuclear pore complex (NPC) structure and nuclear import at the restrictive temperature. Although defective in nuclear import, act2-1 cells still selectively retain reporter proteins in the nucleus, and by indirect immunofluorescence the actin cytoskeleton appears normal. Previous studies in Acanthamoeba and Saccharomyces cerevisiae reported that the cellular location of Act2p partially overlaps that of conventional actin, indicating that it has a cytoskeletal function. In this study, both immunofluorescence localization and cellular fractionation of different epitope-tagged versions of Act2p also reveal an association with the nucleus, suggesting an independent nuclear function for Act2p. Analysis of act2-1 by electron microscopy, 30 min after a shift to the restrictive temperature (37 degrees C), reveals a striking aberration in NPC morphology; NPCs appear as abnormal densities on either side of, rather than spanning, the nuclear envelope. Immunoelectron microscopy confirms that these densities contain XFXFG nucleoporins. act2-1 is synthetically lethal in combination with a deletion in the XFXFG nucleoporin gene, NUP1, or a mutation in the nuclear localization sequence receptor gene, SRP1. Act2p and Srp1p co-immunoprecipitate, suggesting that the proteins exist in a complex. Together our data argue that Act2p plays an important role in NPC structure and function.

Actins↗
PubMed1999

Identification and characterization of nuclear pore subcomplexes in mitotic extract of human somatic cells.

In higher eukaryotic cells, it is generally thought that nuclear pore complex disassembles at the beginning of mitosis and reassembles at the end. Using a monoclonal antibody that recognizes nuclear pore antigens, we found that, at mitosis of mammalian somatic Hela cells, the nuclear pore complex disassembles into at least three subcomplexes, termed subcomplexes A, B and C (molecular mass; 2 MDa<, approximately 660 kDa, and approximately 440 kDa, respectively). The direct partial amino acid sequencing of the components of these subcomplexes indicates that the A subcomplex contains CAN/Nup214/p250 and p62 and the B subcomplex also contains p62, indicating that p62 is contained in two different subcomplexes. Subcomplex C was shown to consist of Nup98 and human RAE1, a human homolog of yeast Rae1p/Gle2p. Since Nup98 and Rae1p/Gle2p have been reported to be involved in mRNA export, this suggests that some of the mitotic subcomplex may be formed by functionally related proteins.

Amino Acid Sequence↗
PubMed2000

Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors.

NTF2 and importin-beta are transport factors that mediate nuclear protein import and which interact with nuclear pore proteins (nucleoporins) during translocation from the cytoplasm to the nucleus through nuclear pore complexes. We employed a native gel electrophoresis method to assess the interaction of nucleoporin constructs that contain FxFG sequence repeats with NTF2 and truncation mutants of importin-beta to determine suitable fragments for crystallization. Based on these data, we obtained crystals of complexes between yeast NTF2 and a construct containing five FxFG nucleoporin repeats from the yeast nucleoporin Nsp1p and between a construct containing residues 1-442 of human importin-beta and the same nucleoporin construct. The yeast NTF2-nucleoporin crystals have trigonal symmetry and diffract past 2.8 A resolution using synchrotron radiation, whereas the importin-beta-nucleoporin complex crystals have P2(1)2(1)2 orthorhombic symmetry and diffract past 3.2 A resolution.

Amino Acid Sequence↗
PubMed2002

Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning.

A targeted silencing screen was performed to identify yeast proteins that, when tethered to a telomere, suppress a telomeric silencing defect caused by truncation of Rap1. A previously uncharacterized protein, Esc1 (establishes silent chromatin), was recovered, in addition to well-characterized proteins Rap1, Sir1, and Rad7. Telomeric silencing was slightly decreased in Deltaesc1 mutants, but silencing of the HM loci was unaffected. On the other hand, targeted silencing by various tethered proteins was greatly weakened in Deltaesc1 mutants. Two-hybrid analysis revealed that Esc1 and Sir4 interact via a 34-amino-acid portion of Esc1 (residues 1440 to 1473) and a carboxyl-terminal domain of Sir4 known as PAD4 (residues 950 to 1262). When tethered to DNA, this Sir4 domain confers efficient partitioning to otherwise unstable plasmids and blocks the ability of bound DNA segments to rotate freely in vivo. Here, both phenomena were shown to require ESC1. Sir protein-mediated partitioning of a telomere-based plasmid also required ESC1. Fluorescence microscopy of cells expressing green fluorescent protein (GFP)-Esc1 showed that the protein localized to the nuclear periphery, a region of the nucleus known to be functionally important for silencing. GFP-Esc1 localization, however, was not entirely coincident with telomeres, the nucleolus, or nuclear pore complexes. Our data suggest that Esc1 is a component of a redundant pathway that functions to localize silencing complexes to the nuclear periphery.

Basic Helix-Loop-Helix Proteins↗
PubMed1997

A nuclear export signal in Kap95p is required for both recycling the import factor and interaction with the nucleoporin GLFG repeat regions of Nup116p and Nup100p.

During nuclear import, cytosolic transport factors move through the nuclear pore complex (NPC) to the nuclear compartment. Kap95p is required during import for docking the nuclear localization signal-receptor and ligand to the NPC. Recycling of this factor back to the cytoplasm is necessary for continued rounds of import; however, the mechanism for Kap95p recycling is unknown. We have determined that recycling of Kap95p requires a nuclear export signal (NES). A region containing the NES in Kap95p was sufficient to mediate active nuclear export in a microinjection assay. Moreover, the NES was necessary for function. Mutation of the NES in Kap95p resulted in a temperaturesensitive import mutant, and immunofluorescence microscopy experiments showed that the mutated Kap95p was not recycled but instead localized in the nucleus and at the nuclear envelope. Srp1p, the yeast nuclear localization signal-receptor, also accumulated in the nuclei of the arrested kap95 mutant cells. Wild-type and NES-mutated Kap95p both bound Gsp1p (the yeast Ran/TC4 homologue), Srp1p, and the FXFG repeat region of the nucleoporin Nup1p. In contrast, the NES mutation abolished Kap95p interaction with the GLFG repeat regions from the nucleoporins Nup116p and Nup100p. In vivo interaction was demonstrated by isolation of Kap95p from yeast nuclear lysates in either protein A-tagged Nup116p or protein A-tagged Nup100p complexes. The protein A-tagged Nup116p complex also specifically contained Gle2p. These results support a model in which a step in the recycling of Kap95p is mediated by interaction of an NES with GLFG regions. Analysis of genetic interactions suggests Nup116p has a primary role in Kap95p recycling, with Nup100p compensating in the absence of Nup116p. This finding highlights an important role for a subfamily of GLFG nucleoporins in nuclear export processes.

Amino Acid Sequence↗
PubMed2000

Live fluorescence imaging reveals early recruitment of emerin, LBR, RanBP2, and Nup153 to reforming functional nuclear envelopes.

We determined the times when the nuclear membrane, nuclear pore complex (NPC) components, and nuclear import function were recovered during telophase in living HeLa cells. Simultaneous observation of fluorescently-labeled NLS-bearing proteins, lamin B receptor (LBR)-GFP, and Hoechst33342-stained chromosomes revealed that nuclear membranes reassembled around chromosomes by 5 minutes after the onset of anaphase (early telophase) whereas nuclear import function was recovered later, at 8 minutes. GFP-tagged emerin also accumulated on chromosomes 5 minutes after the onset of anaphase. Interestingly, emerin and LBR initially accumulated at distinct, separate locations, but then became uniform 8 minutes after the onset of anaphase, concurrent with the recovery of nuclear import function. We further determined the timing of NPC assembly by immunofluorescence staining of cells fixed at precise times after the onset of anaphase. Taken together, these results showed that emerin, LBR, and several NPC components (RanBP2, Nup153, p62), but not Tpr, reconstitute around chromosomes very early in telophase prior to the recovery of nuclear import activity.

Biological Transport↗
PubMed1999

Autoimmune cholangitis and primary biliary cirrhosis--an autoimmune enigma.

AIMS/BACKGROUND: Autoimmune cholangitis/cholangiopathy (AIC) is an enigmatic disease marked by chronic cholangitis, antinuclear antibodies (ANA) and sero-negativity for conventionally detected antimitochondrial antibodies (AMA). We examined whether AIC is a distinct entity, an AMA-negative variant of primary biliary cirrhosis (PBC), or a cholangiopathic variant of autoimmune hepatitis (AIH) by comparing the clinical, laboratory and autoantibody profiles of 21 cases of AIC, 37 cases PBC and 16 cases of AIH from selected Japanese patients. METHODS: The specificities of AMA and ANA were determined by immunofluorescence, immunoblotting and enzyme inhibition assays using various mitochondrial and nuclear autoantigens, and the frequencies for these groups were compared. RESULTS: By clinical, biochemical and histological data, AIC and PBC were similar and both were clearly distinct from AIH. Serologically, by immunofluorescence of AMA and ANA, there was polarisation. By immunoblotting, and notwithstanding the negative test for AMA, a proportion of the AIC sera reacted with the E2 subunits of the 2-oxo-acid dehydrogenase enzyme complexes, but more particularly with the lower molecular weight E2 subunits. The antinuclear reactivity in AIC was with centromere, Sp100 and nuclear pore complex proteins as in PBC, but preferentially with the nuclear pore complex. CONCLUSION: Our results demonstrate that AIC and PBC are similar diseases. However this duo is of interest because, usually, among sets of autoimmune syndromes, differences in serological targetting are matched by differences in clinical presentation: AIC and PBC are an exception to this.

Adult↗
PubMed1994

A novel nuclear pore protein Nup133p with distinct roles in poly(A)+ RNA transport and nuclear pore distribution.

Temperature-sensitive nucleoporin nup49-316 mutant cells accumulate poly(A)+ RNA inside the nucleus when shifted to restrictive temperature. We performed a synthetic lethal screen with this mutant allele to identify further components of the mRNA export machinery. A synthetic lethal mutant slv21 was isolated, which exhibited a ts phenotype and showed nuclear accumulation of poly(A)+ RNA at 37 degrees C. The wild-type gene complementing slv21 was cloned and sequenced. It encodes a novel protein Nup133p which is located at the nuclear pore complex. NUP133 is not an essential gene, but cells in which NUP133 is disrupted grow slowly at permissive temperatures and stop growing at 37 degrees C. Concomitant with the growth inhibition, nup133- cells accumulate poly(A)+ RNA inside the nucleus whereas nuclear import of a karyophilic reporter protein is not altered. Strikingly, nup133- cells display extensive clustering of nuclear pore complexes at a few sites on the nuclear envelope. However, the nuclear pore clustering phenotype and intranuclear accumulation of poly(A)+ RNA are not obligatorily linked, since an amino-terminally truncated Nup133p allows normal poly(A)+ RNA export, but does not complement the clustering phenotype of nup133- cells.

Amino Acid Sequence↗
PubMed1999

Three distinct classes of the alpha-subunit of the nuclear pore-targeting complex (importin-alpha) are differentially expressed in adult mouse tissues.

The process of active nuclear protein transport is mediated by the nuclear localization signal (NLS). An NLS-containing karyophile forms a stable complex, termed the nuclear pore-targeting complex, to target nuclear pores. The alpha-subunit of the complex (importin-alpha) binds to the NLS and the beta-subunit (importin-beta) carries the alpha-subunit, bound to the NLS substrate, into the nucleus. To date, five mouse alpha-subunits have been identified and classified into three subfamilies (alpha-P, alpha-Q, and alpha-S). The expression of these alpha-subunits and the beta-subunit in various adult mouse tissues was examined by immunoblotting and immunohistochemistry using antibodies specific for each subfamily of the alpha-subunit or the beta-subunit. The beta-subunit was found to be ubiquitously expressed, whereas each subfamily of the alpha-subunit showed a unique expression pattern in various tissues, especially in brain and testis. In brain, the expression of alpha-P was not observed, whereas alpha-S was significantly expressed in Purkinje cells, and pyramidal cells of the hippocampus and cerebral cortex. In testis, alpha-P was expressed predominantly in primary spermatocytes, whereas alpha-Q was found mainly in Leydig cells. Expression of alpha-S was detected in almost all cells in convoluted seminiferous tubules and Leydig cells to a similar extent. These results suggest that nuclear protein import may be controlled in a tissue-specific manner by alpha-subunit family proteins.

Animals↗
PubMed2003

Intracellular calcium: a prerequisite for aldosterone action.

Transport of salt and water in various tissues is under control of the mineralocorticoid hormone aldosterone. As a liphophilic hormone, aldosterone diffuses through the plasma membrane and, then, binds to cytosolic mineralocorticoid receptors in the target cells. After binding to nuclear pore complexes, the activated receptor is translocated to the nucleus where transcription processes are initiated. After a lag period of about 20 minutes hormone-specific early mRNA transcripts leave the nucleus through nuclear pores. Some of the steps in this cascade can be followed by electrophysiology in Xenopus laevis oocyte nuclei. In addition to the genomic pathway, aldosterone exerts a rapid pre-genomic response that involves an increase in intracellular calcium. In this study, we tested for the potential role of Ca(2+) in the genomic response of the hormone. We measured the electrical resistance across the nuclear envelope in response to aldosterone, in presence and absence of intracellular Ca(2+). Nuclear envelope electrical resistance reflects receptor binding to the nuclear pore complexes ("early" resistance peak, 2 minutes after aldosterone), ongoing transcription ("transient" resistance drop, 5-15 minutes after aldosterone) and mRNA export ("late" resistance peak, 20 minutes after aldosterone). Pre-injection of the Ca(2+) chelator EGTA eliminated all electrical responses evoked by aldosterone. The transient resistance drop and the late resistance peak, induced by the hormone, were prevented by the transcription inhibitor actinomycin D, coinjected with aldosterone, while the early resistance peak remained unaffected. We conclude that (i). the presence of intracellular Ca(2+) is a prerequisite for the genomic action of aldosterone. (ii). Intracellular calcium plays a role early in the signaling cascade, either in agonist-receptor interaction, or receptor transport/docking to the nuclear pore complexes.

Aldosterone↗
PubMed1983

Nuclear ribonucleoprotein release and nucleoside triphosphatase activity are inhibited by antibodies directed against one nuclear matrix glycoprotein.

Circumstantial evidence suggests that nucleocytoplasmic exchange or transport is an active process involving the nuclear pore complex of the nuclear envelope. To test this hypothesis, antibodies were generated against nuclear envelope components from a highly enriched pore complex fraction from Spisula solidissima oocytes. Some of these antibodies inhibited ATP-dependent ribonucleoprotein release from prelabeled, isolated rat nuclei and inhibited nucleoside triphosphatase activity essential in nucleocytoplasmic transport. Inhibition of both functions by lectins indicated that the antigen was a glycoprotein. It was identified as lamin B, a major component of the nuclear envelope and nuclear matrix. This glycoprotein may not only be a structural nuclear protein but also may have nucleoside triphosphatase activity. We speculate that lamin B represents the solid support for ribonucleoprotein transport. This protein is expected to be highly conserved if active transport in and out of the nucleus is essential in the eukaryotic system.

Adenosine Triphosphate↗
PubMed2003

Intracellular transport of human immunodeficiency virus type 1 integrase.

The human immunodeficiency virus type 1 integrase protein has karyophilic properties; that is, it localizes to the cell nucleus according to a range of assays. As an essential component of the preintegration complex, it has been suggested that the karyophilic properties of integrase might facilitate transport of the preintegration complex through the nuclear pore complexes of nondividing cells. However, no experiments have satisfactorily identified a nuclear localization signal within integrase. In this work, we investigated the karyophilic properties of integrase in intact cells with hopes of identifying a genuine transferable nuclear localization signal. Our results confirm that integrase tightly binds chromosomal DNA in vivo. However, our analysis determined that large integrase fusion proteins are unable to access the nucleus, indicating that integrase might lack a transferable nuclear localization signal. In addition, we present several lines of evidence to indicate that DNA binding might facilitate integrase nuclear accumulation. Furthermore, our data indicate integrase is degraded in the cytoplasm by a proteasome-dependent process, an event that probably contributes to the apparent nuclear accumulation of integrase. These results provide new insight into human immunodeficiency virus type 1 integrase intracellular dynamics.

Cell Nucleus↗
PubMed1988

Preparation of shadowed nuclear envelopes from Xenopus oocyte germinal vesicles for electron microscopy.

Methods for examining the structure of the nuclear envelope of oocytes of Xenopus laevis by electron microscopy using metal shadowing have been developed and evaluated. Minor modifications were made to existing methods for preparing specimens by freeze drying, mainly to eliminate unnecessary steps and a rapid method for examining the structure and arrangement of nuclear envelope components, based on dehydration in an ethanol series followed by amyl acetate and then air drying, was also developed. The preservation of the lamina and connections between the nuclear pore complexes using the rapid air drying method was satisfactory for observing the fibrous components of the envelope and their attachment to the pores. Furthermore, air drying required only simple laboratory apparatus and, moreover, offered several advantages compared to freeze drying when assessing the effect of various disruptive treatments on the nuclear envelope or examining the connections between its components. In specimens prepared by either the more rapid air drying method or by freeze drying, the lamina meshwork beneath the nuclear face of the envelope was clear, but the fine structure of the nuclear pore complexes was superior in freeze dried preparations. In views of the nucleoplasmic face of the envelope, the lamina meshwork was suspended above the support film in freeze dried preparations, but collapsed in most air dried specimens. This collapse was not without its advantages, however, as it facilitated observation of the connections between nuclear pore complexes and lamina fibres, which were often masked in freeze dried preparations.

Animals↗
PubMed2002

Docking of HIV-1 Vpr to the nuclear envelope is mediated by the interaction with the nucleoporin hCG1.

The HIV-1 genome contains several genes coding for auxiliary proteins, including the small Vpr protein. Vpr affects the integrity of the nuclear envelope and participates in the nuclear translocation of the preintegration complex containing the viral DNA. Here, we show by photobleaching experiments performed on living cells expressing a Vpr-green fluorescent protein fusion that the protein shuttles between the nucleus and the cytoplasm, but a significant fraction is concentrated at the nuclear envelope, supporting the hypothesis that Vpr interacts with components of the nuclear pore complex. An interaction between HIV-1 Vpr and the human nucleoporin CG1 (hCG1) was revealed in the yeast two-hybrid system, and then confirmed both in vitro and in transfected cells. This interaction does not involve the FG repeat domain of hCG1 but rather the N-terminal region of the protein. Using a nuclear import assay based on digitonin-permeabilized cells, we demonstrate that hCG1 participates in the docking of Vpr at the nuclear envelope. This association of Vpr with a component of the nuclear pore complex may contribute to the disruption of the nuclear envelope and to the nuclear import of the viral DNA.

Active Transport, Cell Nucleus↗