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.

Arabidopsis At2g40730 encodes a cytoplasmic protein involved in nuclear tRNA export

Botany ◽  
2011 ◽  
Vol 89 (3) ◽  
pp. 175-190 ◽  
Author(s):  
Aaron D. Johnstone ◽  
Robert T. Mullen ◽  
Dev Mangroo
Keyword(s):  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Pore ◽  
Cytoplasmic Protein ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Gtpase Ran ◽  
Cytoplasmic Side

Nuclear tRNA export plays an essential role in several key cellular processes, such as regulation of protein synthesis, cell cycle progression, response to nutrient availability and DNA damage, and development. While the overall mechanism of nuclear tRNA export is, in general, poorly understood, the details of specific steps are emerging from studies conducted in different organisms aimed at identifying and characterizing components involved in the process. Here, we report that Arabidopsis thaliana (L.) Heynh At2g40730 encodes CTEXP, a cytoplasmic protein component of the nuclear tRNA export process. CTEXP bound tRNA directly and saturably, and like the nuclear tRNA export receptor PAUSED, overexpression of CTEXP restored export of a nuclear export-defective lysine amber suppressor tRNA in tobacco cells. CTEXP was also found to associate with nucleoporins of the nuclear pore complex (NPC), PAUSED, and the GTPase Ran in vivo. CTEXP interacted directly with PAUSED in vitro and RanGTP, but not RanGDP. Furthermore, a portion of CTEXP appeared to associate with the NPC. Taken together, the data suggest that CTEXP assists with unloading of tRNAs from PAUSED at the cytoplasmic side of the NPC in plant cells.

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 Nuclear Import of IκBα Is Accomplished by a Ran-Independent Transport Pathway

2000 ◽  
Vol 20 (5) ◽  
pp. 1571-1582 ◽  
Author(s):  
Shrikesh Sachdev ◽  
Sriparna Bagchi ◽  
Donna D. Zhang ◽  
Angela C. Mings ◽  
Mark Hannink
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Nuclear Pore ◽  
Transport Pathway ◽  
Repeat Domain ◽  
Nuclear Export Receptor

ABSTRACT The inhibitor of kappa B alpha (IκBα) protein is able to shuttle between the cytoplasm and the nucleus. We have utilized a combination of in vivo and in vitro approaches to provide mechanistic insight into nucleocytoplasmic shuttling by IκBα. IκBα contains multiple functional domains that contribute to shuttling of IκBα between the cytoplasm and the nucleus. Nuclear import of IκBα is mediated by the central ankyrin repeat domain. Similar to previously described nuclear import pathways, nuclear import of IκBα is temperature and ATP dependent and is blocked by a dominant-negative mutant of importin β. However, in contrast to classical nuclear import pathways, nuclear import of IκBα is independent of soluble cytosolic factors and is not blocked by the dominant-negative RanQ69L protein. Nuclear export of IκBα is mediated by an N-terminal nuclear export sequence. Nuclear export of IκBα requires the CRM1 nuclear export receptor and is blocked by the dominant-negative RanQ69L protein. Our results are consistent with a model in which nuclear import of IκBα is mediated through direct interactions with components of the nuclear pore complex, while nuclear export of IκBα is mediated via a CRM1-dependent pathway.

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 Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

2000 ◽  
Vol 11 (2) ◽  
pp. 703-719 ◽  
Author(s):  
Susanne M. Steggerda ◽  
Ben E. Black ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Living Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Permeabilized Cells ◽  
Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

scholarly journals A Role for RanBP1 in the Release of CRM1 from the Nuclear Pore Complex in a Terminal Step of Nuclear Export

1999 ◽  
Vol 145 (4) ◽  
pp. 645-657 ◽  
Author(s):  
Ralph H. Kehlenbach ◽  
Achim Dickmanns ◽  
Angelika Kehlenbach ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Activated T Cells ◽  
Permeabilized Cells ◽  
Binding Domains ◽  
Biochemical Fractionation ◽  
Nuclear Export Receptor ◽  
Gtpase Ran

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

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 In vivo BiFC analysis of Y14 and NXF1 mRNA export complexes: preferential localization within and around SC35 domains

2006 ◽  
Vol 172 (3) ◽  
pp. 373-381 ◽  
Author(s):  
Ute Schmidt ◽  
Karsten Richter ◽  
Axel Bernhard Berger ◽  
Peter Lichter
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Specific Binding ◽  
Yellow Fluorescent Protein ◽  
Bimolecular Fluorescence Complementation ◽  
Nuclear Speckles ◽  
Mcf7 Cells ◽  
Permeabilized Cells ◽  
Preferential Localization

The bimolecular fluorescence complementation (BiFC) assay, which allows the investigation of interacting molecules in vivo, was applied to study complex formation between the splicing factor Y14 and nuclear export factor 1 (NXF1), which evidence indicates are functionally associated with nuclear mRNA. Y14 linked to the COOH terminus of yellow fluorescent protein (YFP; YC-Y14), and NXF1 fused to the NH2 terminus of YFP (YN-NXF1) expressed in MCF7 cells yielded BiFC upon specific binding. Fluorescence accumulated within and around nuclear speckles, suggesting the involvement of speckles in mRNA processing and export. Accordingly, BiFC depended on transcription and full-length NXF1. Coimmunoprecipitation of YC-Y14 with YN-NXF1, NXF1, Y14, and RNA indicated that YC-Y14 and YN-NXF1 functionally associate with RNA. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching revealed that roughly half of the accumulated BiFC complexes were immobile in vivo. This immobile fraction was readily depleted by adenosine triphosphate (ATP) administration in permeabilized cells. These results suggest that a fraction of RNA, which remains in the nucleus for several hours despite its association with splicing and export proteins, accumulates in speckles because of an ATP-dependent mechanism.

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