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 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.

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 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 The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Lentiviral Bipartite Nuclear Localization Signal Harboring an Atypical Spacer Sequence

2009 ◽  
Vol 83 (24) ◽  
pp. 12842-12853 ◽  
Author(s):  
Andrea Gomez Corredor ◽  
Denis Archambault
Keyword(s):  
Protein Identification ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
Bovine Immunodeficiency Virus ◽  
Immunodeficiency Virus ◽  
Arginine Residues ◽  
Localization Signal ◽  
Infected Cells ◽  
Green Fluorescent ◽  
Rev Protein

ABSTRACT The bovine immunodeficiency virus (BIV) Rev protein (186 amino acids [aa] in length) is involved in the nuclear exportation of partially spliced and unspliced viral RNAs. Previous studies have shown that BIV Rev localizes in the nucleus and nucleolus of infected cells. Here we report the characterization of the nuclear/nucleolar localization signals (NLS/NoLS) of this protein. Through transfection of a series of deletion mutants of BIV Rev fused to enhanced green fluorescent protein and fluorescence microscopy analyses, we were able to map the NLS region between aa 71 and 110 of the protein. Remarkably, by conducting alanine substitution of basic residues within the aa 71 to 110 sequence, we demonstrated that the BIV Rev NLS is bipartite, maps to aa 71 to 74 and 95 to 101, and is predominantly composed of arginine residues. This is the first report of a bipartite Rev (or Rev-like) NLS in a lentivirus/retrovirus. Moreover, this NLS is atypical, as the length of the sequence between the motifs composing the bipartite NLS, e.g., the spacer sequence, is 20 aa. Further mutagenesis experiments also identified the NoLS region of BIV Rev. It localizes mainly within the NLS spacer sequence. In addition, the BIV Rev NoLS sequence differs from the consensus sequence reported for other viral and cellular nucleolar proteins. In summary, we conclude that the nucleolar and nuclear localizations of BIV Rev are mediated via novel NLS and NoLS motifs.

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 Caenorhabditis elegans UNC-98, a C2H2 Zn Finger Protein, Is a Novel Partner of UNC-97/PINCH in Muscle Adhesion Complexes

2003 ◽  
Vol 14 (6) ◽  
pp. 2492-2507 ◽  
Author(s):  
Kristina B. Mercer ◽  
Denise B. Flaherty ◽  
Rachel K. Miller ◽  
Hiroshi Qadota ◽  
Tina L. Tinley ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Focal Adhesions ◽  
Cell Nuclei ◽  
C Elegans ◽  
Dense Bodies ◽  
Green Fluorescent

To further understand the assembly and maintenance of the muscle contractile apparatus, we have identified a new protein, UNC-98, in the muscle of Caenorhabditis elegans. unc-98 mutants display reduced motility and a characteristic defect in muscle structure. We show that the major defect in the mutant muscle is in the M-lines and dense bodies (Z-line analogs). Both functionally and compositionally, nematode M-lines and dense bodies are analogous to focal adhesions of nonmuscle cells. UNC-98 is a novel 310-residue polypeptide consisting of four C2H2 Zn fingers and several possible nuclear localization signal and nuclear export signal sequences. By use of UNC-98 antibodies and green fluorescent protein fusions (to full-length UNC-98 and UNC-98 fragments), we have shown that UNC-98 resides at M-lines, muscle cell nuclei, and possibly at dense bodies. Furthermore, we demonstrated that 1) the N-terminal 106 amino acids are both necessary and sufficient for nuclear localization, and 2) the C-terminal (fourth) Zn finger is required for localization to M-lines and dense bodies. UNC-98 interacts with UNC-97, a C. elegans homolog of PINCH. We propose that UNC-98 is both a structural component of muscle focal adhesions and a nuclear protein that influences gene expression.

scholarly journals Cell Cycle-dependent Dynamics and Regulation of Mitotic Kinesins in Drosophila S2 Cells

2005 ◽  
Vol 16 (8) ◽  
pp. 3896-3907 ◽  
Author(s):  
Gohta Goshima ◽  
Ronald D. Vale
Keyword(s):  
Cell Cycle ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Active Form ◽  
Nuclear Export Signal ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Cycle Dependent ◽  
Green Fluorescent ◽  
And Function

Constructing a mitotic spindle requires the coordinated actions of several kinesin motor proteins. Here, we have visualized the dynamics of five green fluorescent protein (GFP)-tagged mitotic kinesins (class 5, 6, 8, 13, and 14) in live Drosophila Schneider cell line (S2), after first demonstrating that the GFP-tag does not interfere with the mitotic functions of these kinesins using an RNA interference (RNAi)-based rescue strategy. Class 8 (Klp67A) and class 14 (Ncd) kinesin are sequestered in an active form in the nucleus during interphase and engage their microtubule targets upon nuclear envelope breakdown (NEB). Relocalization of Klp67A to the cytoplasm using a nuclear export signal resulted in the disassembly of the interphase microtubule array, providing support for the hypothesis that this kinesin class possesses microtubule-destabilizing activity. The interactions of Kinesin-5 (Klp61F) and -6 (Pavarotti) with microtubules, on the other hand, are activated and inactivated by Cdc2 phosphorylation, respectively, as shown by examining localization after mutating Cdc2 consensus sites. The actions of microtubule-destabilizing kinesins (class 8 and 13 [Klp10A]) seem to be controlled by cell cycle-dependent changes in their localizations. Klp10A, concentrated on microtubule plus ends in interphase and prophase, relocalizes to centromeres and spindle poles upon NEB and remains at these sites throughout anaphase. Consistent with this localization, RNAi analysis showed that this kinesin contributes to chromosome-to-pole movement during anaphase A. Klp67A also becomes kinetochore associated upon NEB, but the majority of the population relocalizes to the central spindle by the timing of anaphase A onset, consistent with our RNAi result showing no effect of depleting this motor on anaphase A. These results reveal a diverse spectrum of regulatory mechanisms for controlling the localization and function of five mitotic kinesins at different stages of the cell cycle.

The Saccharomyces cerevisiae cyclin Clb2p is targeted to multiple subcellular locations by cis- and trans-acting determinants

2001 ◽  
Vol 114 (3) ◽  
pp. 589-597 ◽  
Author(s):  
J.K. Hood ◽  
W.W. Hwang ◽  
P.A. Silver
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Cell Cycle Regulatory Proteins ◽  
Localization Signal ◽  
Green Fluorescent

The cyclin-dependent kinase Cdc28p associates with the cyclin Clb2p to induce mitosis in the yeast Saccharomyces cerevisiae. Several cell cycle regulatory proteins have been shown to require specific nuclear transport events to exert their regulatory functions. Therefore, we investigated the subcellular localization of wild-type Clb2p and several mutant versions of the protein using green fluorescent protein (GFP) fusion constructs. Wild-type Clb2p is primarily nuclear at all points of the cell. A point mutation in a potential leucine-rich nuclear export signal (NES) enhances the nuclear localization of the protein, and delta-yrb2 cells exhibit an apparent Clb2p nuclear export defect. Clb2p contains a bipartite nuclear localization signal (NLS), and its nuclear localization requires the alpha and beta importins (Srp1p and Kap95p), as well as the yeast Ran GTPase and its regulators. Deletion of the Clb2p NLS causes increased cytoplasmic localization of the protein, as well as accumulation at the bud neck. These data indicate that Clb2p exists in multiple places in the yeast cell, possibly allowing Cdc28p to locally phosphorylate substrates at distinct subcellular sites.

scholarly journals Both Binding and Activation of p38 Mitogen-Activated Protein Kinase (MAPK) Play Essential Roles in Regulation of the Nucleocytoplasmic Distribution of MAPK-Activated Protein Kinase 5 by Cellular Stress

2002 ◽  
Vol 22 (20) ◽  
pp. 6931-6945 ◽  
Author(s):  
Ole Morten Seternes ◽  
Bjarne Johansen ◽  
Beate Hegge ◽  
Mona Johannessen ◽  
Stephen M. Keyse ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mapk Pathway ◽  
Nuclear Export Signal ◽  
Mitogen Activated Protein Kinase ◽  
Protein Fusion ◽  
Translational Machinery ◽  
Mitogen Activated Protein

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.

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