scholarly journals Identification of Novel Import and Export Signals of Human TAP, the Protein That Binds to the Constitutive Transport Element of the Type D Retrovirus mRNAs

1999 ◽  
Vol 19 (9) ◽  
pp. 6306-6317 ◽  
Author(s):  
Jenifer Bear ◽  
Wei Tan ◽  
Andrei S. Zolotukhin ◽  
Carlos Tabernero ◽  
Eric A. Hudson ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Protein ◽  
Terminal Portion ◽  
Nuclear Retention ◽  
Constitutive Transport Element ◽  
Type D ◽  
Acid Protein ◽  
Import And Export ◽  
Amino Acid Protein

ABSTRACT The nuclear export of the unspliced type D retrovirus mRNA depends on the cis-acting constitutive transport RNA element (CTE) that has been shown to interact with the human TAP (hTAP) protein promoting the export of the CTE-containing mRNAs. We report here that hTAP is a 619-amino-acid protein extending the previously identified protein by another 60 residues at the N terminus and that hTAP shares high homology with the predicted rat and mouse TAP proteins. We found that hTAP is a nuclear protein that accumulates in the nuclear rim and the nucleoplasm. We further demonstrated that hTAP is able to shuttle between the nucleus and the cytoplasm. Identification of the signals responsible for nuclear import (NLS) and export (NES) revealed that they are distinct but partially overlapping. NLS and NES of hTAP are active transferable signals that do not share similarities with known elements. The C-terminal portion contributes further to hTAP’s nuclear retention and contains a signal(s) for nuclear rim association. Taken together, our data show that hTAP is a dynamic protein capable of bidirectional trafficking across the nuclear envelope. These data further support hTAP’s role as an export factor of the CTE-containing mRNAs.

scholarly journals Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear anchor dTCF/Pan

Development ◽  
2001 ◽  
Vol 128 (11) ◽  
pp. 2107-2117 ◽  
Author(s):  
Nicholas S. Tolwinski ◽  
Eric Wieschaus
Keyword(s):  
Drosophila Melanogaster ◽  
Plasma Membrane ◽  
Nuclear Import ◽  
Current Model ◽  
Cellular Distribution ◽  
Nuclear Retention ◽  
Binding Partners ◽  
Wingless Signaling ◽  
Anchoring System ◽  
Import And Export

Drosophila melanogaster Armadillo plays two distinct roles during development. It is a component of adherens junctions, and functions as a transcriptional activator in response to Wingless signaling. In the current model, Wingless signal causes stabilization of cytoplasmic Armadillo allowing it to enter the nucleus where it can activate transcription. However, the mechanism of nuclear import and export remains to be elucidated. In this study, we show that two gain-of-function alleles of Armadillo activate Wingless signaling by different mechanisms. The S10 allele was previously found to localize to the nucleus, where it activates transcription. In contrast, the ΔArm allele localizes to the plasma membrane, and forces endogenous Arm into the nucleus. Therefore, ΔArm is dependent on the presence of a functional endogenous allele of arm to activate transcription. We show that ΔArm may function by titrating Axin protein to the membrane, suggesting that it acts as a cytoplasmic anchor keeping Arm out of the nucleus. In axin mutants, Arm is localized to the nuclei. We find that nuclear retention is dependent on dTCF/Pangolin. This suggests that cellular distribution of Arm is controlled by an anchoring system, where various nuclear and cytoplasmic binding partners determine its localization.

scholarly journals Discrimination between NL1- and NL2-Mediated Nuclear Localization of the Glucocorticoid Receptor

1999 ◽  
Vol 19 (2) ◽  
pp. 1025-1037 ◽  
Author(s):  
Joanne G. A. Savory ◽  
Brian Hsu ◽  
Ian R. Laquian ◽  
Ward Giffin ◽  
Terry Reich ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Binding Domain ◽  
Leptomycin B ◽  
Importin Α ◽  
Agonist And Antagonist ◽  
Import And Export ◽  
Pathway Inhibition

ABSTRACT Glucocorticoid receptor (GR) cycles between a free liganded form that is localized to the nucleus and a heat shock protein (hsp)-immunophilin-complexed, unliganded form that is usually localized to the cytoplasm but that can also be nuclear. In addition, rapid nucleocytoplasmic exchange or shuttling of the receptor underlies its localization. Nuclear import of liganded GR is mediated through a well-characterized sequence, NL1, adjacent to the receptor DNA binding domain and a second, uncharacterized motif, NL2, that overlaps with the ligand binding domain. In this study we report that rapid nuclear import (half-life [t 1/2] of 4 to 6 min) of agonist- and antagonist-treated GR and the localization of unliganded, hsp-associated GRs to the nucleus in G0 are mediated through NL1 and correlate with the binding of GR to pendulin/importin α. By contrast, NL2-mediated nuclear transfer of GR occurred more slowly (t 1/2 = 45 min to 1 h), was agonist specific, and appeared to be independent of binding to importin α. Together, these results suggest that NL2 mediates the nuclear import of GR through an alternative nuclear import pathway. Nuclear export of GR was inhibited by leptomycin B, suggesting that the transfer of GR to the cytoplasm is mediated through the CRM1-dependent pathway. Inhibition of GR nuclear export by leptomycin B enhanced the nuclear localization of both unliganded, wild-type GR and hormone-treated NL1− GR. These results highlight that the subcellular localization of both liganded and unliganded GRs is determined, at least in part, by a flexible equilibrium between the rates of nuclear import and export.

scholarly journals Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activation

1994 ◽  
Vol 14 (10) ◽  
pp. 6411-6418
Author(s):  
A Feller ◽  
E Dubois ◽  
F Ramos ◽  
A Piérard
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Binding ◽  
Transcriptional Activation ◽  
Activation Function ◽  
Lysine Biosynthesis ◽  
Binding Motif ◽  
Terminal Portion ◽  
Rich Domain ◽  
Acid Protein ◽  
Amino Acid Protein

The product of the LYS14 gene of Saccharomyces cerevisiae activates the transcription of at least four genes involved in lysine biosynthesis. Physiological and genetic studies indicate that this activation is dependent on the inducer alpha-aminoadipate semialdehyde, an intermediate of the pathway. The gene LYS14 was sequenced and, from its nucleotide sequence, predicted to encode a 790-amino-acid protein carrying a cysteine-rich DNA-binding motif of the Zn(II)2Cys6 type in its N-terminal portion. Deletion of this N-terminal portion including the cysteine-rich domain resulted in the loss of LYS14 function. To test the function of Lys14 as a transcriptional activator, this protein without its DNA-binding motif was fused to the DNA-binding domain of the Escherichia coli LexA protein. The resulting LexA-Lys14 hybrid protein was capable of activating transcription from a promoter containing a lexA operator, thus confirming the transcriptional activation function of Lys14. Furthermore, evidence that this function, which is dependent on the presence of alpha-aminoadipate semialdehyde, is antagonized by lysine was obtained. Such findings suggest that activation by alpha-aminoadipate semialdehyde and the apparent repression by lysine are related mechanisms. Lysine possibly acts by limiting the supply of the coinducer, alpha-aminoadipate semialdehyde.

scholarly journals Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export

2002 ◽  
Vol 156 (4) ◽  
pp. 617-630 ◽  
Author(s):  
Phyllis Frosst ◽  
Tinglu Guan ◽  
Cecilia Subauste ◽  
Klaus Hahn ◽  
Larry Gerace
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Protein ◽  
Coiled Coil ◽  
Basic Amino Acid ◽  
Living Cells ◽  
Protein Export ◽  
Interphase Cells ◽  
Nuclear Injection ◽  
Amino Acid Signal

Tpr is a coiled-coil protein found near the nucleoplasmic side of the pore complex. Since neither the precise localization of Tpr nor its functions are well defined, we generated antibodies to three regions of Tpr to clarify these issues. Using light and EM immunolocalization, we determined that mammalian Tpr is concentrated within the nuclear basket of the pore complex in a distribution similar to Nup153 and Nup98. Antibody localization together with imaging of GFP-Tpr in living cells revealed that Tpr is in discrete foci inside the nucleus similar to several other nucleoporins but is not present in intranuclear filamentous networks (Zimowska et al., 1997) or in long filaments extending from the pore complex (Cordes et al., 1997) as proposed. Injection of anti-Tpr antibodies into mitotic cells resulted in depletion of Tpr from the nuclear envelope without loss of other pore complex basket proteins. Whereas nuclear import mediated by a basic amino acid signal was unaffected, nuclear export mediated by a leucine-rich signal was retarded significantly. Nuclear injection of anti-Tpr antibodies in interphase cells similarly yielded inhibition of protein export but not import. These results indicate that Tpr is a nucleoporin of the nuclear basket with a role in nuclear protein export.

scholarly journals Histone Deacetylase 4 Possesses Intrinsic Nuclear Import and Export Signals

2001 ◽  
Vol 21 (17) ◽  
pp. 5992-6005 ◽  
Author(s):  
Audrey H. Wang ◽  
Xiang-Jiao Yang
Keyword(s):  
Histone Deacetylase ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Mutational Analysis ◽  
Nuclear Export Signal ◽  
Middle Part ◽  
Nucleocytoplasmic Trafficking ◽  
Histone Deacetylase 4 ◽  
Import And Export

ABSTRACT Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.

scholarly journals Involvement of Nuclear Import and Export Factors in U8 Box C/D snoRNP Biogenesis

2007 ◽  
Vol 27 (20) ◽  
pp. 7018-7027 ◽  
Author(s):  
Nicholas J. Watkins ◽  
Ira Lemm ◽  
Reinhard Lührmann
Keyword(s):  
Rna Interference ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Nucleocytoplasmic Transport ◽  
Differential Association ◽  
Import And Export ◽  
Snornp Biogenesis ◽  
Cellular Compartments ◽  
Distinct Distribution

ABSTRACT Box C/D snoRNPs, factors essential for ribosome biogenesis, are proposed to be assembled in the nucleoplasm before localizing to the nucleolus. However, recent work demonstrated the involvement of nuclear export factors in this process, suggesting that export may take place. Here we show that there are distinct distributions of U8 pre-snoRNAs and pre-snoRNP complexes in HeLa cell nuclear and cytoplasmic extracts. We observed differential association of nuclear export (PHAX, CRM1, and Ran) factors with complexes in the two extracts, consistent with nucleocytoplasmic transport. Furthermore, we show that the U8 pre-snoRNA in one of the cytoplasmic complexes contains an m3G cap and is associated with the nuclear import factor Snurportin1. Using RNA interference, we show that loss of either PHAX or Snurportin1 results in the incorrect localization of the U8 snoRNA. Our data therefore show that nuclear export and import factors are directly involved in U8 box C/D snoRNP biogenesis. The distinct distribution of U8 pre-snoRNP complexes between the two cellular compartments together with the association of both nuclear import and export factors with the precursor complex suggests that the mammalian U8 snoRNP is exported during biogenesis.

scholarly journals The Balance of Nuclear Import and Export Determines the Intracellular Distribution and Function of Tomato Heat Stress Transcription Factor HsfA2

2001 ◽  
Vol 21 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Dirk Heerklotz ◽  
Pascal Döring ◽  
Frank Bonzelius ◽  
Sybille Winkelhaus ◽  
Lutz Nover
Keyword(s):  
Transcription Factor ◽  
Heat Stress ◽  
Nuclear Import ◽  
Cho Cells ◽  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Chinese Hamster ◽  
Intracellular Distribution ◽  
Import And Export ◽  
Oligomerization Domain

ABSTRACT Tomato heat stress transcription factor HsfA2 is a shuttling protein with dominant cytoplasmic localization as a result of a nuclear import combined with an efficient export. Besides the nuclear localization signal (NLS) adjacent to the oligomerization domain, a C-terminal leucine-rich motif functions as a nuclear export signal (NES). Mutant forms of HsfA2 with a defective or an absent NES are nuclear proteins. The same is true for the wild-type HsfA2 if coexpressed with HsfA1 or in the presence of export inhibitor leptomycin B (LMB). Fusion of the NES domain of HsfA2 to HsfB1, which is a nuclear protein, caused export of the HsfB1-A2NES hybrid protein, and this effect was reversed by the addition of LMB. Due to the lack of background problems, Chinese hamster ovary (CHO) cells represent an excellent system for expression and functional analysis of tomato Hsfs. The results faithfully reflect the situation found in plant cells (tobacco protoplasts). The intriguing role of NLS and NES accessibility for the intracellular distribution of HsfA2 is underlined by the results of heat stress treatments of CHO cells (41°C). Despite the fact that nuclear import and export are not markedly affected, HsfA2 remains completely cytoplasmic at 41°C even in the presence of LMB. The temperature-dependent conformational transition of HsfA2 with shielding of the NLS evidently needs intramolecular interaction between the internal HR-A/B and the C-terminal HR-C regions. It is not observed with the HR oligomerization domain (HR-A/B region) deletion form of HsfA2 or in HsfA2-HsfA1 hetero-oligomers.

scholarly journals Yeast Ran-Binding Protein 1 (Yrb1) Shuttles between the Nucleus and Cytoplasm and Is Exported from the Nucleus via a CRM1(XPO1)-Dependent Pathway

2000 ◽  
Vol 20 (12) ◽  
pp. 4295-4308 ◽  
Author(s):  
Markus Künzler ◽  
Thomas Gerstberger ◽  
Françoise Stutz ◽  
F. Ralf Bischoff ◽  
Ed Hurt
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Binding Protein ◽  
Nuclear Pore ◽  
Leptomycin B ◽  
Import And Export ◽  
Yeast Homolog ◽  
Dependent Pathway ◽  
Cytoplasmic Side

ABSTRACT The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.

scholarly journals Cellular stresses induce the nuclear accumulation of importin α and cause a conventional nuclear import block

2004 ◽  
Vol 165 (5) ◽  
pp. 617-623 ◽  
Author(s):  
Yoichi Miyamoto ◽  
Takuya Saiwaki ◽  
Junichi Yamashita ◽  
Yoshinari Yasuda ◽  
Ippei Kotera ◽  
...  
Keyword(s):  
Heat Shock ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Stress Conditions ◽  
Nuclear Accumulation ◽  
Nuclear Retention ◽  
Heat Shock Cognate 70 ◽  
Importin Α ◽  
Response To Stress ◽  
Cellular Stresses

We report here that importin α accumulates reversibly in the nucleus in response to cellular stresses including UV irradiation, oxidative stress, and heat shock. The nuclear accumulation of importin α appears to be triggered by a collapse in the Ran gradient, resulting in the suppression of the nuclear export of importin α. In addition, nuclear retention and the importin β/Ran-independent import of importin α also facilitate its rapid nuclear accumulation. The findings herein show that the classical nuclear import pathway is down-regulated via the removal of importin α from the cytoplasm in response to stress. Moreover, whereas the nuclear accumulation of heat shock cognate 70 is more sensitive to heat shock than the other stresses, importin α is able to accumulate in the nucleus at all the stress conditions tested. These findings suggest that the stress-induced nuclear accumulation of importin α can be involved in a common physiological response to various stress conditions.

scholarly journals An Investigation of the Mechanism for a Novel Influenza Inhibitor Designed to Target the Import of PB1

2021 ◽  
Author(s):  
Alexander Dorius ◽  
David D. Busath
Keyword(s):  
Complex Formation ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Nuclear Factor Kappa B ◽  
Nuclear Factor ◽  
Direct Inhibition ◽  
Nuclear Retention ◽  
Nuclear Factor Kappa

AbstractA novel inhibitor of PB1 import, referred to here as GM30, previously displayed nuclear retention of viral nucleoprotein (NP), which spurred the question of whether the compound directly or indirectly blocked viral ribonucleoprotein (vRNP) complex export. vRNPs are exported from the nucleus via exportin 1 (XPO1). Using verdinexor (VNXR), a direct inhibitor of vRNP export that binds to XPO1 and also blocks nuclear factor kappa B (NF-κB) export, we found that GM30 does not block nuclear export of NF-κB. GM30 likewise demonstrated high nuclear retention of NP but not as much as the direct inhibition of nuclear export by VNXR. When the compound was added hours after infection, the compound lost its ability to block nuclear export but VNXR retained its ability to block nuclear export. GM30 is therefore likely an indirect inhibitor of nuclear export because it disrupts the vRNP complex formation and impedes export from the nucleus by reducing nuclear import of PB1.

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