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 O-GlcNAc modification of nuclear pore complexes accelerates bi-directional transport

2020 ◽  
Author(s):  
Tae Yeon Yoo ◽  
Timothy J Mitchison
Keyword(s):  
Nuclear Import ◽  
Nuclear Transport ◽  
Super Resolution ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nuclear Pore Complexes ◽  
Facilitated Diffusion ◽  
Import And Export ◽  
Pore Complexes

AbstractMacromolecular transport across the nuclear envelope depends on facilitated diffusion through nuclear pore complexes (NPCs). The interior of NPCs contains a permeability barrier made of phenylalanine-glycine (FG) repeat domains that selectively facilitates the permeation of cargoes bound to nuclear transport receptors (NTRs). FG repeats in NPC are a major site of O-linked N-acetylglucosamine (O-GlcNAc) modification, but the functional role of this modification in nucleocytoplasmic transport is unclear. We developed high-throughput assays based on optogenetic probes to quantify the kinetics of nuclear import and export in living human cells. We found that increasing O-GlcNAc modification of the NPC accelerated NTR-facilitated nucleocytoplasmic transport of proteins in both directions, and decreasing modification slowed transport. Super-resolution imaging revealed strong enrichment of O-GlcNAc at the FG-repeat barrier. O-GlcNAc modification also accelerated passive permeation of a small, inert protein through NPCs. We conclude that O-GlcNAc modification accelerates nucleocytoplasmic transport by enhancing the non-specific permeability the FG-repeat barrier, perhaps by steric inhibition of interactions between FG repeats.SummaryNuclear pore complexes mediate nuclear transport and are highly modified with O-linked N-acetylglucosamine (O-GlcNAc) on FG repeat domains. Using a new quantitative live-cell imaging assay, Yoo and Mitchison demonstrate acceleration of nuclear import and export by O-GlcNAc modification.

scholarly journals Nucleocytoplasmic Trafficking of G2/M Regulators in Yeast

2008 ◽  
Vol 19 (9) ◽  
pp. 4006-4018 ◽  
Author(s):  
Mignon A. Keaton ◽  
Lee Szkotnicki ◽  
Aron R. Marquitz ◽  
Jake Harrison ◽  
Trevin R. Zyla ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Cell Cycle Regulators ◽  
Cyclin Dependent Kinase ◽  
Nucleocytoplasmic Trafficking ◽  
Nucleocytoplasmic Shuttling ◽  
Upstream Regulator ◽  
Cellular Compartments ◽  
Cdk Regulation

Nucleocytoplasmic shuttling is prevalent among many cell cycle regulators controlling the G2/M transition. Shuttling of cyclin/cyclin-dependent kinase (CDK) complexes is thought to provide access to substrates stably located in either compartment. Because cyclin/CDK shuttles between cellular compartments, an upstream regulator that is fixed in one compartment could in principle affect the entire cyclin/CDK pool. Alternatively, the regulators themselves may need to shuttle to effectively regulate their moving target. Here, we identify localization motifs in the budding yeast Swe1p (Wee1) and Mih1p (Cdc25) cell cycle regulators. Replacement of endogenous Swe1p or Mih1p with mutants impaired in nuclear import or export revealed that the nuclear pools of Swe1p and Mih1p were more effective in CDK regulation than were the cytoplasmic pools. Nevertheless, shuttling of cyclin/CDK complexes was sufficiently rapid to coordinate nuclear and cytoplasmic events even when Swe1p or Mih1p were restricted to one compartment. Additionally, we found that Swe1p nuclear export was important for its degradation. Because Swe1p degradation is regulated by cytoskeletal stress, shuttling of Swe1p between nucleus and cytoplasm serves to couple cytoplasmic stress to nuclear cyclin/CDK inhibition.

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 The Ebola Virus Interferon Antagonist VP24 Undergoes Active Nucleocytoplasmic Trafficking

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1650
Author(s):  
Angela R. Harrison ◽  
Cassandra T. David ◽  
Stephen M. Rawlinson ◽  
Gregory W. Moseley
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Ebola Virus ◽  
Molecular Mapping ◽  
Nucleocytoplasmic Transport ◽  
Cytoplasmic Localization ◽  
Nucleocytoplasmic Trafficking ◽  
Nuclear Trafficking ◽  
C Terminus ◽  
Import Receptors

Viral interferon (IFN) antagonist proteins mediate evasion of IFN-mediated innate immunity and are often multifunctional, with distinct roles in viral replication. The Ebola virus IFN antagonist VP24 mediates nucleocapsid assembly, and inhibits IFN-activated signaling by preventing nuclear import of STAT1 via competitive binding to nuclear import receptors (karyopherins). Proteins of many viruses, including viruses with cytoplasmic replication cycles, interact with nuclear trafficking machinery to undergo nucleocytoplasmic transport, with key roles in pathogenesis; however, despite established karyopherin interaction, potential nuclear trafficking of VP24 has not been investigated. We find that inhibition of nuclear export pathways or overexpression of VP24-binding karyopherin results in nuclear localization of VP24. Molecular mapping indicates that cytoplasmic localization of VP24 depends on a CRM1-dependent nuclear export sequence at the VP24 C-terminus. Nuclear export is not required for STAT1 antagonism, consistent with competitive karyopherin binding being the principal antagonistic mechanism, while export mediates return of nuclear VP24 to the cytoplasm where replication/nucleocapsid assembly occurs.

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 C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Joni Vanneste ◽  
Thomas Vercruysse ◽  
Steven Boeynaems ◽  
Adria Sicart ◽  
Philip Van Damme ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Hela Cells ◽  
Nuclear Import ◽  
Nucleocytoplasmic Transport ◽  
Repeat Expansions ◽  
Import And Export ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

Abstract Repeat expansions in the C9orf72 gene cause amyotrophic lateral sclerosis and frontotemporal dementia characterized by dipeptide-repeat protein (DPR) inclusions. The toxicity associated with two of these DPRs, poly-GR and poly-PR, has been associated with nucleocytoplasmic transport. To investigate the causal role of poly-GR or poly-PR on active nucleocytoplasmic transport, we measured nuclear import and export in poly-GR or poly-PR expressing Hela cells, neuronal-like SH-SY5Y cells and iPSC-derived motor neurons. Our data strongly indicate that poly-GR and poly-PR do not directly impede active nucleocytoplasmic transport.

scholarly journals The Ran GTPase Gradient Protects the Nucleolus from Aging-Associated Morphological Changes

2020 ◽  
Author(s):  
Bartlomiej Remlein ◽  
Bryce M. Paschal
Keyword(s):  
Molecular Mass ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Cultured Cells ◽  
Nucleocytoplasmic Transport ◽  
High Molecular Mass ◽  
Regulatory Function ◽  
Chemical Induction ◽  
Ran Gtpase ◽  
Passage Number

ABSTRACTIn the context of its regulatory function for nucleocytoplasmic transport, the Ran GTPase undergoes cycles of nuclear import, GTP loading, nuclear export, and GTP hydrolysis. These reactions give rise to a nuclear:cytoplasmic (N:C) Ran gradient. In Hutchinson-Gilford Progeria Syndrome, disruption of the Ran gradient suppresses nuclear import of high molecular mass complexes by reducing the nuclear concentration of Ran. Here, we report that cells undergoing senescence, as a consequence of passage number, chemical induction, and altered nuclear lamina structure, all display a Ran gradient disruption quantitatively similar to that observed in Progeria patient cells. We found that the Ran gradient is critical for maintenance of nucleolar structure, as its disruption increases the size and decreases the average number of nucleoli per cell. Nucleolar number and size are biomarkers of longevity in diverse organisms, thus the nuclear level of Ran may be important for the nucleolar morphology in aging. The contribution of the Ran gradient includes regulating import of nucleolin and nucleophosmin, nucleolar proteins that assemble into high molecular mass complexes. The steepness of the Ran gradient is highly dependent on nuclear heterochromatin, which is reduced by passage number and chemical induction of senescence in cultured cells, and is known to decline during normal aging. Our data suggest that the Ran gradient senses nuclear heterochromatin, and through its function as a transport regulator, helps maintain the protein composition and structure of the nucleolus.

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 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.

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