scholarly journals Facilitated Nucleocytoplasmic Shuttling of the Ran Binding Protein RanBP1

2000 ◽  
Vol 20 (10) ◽  
pp. 3510-3521 ◽  
Author(s):  
Kendra Plafker ◽  
Ian G. Macara
Keyword(s):  
Nuclear Export ◽  
Binding Protein ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Simple Diffusion ◽  
Permeabilized Cells ◽  
C Terminus ◽  
Transport Receptors ◽  
Export Signal

ABSTRACT The Ran binding protein RanBP1 is localized to the cytosol of interphase cells. A leucine-rich nuclear export signal (NES) near the C terminus of RanBP1 is essential to maintain this distribution. We now show that RanBP1 accumulates in nuclei of cells treated with the export inhibitor, leptomycin B, and collapse of the nucleocytoplasmic Ran:GTP gradient leads to equilibration of RanBP1 across the nuclear envelope. Low temperature prevents nuclear accumulation of RanBP1, suggesting that import does not occur via simple diffusion. Glutathione S-transferase (GST)–RanBP1(1-161), which lacks the NES, accumulates in the nucleus after cytoplasmic microinjection. In permeabilized cells, nuclear accumulation of GST-RanBP1(1-161) requires nuclear Ran:GTP but is not inhibited by a dominant interfering G19V mutant of Ran. Nuclear accumulation is enhanced by addition of exogenous karyopherins/importins or RCC1, both of which also enhance nuclear Ran accumulation. Import correlates with Ran concentration. Remarkably, an E37K mutant of RanBP1 does not import into the nuclei under any conditions tested despite the fact that it can form a ternary complex with Ran and importin β. These data indicate that RanBP1 translocates through the pores by an active, nonclassical mechanism and requires Ran:GTP for nuclear accumulation. Shuttling of RanBP1 may function to clear nuclear pores of Ran:GTP, to prevent premature release of import cargo from transport receptors.

scholarly journals The Nuclear Export Receptor Xpo1p Forms Distinct Complexes with NES Transport Substrates and the Yeast Ran Binding Protein 1 (Yrb1p)

2001 ◽  
Vol 12 (3) ◽  
pp. 539-549 ◽  
Author(s):  
Patrick Maurer ◽  
Michael Redd ◽  
Jens Solsbacher ◽  
F. Ralf Bischoff ◽  
Markus Greiner ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Binding Protein ◽  
Nuclear Export Signal ◽  
Nuclear Pore ◽  
Nuclear Targeting ◽  
Gtp Hydrolysis ◽  
C Terminus ◽  
Transport Receptors ◽  
Nuclear Export Receptor ◽  
Export Signal

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin β-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.

scholarly journals Reconstitution of nuclear protein export in isolated nuclear envelopes

2002 ◽  
Vol 158 (5) ◽  
pp. 849-854 ◽  
Author(s):  
Jan Peter Siebrasse ◽  
Elias Coutavas ◽  
Reiner Peters
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Xenopus Oocytes ◽  
Nuclear Protein ◽  
Nuclear Export Signal ◽  
Protein Export ◽  
Leptomycin B ◽  
Permeabilized Cells ◽  
Export Signal ◽  
Kinase A

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.

scholarly journals Shuttling Imbalance of MLF1 Results in p53 Instability and Increases Susceptibility to Oncogenic Transformation

2007 ◽  
Vol 28 (1) ◽  
pp. 422-434 ◽  
Author(s):  
Noriko Yoneda-Kato ◽  
Jun-ya Kato
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chromosomal Translocation ◽  
Nuclear Accumulation ◽  
Tumor Suppressor P53 ◽  
Oncogenic Transformation ◽  
Early Passage ◽  
Leptomycin B ◽  
Nuclear Shuttling ◽  
Export Signal

ABSTRACT Myeloid leukemia factor 1 (MLF1) stabilizes the activity of the tumor suppressor p53 by suppressing its E3 ubiquitin ligase, COP1, through a third component of the COP9 signalosome (CSN3). However, little is known about how MLF1 functions upstream of the CSN3-COP1-p53 pathway and how its deregulation by the formation of the fusion protein nucleophosmin (NPM)-MLF1, generated by t(3;5)(q25.1;q34) chromosomal translocation, leads to leukemogenesis. Here we show that MLF1 is a cytoplasmic-nuclear-shuttling protein and that its nucleolar localization on fusing with NPM prevents the full induction of p53 by both genotoxic and oncogenic cellular stress. The majority of MLF1 was located in the cytoplasm, but the treatment of cells with leptomycin B rapidly induced a nuclear accumulation of MLF1. A mutation of the nuclear export signal (NES) motif identified in the MLF1 sequence enhanced the antiproliferative activity of MLF1. The fusion of MLF1 with NPM translocated MLF1 to the nucleolus and abolished the growth-suppressing activity. The introduction of NPM-MLF1 into early-passage murine embryonic fibroblasts allowed the cells to escape from cellular senescence at a markedly earlier stage and induced neoplastic transformation in collaboration with the oncogenic form of Ras. Interestingly, disruption of the MLF1-derived NES sequence completely abolished the growth-promoting activity of NPM-MLF1 in murine fibroblasts and hematopoietic cells. Thus, our results provide important evidence that the shuttling of MLF1 is critical for the regulation of cell proliferation and a disturbance in the shuttling balance increases the cell's susceptibility to oncogenic transformation.

scholarly journals A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1.

1996 ◽  
Vol 134 (5) ◽  
pp. 1157-1168 ◽  
Author(s):  
S A Richards ◽  
K M Lounsbury ◽  
K L Carey ◽  
I G Macara
Keyword(s):  
Nuclear Export ◽  
Protein Import ◽  
Nuclear Protein ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Nuclear Export Signal ◽  
Cyclin B1 ◽  
Nuclear Accumulation ◽  
Protein Fusion ◽  
Export Signal

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.

scholarly journals Keap1 Regulates the Oxidation-Sensitive Shuttling of Nrf2 into and out of the Nucleus via a Crm1-Dependent Nuclear Export Mechanism

2005 ◽  
Vol 25 (11) ◽  
pp. 4501-4513 ◽  
Author(s):  
Michaella Velichkova ◽  
Tama Hasson
Keyword(s):  
Nuclear Export ◽  
Consensus Sequence ◽  
Nuclear Export Signal ◽  
Antioxidant Response ◽  
Negative Regulator ◽  
Nuclear Accumulation ◽  
Leptomycin B ◽  
Hydrophobic Amino Acids ◽  
Export Signal ◽  
Full Activation

ABSTRACT Keap1 is a negative regulator of Nrf2, a transcription factor essential for antioxidant response element (ARE)-mediated gene expression. We find that Keap1 sequesters Nrf2 in the cytoplasm, not by docking it to the actin cytoskeleton but instead through an active Crm1/exportin-dependent nuclear export mechanism. Deletion and mutagenesis studies identified a nuclear export signal (NES) in the intervening region of Keap1 comprised of hydrophobic leucine and isoleucine residues in agreement with a traditional NES consensus sequence. Mutation of the hydrophobic amino acids resulted in nuclear accumulation of both Keap1 and Nrf2, as did treatment with the drug leptomycin B, which inactivates Crm1/exportin. ARE genes were partially activated under these conditions, suggesting that additional oxidation-sensitive elements are required for full activation of the antioxidant response. Based on these data, we propose a new model for regulation of Nrf2 by Keap1. Under normal conditions, Keap1 and Nrf2 are complexed in the cytoplasm where they are targeted for degradation. Oxidative stress inactivates Keap1's NES, allowing entry of both Keap1 and Nrf2 into the nucleus and transcriptional transactivation of ARE genes.

scholarly journals Regulation of STAT1 Nuclear Export by Jak1

2000 ◽  
Vol 20 (19) ◽  
pp. 7273-7281 ◽  
Author(s):  
Kerri Mowen ◽  
Michael David
Keyword(s):  
Tyrosine Kinases ◽  
Nuclear Export ◽  
Kinase Activity ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Transcriptional Coactivator ◽  
Leptomycin B ◽  
Nuclear Retention ◽  
Export Signal ◽  
Impaired Binding

ABSTRACT Signal transducer and activator of transcription 1 (STAT1) mediates gene expression in response to cytokines and growth factors. Activation of STAT1 is achieved through its tyrosine phosphorylation, a process that involves Jak tyrosine kinases. Here we show that STAT1, although phosphorylated on Y701, is unable to localize in the nucleus in the absence of Jak1 or Jak1 kinase activity. In contrast, the nuclear accumulation of STAT1 in Tyk2-deficient cells remains intact. Nuclear presence of tyrosine-phosphorylated STAT1 could be restored in Jak1-deficient cells by leptomycin B, an inhibitor of nuclear export. Amino acids 197 to 205 of STAT1 were found to encode a leucine-rich nuclear export signal (NES). An L→A mutation within the NES restored nuclear retention of STAT1 in Jak1-deficient cells. Impaired binding of the transcriptional coactivator CBP to tyrosine-phosphorylated STAT1 derived from Jak1-deficient cells offers a model for the intermolecular regulation of the nuclear export sequence.

scholarly journals Identification of the Nuclear Export Signal and STAT-Binding Domains of the Nipah Virus V Protein Reveals Mechanisms Underlying Interferon Evasion

2004 ◽  
Vol 78 (10) ◽  
pp. 5358-5367 ◽  
Author(s):  
Jason J. Rodriguez ◽  
Cristian D. Cruz ◽  
Curt M. Horvath
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Nuclear Export ◽  
Nipah Virus ◽  
Nuclear Export Signal ◽  
Hendra Virus ◽  
Nuclear Accumulation ◽  
V Protein ◽  
Binding Domains ◽  
Export Signal

ABSTRACT The V proteins of Nipah virus and Hendra virus have been demonstrated to bind to cellular STAT1 and STAT2 proteins to form high-molecular-weight complexes that inhibit interferon (IFN)-induced antiviral transcription by preventing STAT nuclear accumulation. Analysis of the Nipah virus V protein has revealed a region between amino acids 174 and 192 that functions as a CRM1-dependent nuclear export signal (NES). This peptide is sufficient to complement an export-defective human immunodeficiency virus Rev protein, and deletion and substitution mutagenesis revealed that this peptide is necessary for both V protein shuttling and cytoplasmic retention of STAT1 and STAT2 proteins. However, the NES is not required for V-dependent IFN signaling inhibition. IFN signaling is blocked primarily by interaction between Nipah virus V residues 100 to 160 and STAT1 residues 509 to 712. Interaction with STAT2 requires a larger Nipah virus V segment between amino acids 100 and 300, but deletion of residues 230 to 237 greatly reduced STAT2 coprecipitation. Further, V protein interactions with cellular STAT1 is a prerequisite for STAT2 binding, and sequential immunoprecipitations demonstrate that V, STAT1, and STAT2 can form a tripartite complex. These findings characterize essential regions for Henipavirus V proteins that represent potential targets for therapeutic intervention.

scholarly journals Characterization of a Novel Transferable CRM-1-Independent Nuclear Export Signal in a Herpesvirus Tegument Protein That Shuttles between the Nucleus and Cytoplasm

2006 ◽  
Vol 80 (20) ◽  
pp. 10021-10035 ◽  
Author(s):  
Janneke Verhagen ◽  
Michelle Donnelly ◽  
Gillian Elliott
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Nuclear Targeting ◽  
Leptomycin B ◽  
Herpesvirus Type ◽  
Close Proximity ◽  
Bovine Herpesvirus Type 1 ◽  
Import And Export ◽  
Export Signal

ABSTRACT A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.

scholarly journals Control of NF–κB transcriptional activation by signal induced proteolysis of IκBα

1999 ◽  
Vol 354 (1389) ◽  
pp. 1601-1609 ◽  
Author(s):  
R. T. Hay ◽  
L. Vuillard ◽  
J. M. P. Desterro ◽  
M. S. Rodriguez
Keyword(s):  
Nuclear Localization ◽  
Transcriptional Activation ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Cytoplasmic Process ◽  
Leptomycin B ◽  
Rapid Degradation ◽  
Localization Signal ◽  
Export Signal

In unstimulated cells the transcription factor NF–κB is held in the cytoplasm in an inactive state by IκB inhibitor proteins. Ultimately activation of NF–κB is achieved by ubiquitination and proteasome–mediated degradation of IκBα and we have therefore investigated factors which control this proteolysis. Signal–induced degradation of IκBα exposes the nuclear localization signal of NF–κB, thus allowing it to translocate into the nucleus and activate transcription from responsive genes. An autoregulatory loop is established when NF–κB induces expression of the IκBα gene and newly synthesized IκBα accumulates in the nucleus where it negatively regulates NF–κB–dependent transcription. As part of this post–induction repression, the nuclear export signal on IκBα mediates transport of NF–κB–IκBα complexes from the nucleus to the cytoplasm. As nuclear export of IκBα is blocked by leptomycin B this drug was used to examine the effect of cellular location on susceptibility of IκBα to signal–induced degradation. In the presence of leptomycin B, IκBα is accumulated in the nucleus and in this compartment is resistant to signal–induced degradation. Thus signal–induced degradation of IκBα is mainly, if not exclusively a cytoplasmic process. An efficient nuclear export of IκBα is therefore essential for maintaining a low level of IκBα in the nucleus and allowing NF–κB to be transcriptionally active upon cell stimulation. We have detected a modified form of IκBα, conjugated to the small ubiquitin–like protein SUMO–1, which is resistant to signal–induced degradation. SUMO–1 modified IκBα remains associated with NF–κB and thus overexpression of SUMO–1 inhibits the signal–induced activation of NF–κB–dependent transcription. Reconstitution of the conjugation reaction with highly purified proteins demonstrated that in the presence of a novel E1 SUMO–1 activating enzyme, Ubch9 directly conjugated SUMO–1 to IκBα on residues K21 and K22, which are also used for ubiquitin modification. Thus, while ubiquitination targets proteins for rapid degradation, SUMO–1 modification acts antagonistically to generate proteins resistant to degradation.

Sign in / Sign up

Export Citation Format

Share Document