scholarly journals The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export

2003 ◽  
Vol 163 (4) ◽  
pp. 701-706 ◽  
Author(s):  
Peggy Roth ◽  
Nikos Xylourgidis ◽  
Nafiseh Sabri ◽  
Anne Uv ◽  
Maarten Fornerod ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Accumulation ◽  
Major Function ◽  
Green Fluorescent ◽  
Mutant Phenotypes

Many cellular responses rely on the control of nucleocytoplasmic transport of transcriptional regulators. The Drosophila nucleoporin Nup88 is selectively required for nuclear accumulation of Rel proteins and full activation of the innate immune response. Here, we investigate the mechanisms underlying its role in nucleocytoplasmic transport. Nuclear import of an nuclear localization signal-enhanced green fluorescent protein (NLS-EGFP) reporter is not affected in DNup88 (members only; mbo) mutants, whereas the level of CRM1-dependent EGFP-nuclear export signal (EGFP-NES) export is increased. We show that the nuclear accumulation of the Drosophila Rel protein Dorsal requires CRM1. DNup88 binds to DNup214 and DCRM1 in vitro, and both proteins become mislocalized from the nuclear rim into the nucleus of mbo mutants. Overexpression of DNup88 is sufficient to relocalize DNup214 and CRM1 on the nuclear envelope and revert the mutant phenotypes. We propose that a major function of DNup88 is to anchor DNup214 and CRM1 on the nuclear envelope and thereby attenuate NES-mediated nuclear export.

scholarly journals CRM1 Mediates the Export of ADAR1 through a Nuclear Export Signal within the Z-DNA Binding Domain

2001 ◽  
Vol 21 (22) ◽  
pp. 7862-7871 ◽  
Author(s):  
Hanne Poulsen ◽  
Jakob Nilsson ◽  
Christian K. Damgaard ◽  
Jan Egebjerg ◽  
Jørgen Kjems
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Adenosine Deaminases ◽  
Editing Activity ◽  
Green Fluorescent ◽  
Export Signal

ABSTRACT RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

scholarly journals LZTS2 Is a Novel β-Catenin-Interacting Protein and Regulates the Nuclear Export of β-Catenin

2006 ◽  
Vol 26 (23) ◽  
pp. 8857-8867 ◽  
Author(s):  
Gregory Thyssen ◽  
Tzu-Huey Li ◽  
Lynn Lehmann ◽  
Ming Zhuo ◽  
Manju Sharma ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Point Mutations ◽  
Cellular Level ◽  
Interacting Protein ◽  
Nuclear Exclusion ◽  
Green Fluorescent ◽  
Wnt Signal

ABSTRACT β-Catenin plays multiple roles in cell-cell adhesion and Wnt signal transduction. Through the Wnt signal, the cellular level of β-catenin is constitutively regulated by the multicomponent destruction complex containing glycogen synthase kinase 3β, axin, and adenomatous polyposis coli. Here, we present multiple lines of evidence to demonstrate that LZTS2 (lucine zipper tumor suppressor 2) interacts with β-catenin, represses the transactivation of β-catenin, and affects the subcellular localization of β-catenin. The LZTS2 gene is located at 10q24.3, which is frequently lost in a variety of human tumors. A functional nuclear export signal (NES) was identified in the C terminus of the protein (amino acids 631 to 641). Appending this motif to green fluorescent protein (GFP) induced nuclear exclusion of the GFP fusion protein. However, introducing point mutations in either one or two leucine residues of this NES sequence abolished the nuclear exclusion of the LZTS2 protein. The nuclear export of LZTS2 can be blocked by leptomycin B (LMB), an inhibitor of the CRM1/exportin-alpha pathway. Intriguingly, β-catenin colocalizes with LZTS2 in the cytoplasm of cells in the absence of LMB but in the nuclei of cells in the presence of LMB. Increasing the LZTS2 protein in cells reduces the level of nuclear β-catenin in SW480 cells. Taken together, these data demonstrate that LZTS2 is a β-catenin-interacting protein that can modulate β-catenin signaling and localization.

scholarly journals Calreticulin Is a Receptor for Nuclear Export

2001 ◽  
Vol 152 (1) ◽  
pp. 127-140 ◽  
Author(s):  
James M. Holaska ◽  
Ben E. Black ◽  
Dona C. Love ◽  
John A. Hanover ◽  
John Leszyk ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Hormone Receptors ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Steroid Hormone ◽  
Nuclear Export Signal ◽  
Steroid Hormone Receptors ◽  
Permeabilized Cell ◽  
Green Fluorescent

In previous work, we used a permeabilized cell assay that reconstitutes nuclear export of protein kinase inhibitor (PKI) to show that cytosol contains an export activity that is distinct from Crm1 (Holaska, J.M., and B.M. Paschal. 1995. Proc. Natl. Acad. Sci. USA. 95: 14739–14744). Here, we describe the purification and characterization of the activity as calreticulin (CRT), a protein previously ascribed to functions in the lumen of the ER. We show that cells contain both ER and cytosolic pools of CRT. The mechanism of CRT-dependent export of PKI requires a functional nuclear export signal (NES) in PKI and involves formation of an export complex that contains RanGTP. Previous studies linking CRT to downregulation of steroid hormone receptor function led us to examine its potential role in nuclear export of the glucocorticoid receptor (GR). We found that CRT mediates nuclear export of GR in permeabilized cell, microinjection, and transfection assays. GR export is insensitive to the Crm1 inhibitor leptomycin B in vivo, and it does not rely on a leucine-rich NES. Rather, GR export is facilitated by its DNA-binding domain, which is shown to function as an NES when transplanted to a green fluorescent protein reporter. CRT defines a new export pathway that may regulate the transcriptional activity of steroid hormone receptors.

scholarly journals Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits

2008 ◽  
Vol 28 (10) ◽  
pp. 3151-3161 ◽  
Author(s):  
Joshua White ◽  
Zhihua Li ◽  
Richa Sardana ◽  
Janusz M. Bujnicki ◽  
Edward M. Marcotte ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Fluorescent Protein ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Nuclear Accumulation ◽  
Methyltransferase Activity ◽  
Late Step ◽  
Green Fluorescent

ABSTRACT BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.

Identification of a functional nuclear export signal in the green fluorescent protein asFP499

2006 ◽  
Vol 342 (4) ◽  
pp. 1178-1182 ◽  
Author(s):  
Huseyin Mustafa ◽  
Bernd Straßer ◽  
Sabine Rauth ◽  
Robert A. Irving ◽  
Kim L. Wark
Keyword(s):  
Green Fluorescent Protein ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Green Fluorescent ◽  
Export Signal

scholarly journals AMPK Facilitates Nuclear Accumulation of Nrf2 by Phosphorylating at Serine 550

2016 ◽  
Vol 36 (14) ◽  
pp. 1931-1942 ◽  
Author(s):  
Min Sung Joo ◽  
Won Dong Kim ◽  
Ki Young Lee ◽  
Ji Hyun Kim ◽  
Ja Hyun Koo ◽  
...  
Keyword(s):  
Cell Survival ◽  
Nuclear Export ◽  
Glycogen Synthase ◽  
Nuclear Export Signal ◽  
Direct Interaction ◽  
Antioxidant Response ◽  
Nuclear Accumulation ◽  
Related Factor ◽  
Ampk Activation

Nrf2 (nuclear factor erythroid 2-related factor 2) is an antioxidant transcription factor. AMP-activated protein kinase (AMPK) functions as a central regulator of cell survival in response to stressful stimuli. Nrf2 should be coordinated with the cell survival pathway controlled by AMPK, but so far the mechanistic connections remain undefined. This study investigated the role of AMPK in Nrf2 trafficking and its activity regulation. A subnetwork integrating neighbor molecules suggested direct interaction between AMPK and Nrf2. In cells, AMPK activation caused nuclear accumulation of Nrf2. In thein vitrokinase and peptide competition assays, AMPK phosphorylated Nrf2 at the Ser558 residue (Ser550 in mouse) located in the canonical nuclear export signal. Nrf2 with an S550A mutation failed to be accumulated in the nucleus after AMPK activation. Leptomycin B, a nuclear export inhibitor, did not enhance nuclear accumulation of wild-type Nrf2 (WT-Nrf2) activated by AMPK or a phospho-Ser550-mimetic Nrf2 mutant, corroborating the finding that AMPK facilitated nuclear accumulation of Nrf2, probably by inhibiting nuclear export. Activated glycogen synthase kinase 3β (GSK3β) diminished the basal nuclear level of Myc-S550A-Nrf2. Taking the data collectively, AMPK phosphorylates Nrf2 at the Ser550 residue, which, in conjunction with AMPK-mediated GSK3β inhibition, promotes nuclear accumulation of Nrf2 for antioxidant response element (ARE)-driven gene transactivation.

scholarly journals The Redox State of SECIS Binding Protein 2 Controls Its Localization and Selenocysteine Incorporation Function

2006 ◽  
Vol 26 (13) ◽  
pp. 4895-4910 ◽  
Author(s):  
Laura V. Papp ◽  
Jun Lu ◽  
Frank Striebel ◽  
Derek Kennedy ◽  
Arne Holmgren ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nuclear Export ◽  
Stop Codon ◽  
Binding Protein ◽  
Redox Homeostasis ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Antioxidant Systems

ABSTRACT Selenoproteins are central controllers of cellular redox homeostasis. Incorporation of selenocysteine (Sec) into selenoproteins employs a unique mechanism to decode the UGA stop codon. The process requires the Sec insertion sequence (SECIS) element, tRNASec, and protein factors including the SECIS binding protein 2 (SBP2). Here, we report the characterization of motifs within SBP2 that regulate its subcellular localization and function. We show that SBP2 shuttles between the nucleus and the cytoplasm via intrinsic, functional nuclear localization signal and nuclear export signal motifs and that its nuclear export is dependent on the CRM1 pathway. Oxidative stress induces nuclear accumulation of SBP2 via oxidation of cysteine residues within a redox-sensitive cysteine-rich domain. These modifications are efficiently reversed in vitro by human thioredoxin and glutaredoxin, suggesting that these antioxidant systems might regulate redox status of SBP2 in vivo. Depletion of SBP2 in cell lines using small interfering RNA results in a decrease in Sec incorporation, providing direct evidence for its requirement for selenoprotein synthesis. Furthermore, Sec incorporation is reduced substantially after treatment of cells with agents that cause oxidative stress, suggesting that nuclear sequestration of SBP2 under such conditions may represent a mechanism to regulate the expression of selenoproteins.

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 Characterization of Signal Sequences Determining the Nuclear/Nucleolar Import and Nuclear Export of the Caprine Arthritis-Encephalitis Virus Rev Protein

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 900
Author(s):  
Marlène Labrecque ◽  
Claude Marchand ◽  
Denis Archambault
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Encephalitis Virus ◽  
Viral Mrnas ◽  
Caprine Arthritis Encephalitis Virus ◽  
Cell Compartments ◽  
Arginine Residues ◽  
Green Fluorescent ◽  
Rev Protein

Caprine arthritis-encephalitis virus (CAEV), a lentivirus, relies on the action of the Rev protein for its replication. The CAEV Rev fulfills its function by allowing the nuclear exportation of partially spliced or unspliced viral mRNAs. In this study, we characterized the nuclear and nucleolar localization signals (NLS and NoLS, respectively) and the nuclear export signal (NES) of the CAEV Rev protein. These signals are key actors in the nucleocytoplasmic shuttling of a lentiviral Rev protein. Several deletion and alanine substitution mutants were generated from a plasmid encoding the CAEV Rev wild-type protein that was fused to the enhanced green fluorescent protein (EGFP). Following cell transfection, images were captured by confocal microscopy and the fluorescence was quantified in the different cell compartments. The results showed that the NLS region is localized between amino acids (aa) 59 to 75, has a monopartite-like structure and is exclusively composed of arginine residues. The NoLS was found to be partially associated with the NLS. Finally, the CAEV Rev protein’s NES mapped between aa 89 to 101, with an aa spacing between the hydrophobic residues that was found to be unconventional as compared to that of other retroviral Rev/Rev-like proteins.

scholarly journals A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

1999 ◽  
Vol 144 (3) ◽  
pp. 389-401 ◽  
Author(s):  
Ed Hurt ◽  
Stefan Hannus ◽  
Birgit Schmelzl ◽  
Denise Lau ◽  
David Tollervey ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
In Vivo Assay ◽  
Green Fluorescent ◽  
Transport Factors

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

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