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 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.

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.

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 A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex

2021 ◽  
Vol 134 (6) ◽  
Author(s):  
Mohamed Hamed ◽  
Birgit Caspar ◽  
Sarah A. Port ◽  
Ralph H. Kehlenbach
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Nuclear Pore ◽  
Wild Type ◽  
Leptomycin B ◽  
Nuclear Export Sequence ◽  
Binding Partners ◽  
Dependent Protein ◽  
Nuclear Export Receptor ◽  
Cytoplasmic Side

ABSTRACT Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

scholarly journals Assembly of an Export-Competent mRNP Is Needed for Efficient Release of the 3′-End Processing Complex after Polyadenylation

2009 ◽  
Vol 29 (19) ◽  
pp. 5327-5338 ◽  
Author(s):  
Xiangping Qu ◽  
Søren Lykke-Andersen ◽  
Tommy Nasser ◽  
Cyril Saguez ◽  
Edouard Bertrand ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Growth Defect ◽  
Nuclear Pore ◽  
Rna Immunoprecipitation ◽  
Cleavage And Polyadenylation ◽  
Severe Growth Defect ◽  
Nuclear Export Receptor ◽  
Polyadenylated Mrna

ABSTRACT Before polyadenylated mRNA is exported from the nucleus, the 3′-end processing complex is removed by a poorly described mechanism. In this study, we asked whether factors involved in mRNP maturation and export are also required for disassembly of the cleavage and polyadenylation complex. An RNA immunoprecipitation assay monitoring the amount of the cleavage factor (CF) IA component Rna15p associated with poly(A)+ RNA reveals defective removal of Rna15p in mutants of the nuclear export receptor Mex67p as well as other factors important for assembly of an export-competent mRNP. In contrast, Rna15p is not retained in mutants of export factors that function primarily on the cytoplasmic side of the nuclear pore. Consistent with a functional interaction between Mex67p and the 3′-end processing complex, a mex67 mutant accumulates unprocessed SSA4 transcripts and exhibits a severe growth defect when this mutation is combined with mutation of Rna15p or another CF IA subunit, Rna14p. RNAs that become processed in a mex67 mutant have longer poly(A) tails both in vivo and in vitro. This influence of Mex67p on 3′-end processing is conserved, as depletion of its human homolog, TAP/NXF1, triggers mRNA hyperadenylation. Our results indicate a function for nuclear mRNP assembly factors in releasing the 3′-end processing complex once polyadenylation is complete.

scholarly journals The deubiquitylase Ubp15 couples transcription to mRNA export

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Fanny Eyboulet ◽  
Célia Jeronimo ◽  
Jacques Côté ◽  
François Robert
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Messenger Rnas ◽  
E3 Ligases ◽  
Nuclear Export Receptor ◽  
Nascent Transcripts

Nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed, and exported. The role of ubiquitylation in this process is increasingly recognized but, while a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here we identified deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts with both RNA polymerase II and the nuclear pore complex, and its deletion reverts the nuclear export defect of E3 ligase Rsp5 mutants. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.

scholarly journals The Deubiquitylase Ubp15 Couples Transcription to mRNA Export

2020 ◽  
Author(s):  
Fanny Eyboulet ◽  
Célia Jeronimo ◽  
Jacques Côté ◽  
François Robert
Keyword(s):  
Rna Polymerase Ii ◽  
Nuclear Pore Complex ◽  
Nuclear Export ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Messenger Rnas ◽  
E3 Ligases ◽  
Nuclear Export Receptor ◽  
Nascent Transcripts

ABSTRACTThe nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed and exported. The role of ubiquitylation in this process is increasingly recognized as the ubiquitylation of many key players have been shown to affect mRNA nuclear export. While a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here, we identified the deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts both with RNA polymerase II and with the nuclear pore complex, and its deletion reverts the nuclear export defect of mutants of the E3 ligase Rsp5. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.

scholarly journals Scyl1 Facilitates Nuclear tRNA Export in Mammalian Cells by Acting at the Nuclear Pore Complex

2010 ◽  
Vol 21 (14) ◽  
pp. 2483-2499 ◽  
Author(s):  
Shawn C. Chafe ◽  
Dev Mangroo
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Trafficking ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Elongation Factor ◽  
Nuclear Pore ◽  
Eukaryotic Elongation Factor ◽  
Quaternary Complex

Scyl1 is an evolutionarily conserved N-terminal protein kinase-like domain protein that plays a role in COP1-mediated retrograde protein trafficking in mammalian cells. Furthermore, loss of Scyl1 function has been shown to result in neurodegenerative disorders in mice. Here, we report that Scyl1 is also a cytoplasmic component of the mammalian nuclear tRNA export machinery. Like exportin-t, overexpression of Scyl1 restored export of a nuclear export-defective serine amber suppressor tRNA mutant in COS-7 cells. Scyl1 binds tRNA saturably, and associates with the nuclear pore complex by interacting, in part, with Nup98. Scyl1 copurifies with the nuclear tRNA export receptors exportin-t and exportin-5, the RanGTPase, and the eukaryotic elongation factor eEF-1A, which transports aminoacyl-tRNAs to the ribosomes. Scyl1 interacts directly with exportin-t and RanGTP but not with eEF-1A or RanGDP in vitro. Moreover, exportin-t containing tRNA, Scyl1, and RanGTP form a quaternary complex in vitro. Biochemical characterization also suggests that the nuclear aminoacylation-dependent pathway is primarily responsible for tRNA export in mammalian cells. These findings together suggest that Scyl1 participates in the nuclear aminoacylation-dependent tRNA export pathway and may unload aminoacyl-tRNAs from the nuclear tRNA export receptor at the cytoplasmic side of the nuclear pore complex and channels them to eEF-1A.

The Formation, Structure, Distribution and Frequency of Nuclear Pores

1971 ◽  
Vol 29 ◽  
pp. 518-519
Author(s):  
G. G. Maul
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Dynamic Structure ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Filter Function ◽  
Etching Technique ◽  
Selective Filter ◽  
Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

scholarly journals Specific Binding of the Karyopherin Kap121p to a Subunit of the Nuclear Pore Complex Containing Nup53p, Nup59p, and Nup170p

1998 ◽  
Vol 143 (7) ◽  
pp. 1813-1830 ◽  
Author(s):  
Marcello Marelli ◽  
John D. Aitchison ◽  
Richard W. Wozniak
Keyword(s):  
Nuclear Pore Complex ◽  
Core Protein ◽  
Specific Binding ◽  
Protein A ◽  
Small Gtpase ◽  
Nuclear Pore ◽  
Binding Assays ◽  
Reporter Protein ◽  
Physiological Relevance

We have identified a specific karyopherin docking complex within the yeast nuclear pore complex (NPC) that contains two novel, structurally related nucleoporins, Nup53p and Nup59p, and the NPC core protein Nup170p. This complex was affinity purified from cells expressing a functional Nup53p–protein A chimera. The localization of Nup53p, Nup59p, and Nup170p within the NPC by immunoelectron microscopy suggests that the Nup53p-containing complex is positioned on both the cytoplasmic and nucleoplasmic faces of the NPC core. In association with the isolated complex, we have also identified the nuclear transport factor Kap121p (Pse1p). Using in vitro binding assays, we showed that each of the nucleoporins interacts with one another. However, the association of Kap121p with the complex is mediated by its interaction with Nup53p. Moreover, Kap121p is the only β-type karyopherin that binds Nup53p suggesting that Nup53p acts as a specific Kap121p docking site. Kap121p can be released from Nup53p by the GTP bound form of the small GTPase Ran. The physiological relevance of the interaction between Nup53p and Kap121p was further underscored by the observation that NUP53 mutations alter the subcellular distribution of Kap121p and the Kap121p- mediated import of a ribosomal L25 reporter protein. Interestingly, Nup53p is specifically phosphorylated during mitosis. This phenomenon is correlated with a transient decrease in perinuclear-associated Kap121p.

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