scholarly journals Nucleocytoplasmic shuttling by nucleoporins Nup153 and Nup214 and CRM1-dependent nuclear export control the subcellular distribution of latent Stat1

2004 ◽  
Vol 165 (6) ◽  
pp. 823-833 ◽  
Author(s):  
Andreas Marg ◽  
Ying Shan ◽  
Thomas Meyer ◽  
Torsten Meissner ◽  
Martin Brandenburg ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Translocation ◽  
Cysteine Residue ◽  
Nuclear Pore ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Shuttling ◽  
Steady State Distribution ◽  
Linker Domain ◽  
Transport Factors ◽  
Stimulation Of

Interferon stimulation of cells leads to the tyrosine phosphorylation of latent Stat1 and subsequent transient accumulation in the nucleus that requires canonical transport factors. However, the mechanisms that control the predominantly cytoplasmic localization in unstimulated cells have not been resolved. We uncovered that constitutive energy- and transport factor-independent nucleocytoplasmic shuttling is a property of unphosphorylated Stat1, Stat3, and Stat5. The NH2- and COOH-terminal Stat domains are generally dispensable, whereas alkylation of a single cysteine residue blocked cytokine-independent nuclear translocation and thus implicated the linker domain into the cycling of Stat1. It is revealed that constitutive nucleocytoplasmic shuttling of Stat1 is mediated by direct interactions with the FG repeat regions of nucleoporin 153 and nucleoporin 214 of the nuclear pore. Concurrent active nuclear export by CRM1 created a nucleocytoplasmic Stat1 concentration gradient that is significantly reduced by the blocking of energy-requiring translocation mechanisms or the specific inactivation of CRM1. Thus, we propose that two independent translocation pathways cooperate to determine the steady-state distribution of Stat1.

scholarly journals Nucleocytoplasmic Shuttling of Endocytic Proteins

2001 ◽  
Vol 153 (7) ◽  
pp. 1511-1518 ◽  
Author(s):  
Manuela Vecchi ◽  
Simona Polo ◽  
Viviane Poupon ◽  
Jan-Willem van de Loo ◽  
Alexandre Benmerah ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Nuclear Export ◽  
Myeloid Leukemia ◽  
Nuclear Translocation ◽  
Initial Rate ◽  
Nucleocytoplasmic Shuttling ◽  
Cellular Processes ◽  
Transactivation Assay ◽  
Ordered Assembly ◽  
Endocytic Proteins

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and α-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

Abstract P217: Nuclear Protein Import as the Missing Link in Phenylephrine-Induced Cardiomyocyte Hypertrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Mirna N Chahine ◽  
Maxime Mioulane ◽  
Gabor Földes ◽  
Alexander Lyon ◽  
Sian E Harding
Keyword(s):  
Gene Expression ◽  
Nuclear Export ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Translocation ◽  
Cardiomyocyte Hypertrophy ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Adult Rat ◽  
Nuclear Protein Import

During cardiac hypertrophy, cardiomyocytes (CM) present alterations in gene expression and increased contractile protein content. Nuclear protein import (NPI) is critical in regulating gene expression, transcription, and subsequently cell hypertrophy. However, it is unknown how the nuclear transport machinery (transport receptors and nuclear pore complex (NPC)) functions to sustain increased demands for nucleocytoplasmic trafficking. The aim of this study was to determine if exposure of adult CM to phenylephrine (PE) affects hypertrophy by altering NPI and NPC density. Comparisons were made to adult failing rat and human CM. Rat myocytes were enzymatically isolated from adult hearts, and used for immunocytochemistry, qPCR and western immunoblotting. Failing CM were obtained from explanted human hearts at the time of transplant and from a rat model of myocardial infarction-induced hypertrophy and failure. Rat adult CM exposed for 48h to PE were injected with a protein import substrate (Alexa488-BSA-NLS) to visually monitor nuclear import with the confocal microscope. The effects of P38 MAPK inhibitor, HDAC inhibitor, Exportin-1 (CRM-1) inhibitor, and GSK-3 β inhibitor were investigated. Cell and nuclear sizes were increased in PE treated-adult rat CM and in the adult failing rat and human CM compared to normal CM. In contrast, PE depressed the rate and maximal NPI (by 65 +/- 3.4 % (3.55 from 5.46), p<0.05) as well as nucleoporin p62 mRNA and protein expression levels in adult rat CM compared to non-treated CM. Nucleoporin p62, cytoplasmic Ranbp1, and nuclear translocation of importins (Imp.α and β) relative densities were also decreased in PE treated-adult rat CM and in adult failing rat CM and human heart tissue compared to normal controls. On the contrary, CRM-1 nuclear export relative density was increased during the same pathological conditions. Thus NPI downregulation is linked to an increased nuclear export required by CM to generate the hypertrophic phenotype. All these effects were P38MAPK, HDAC and CRM-1 dependent but GSK-3Beta independent in rat CM. Our results show that alterations in NPI and NPC density occur in failing CM as well as in CM under hypertrophic stimuli. NPI may represent a critical therapeutic target in hypertrophic conditions.

scholarly journals Mutations in Tap Uncouple RNA Export Activity from Translocation through the Nuclear Pore Complex

2006 ◽  
Vol 17 (2) ◽  
pp. 931-943 ◽  
Author(s):  
Lyne Lévesque ◽  
Yeou-Cherng Bor ◽  
Leah H. Matzat ◽  
Li Jin ◽  
Stephen Berberoglu ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Point Mutations ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Transport Receptors ◽  
Cellular Mrnas ◽  
Rna Export ◽  
Rna Complex ◽  
Transport Receptor

Interactions between transport receptors and phenylalanine-glycine (FG) repeats on nucleoporins drive the translocation of receptor-cargo complexes through nuclear pores. Tap, a transport receptor that mediates nuclear export of cellular mRNAs, contains a UBA-like and NTF2-like folds that can associate directly with FG repeats. In addition, two nuclear export sequences (NESs) within the NTF2-like region can also interact with nucleoporins. The Tap-RNA complex was shown to bind to three nucleoporins, Nup98, p62, and RanBP2, and these interactions were enhanced by Nxt1. Mutations in the Tap-UBA region abolished interactions with all three nucleoporins, whereas the effect of point mutations within the NTF2-like domain of Tap known to disrupt Nxt1 binding or nucleoporin binding were nucleoporin dependent. A mutation in any of these Tap domains was sufficient to reduce RNA export but was not sufficient to disrupt Tap interaction with the NPC in vivo or its nucleocytoplasmic shuttling. However, shuttling activity was reduced or abolished by combined mutations within the UBA and either the Nxt1-binding domain or NESs. These data suggest that Tap requires both the UBA- and NTF2-like domains to mediate the export of RNA cargo, but can move through the pores independently of these domains when free of RNA cargo.

scholarly journals Nxt1 Is Necessary for the Terminal Step of Crm1-Mediated Nuclear Export

2001 ◽  
Vol 152 (1) ◽  
pp. 141-156 ◽  
Author(s):  
Ben E. Black ◽  
James M. Holaska ◽  
Lyne Lévesque ◽  
Batool Ossareh-Nazari ◽  
Carol Gwizdek ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Pore ◽  
Reporter Protein ◽  
Soluble Factors ◽  
Permeabilized Cells ◽  
Export Pathway ◽  
A Site ◽  
Cytoplasmic Side

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor–substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616–8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.

scholarly journals Cytoplasmic Localization of Wis1 MAPKK by Nuclear Export Signal Is Important for Nuclear Targeting of Spc1/Sty1 MAPK in Fission Yeast

2002 ◽  
Vol 13 (8) ◽  
pp. 2651-2663 ◽  
Author(s):  
Aaron Ngocky Nguyen ◽  
Aminah D. Ikner ◽  
Mitsue Shiozaki ◽  
Sasha M. Warren ◽  
Kazuhiro Shiozaki
Keyword(s):  
Fission Yeast ◽  
Nuclear Export ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
Cytoplasmic Localization ◽  
Nuclear Targeting ◽  
Export Signal

Mitogen-activated protein kinase (MAPK) cascade is a ubiquitous signaling module that transmits extracellular stimuli through the cytoplasm to the nucleus; in response to activating stimuli, MAPKs translocate into the nucleus. Mammalian MEK MAPK kinases (MAPKKs) have in their N termini an MAPK-docking site and a nuclear export signal (NES) sequence, which are known to play critical roles in maintaining ERK MAPKs in the cytoplasm of unstimulated cells. Herein, we show that the Wis1 MAPKK of the stress-activated Spc1 MAPK cascade in fission yeast also has a MAPK-docking site and an NES sequence in its N-terminal domain. Unexpectedly, an inactivating mutation to the NES of chromosomal wis1 + does not affect the subcellular localization of Spc1 MAPK, whereas this NES mutation disturbs the cytoplasmic localization of Wis1. However, when Wis1 is targeted to the nucleus by fusing to a nuclear localization signal sequence, stress-induced nuclear translocation of Spc1 is abrogated, indicating that cytoplasmic Wis1 is required for nuclear transport of Spc1 upon stress. Moreover, we have observed that a fraction of Wis1 translocates into the nucleus in response to stress. These results suggest that cytoplasmic localization of Wis1 MAPKK by its NES is important for stress signaling to the nucleus.

scholarly journals Analysis of Cellular Factors That Mediate Nuclear Export of RNAs Bearing the Mason-Pfizer Monkey Virus Constitutive Transport Element

2000 ◽  
Vol 74 (13) ◽  
pp. 5863-5871 ◽  
Author(s):  
Yibin Kang ◽  
Hal P. Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Export ◽  
Biological Activities ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Rna Molecules ◽  
Constitutive Transport Element ◽  
Rna Export

ABSTRACT There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)+ RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin β-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.

scholarly journals Unconventional tonicity-regulated nuclear trafficking of NFAT5 mediated by KPNB1, XPOT and RUVBL2

2021 ◽  
Author(s):  
Chris Y. Cheung ◽  
Ting-Ting Huang ◽  
Ning Chow ◽  
Shuqi Zhang ◽  
Yanxiang Zhao ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Shuttling ◽  
Proteomics Analysis ◽  
Nuclear Trafficking ◽  
Localization Signal ◽  
Underlying Mechanisms ◽  
Sirna Screening

NFAT5 is the only known mammalian tonicity-responsive transcription factor functionally implicated in diverse physiological and pathological processes. NFAT5 activity is tightly regulated by extracellular tonicity but the underlying mechanisms remain elusive. We demonstrated that NFAT5 enters the nucleus via the nuclear pore complex. We also found that NFAT5 utilizes a non-canonical nuclear localization signal (NFAT5-NLS) for nuclear imports. siRNA screening revealed that karyopherin beta-1 (KPNB1) drives nuclear import of NFAT5 via directly interacting with NFAT5-NLS. Proteomics analysis and siRNA screening further revealed that nuclear export of NFAT5 under hypotonicity is mediated by Exportin-T, and that it requires RuvB-Like AAA type ATPase 2 (RUVBL2) as an indispensable chaperone. Our findings have identified KPNB1 and RUVBL2 as key molecules responsible for the unconventional tonicity-regulated nucleocytoplasmic shuttling of NFAT5. These findings offer an opportunity for developing novel NFAT5 targeting strategies that are potentially useful for the treatment of diseases associated with NFAT5 dysregulation.

Nucleocytoplasmic Shuttling of Porcine Parvovirus NS1 Protein Mediated by the CRM1 Nuclear Export Pathway and the Importin α/β Nuclear Import Pathway

2021 ◽  
Author(s):  
Liyan Cao ◽  
Fang Fu ◽  
Jianfei Chen ◽  
Hongyan Shi ◽  
Xin Zhang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nonstructural Protein ◽  
Nuclear Pore ◽  
Porcine Parvovirus ◽  
Ns1 Protein ◽  
Nucleocytoplasmic Shuttling ◽  
Export Pathway ◽  
Importin Α

Porcine parvovirus (PPV) NS1, the major nonstructural protein of this virus, plays an important role in PPV replication. We show, for the first time, that NS1 dynamically shuttles between the nucleus and cytoplasm, although its subcellular localization is predominantly nuclear. NS1 contains two nuclear export signals (NESs) at amino acids 283–291 (designated NES2) and 602–608 (designated NES1). NES1 and NES2 are both functional and transferable NESs, and their nuclear export activity is blocked by leptomycin B (LMB), suggesting that the export of NS1 from the nucleus is dependent upon the chromosome region maintenance 1 (CRM1) pathway. Deletion and site-directed mutational analyses showed that NS1 contains a bipartite nuclear localization signal (NLS) at amino acids 256–274. Coimmunoprecipitation assays showed that NS1 interacts with importins α5 and α7 through its NLS. The overexpression of CRM1, importins α5 and α7 significantly promoted PPV replication, whereas the inhibition of CRM1 and importin α/β-mediated transport by specific inhibitors (LMB, importazole and ivermectin) clearly blocked PPV replication. The mutant viruses of delete NESs or NLS motif of the NS1 by using reverse genetics could not be rescued, suggesting that NESs and NLS are essential for PPV replication. Collectively, these findings suggest that NS1 shuttles between the nucleus and cytoplasm, mediated by its functional NESs and NLS, via the CRM1-dependent nuclear export pathway and the importin α/β-mediated nuclear import pathway, and PPV proliferation was inhibited if blocking NS1 nuclear import or export. Importance PPV replicates in the nucleus, and the nuclear envelope is a barrier to its entry into and egress from the nucleus. PPV NS1 is a nucleus-targeting protein that is important for viral DNA replication. Because the NS1 molecule is large (> 50 kDa), it cannot pass through the nuclear pore complex by diffusion alone, and requires specific transport receptors to permit its nucleocytoplasmic shuttling. In this study, the two functional NESs in the NS1 protein were identified, and its dependence on the CRM1 pathway for nuclear export demonstrated. The nuclear import of NS1 utilizes importins α5 and α7 in the importin α/β nuclear import pathway.

scholarly journals The hsp90-FKBP52 Complex Links the Mineralocorticoid Receptor to Motor Proteins and Persists Bound to the Receptor in Early Nuclear Events

2009 ◽  
Vol 30 (5) ◽  
pp. 1285-1298 ◽  
Author(s):  
Mario D. Galigniana ◽  
Alejandra G. Erlejman ◽  
Martín Monte ◽  
Celso Gomez-Sanchez ◽  
Graciela Piwien-Pilipuk
Keyword(s):  
Nuclear Export ◽  
Mineralocorticoid Receptor ◽  
Soluble Fraction ◽  
Molecular System ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Trafficking ◽  
Cytoplasmic Transport ◽  
Nuclear Events

ABSTRACT In this study, we demonstrate that the subcellular localization of the mineralocorticoid receptor (MR) is regulated by tetratricopeptide domain (TPR) proteins. The high-molecular-weight immunophilin (IMM) FKBP52 links the MR-hsp90 complex to dynein/dynactin motors favoring the cytoplasmic transport of MR to the nucleus. Replacement of this hsp90-binding IMM by FKBP51 or the TPR peptide favored the cytoplasmic localization of MR. The complete movement machinery, including dynein and tubulin, could be recovered from paclitaxel/GTP-stabilized cytosol and was fully reassembled on stripped MR immune pellets. The whole MR-hsp90-based heterocomplex was transiently recovered in the soluble fraction of the nucleus after 10 min of incubation with aldosterone. Moreover, cross-linked MR-hsp90 heterocomplexes accumulated in the nucleus in a hormone-dependent manner, demonstrating that the heterocomplex can pass undissociated through the nuclear pore. On the other hand, a peptide that comprises the DNA-binding domain of MR impaired the nuclear export of MR, suggesting the involvement of this domain in the process. This study represents the first report describing the entire molecular system that commands MR nucleocytoplasmic trafficking and proposes that the MR-hsp90-TPR protein heterocomplex is dissociated in the nucleus rather than in the cytoplasm.

scholarly journals Nup98 Is a Mobile Nucleoporin with Transcription-dependent Dynamics

2002 ◽  
Vol 13 (4) ◽  
pp. 1282-1297 ◽  
Author(s):  
Eric R. Griffis ◽  
Nihal Altan ◽  
Jennifer Lippincott-Schwartz ◽  
Maureen A. Powers
Keyword(s):  
Binding Sites ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Amino Acid Repeat ◽  
Nuclear Trafficking ◽  
Rate Of Recovery ◽  
Green Fluorescent ◽  
Transport Factors

Nucleoporin 98 (Nup98), a glycine-leucine-phenylalanine-glycine (GLFG) amino acid repeat-containing nucleoporin, plays a critical part in nuclear trafficking. Injection of antibodies to Nup98 into the nucleus blocks the export of most RNAs. Nup98 contains binding sites for several transport factors; however, the mechanism by which this nucleoporin functions has remained unclear. Multiple subcellular localizations have been suggested for Nup98. Here we show that Nup98 is indeed found both at the nuclear pore complex and within the nucleus. Inside the nucleus, Nup98 associates with a novel nuclear structure that we term the GLFG body because the GLFG domain of Nup98 is required for targeting to this structure. Photobleaching of green fluorescent protein-Nup98 in living cells reveals that Nup98 is mobile and moves between these different localizations. The rate of recovery after photobleaching indicates that Nup98 interacts with other, less mobile, components in the nucleoplasm. Strikingly, given the previous link to nuclear export, the mobility of Nup98 within the nucleus and at the pore is dependent on ongoing transcription by RNA polymerases I and II. These data give rise to a model in which Nup98 aids in direction of RNAs to the nuclear pore and provide the first potential mechanism for the role of a mobile nucleoporin.

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