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

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.145.4.645 ◽

1999 ◽

Vol 145 (4) ◽

pp. 645-657 ◽

Cited By ~ 158

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.

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The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

Molecular Biology of the Cell ◽

10.1091/mbc.e09-12-1041 ◽

2010 ◽

Vol 21 (11) ◽

pp. 1885-1896 ◽

Cited By ~ 49

Author(s):

Masahiro Oka ◽

Munehiro Asally ◽

Yoshinari Yasuda ◽

Yutaka Ogawa ◽

Taro Tachibana ◽

...

Keyword(s):

Nuclear Export ◽

Nuclear Protein ◽

Mutational Analysis ◽

Specific Inhibitor ◽

Protein Export ◽

Dependent Manner ◽

Repeat Region ◽

Leptomycin B ◽

Dependent Protein ◽

Nuclear Export Receptor

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1. Mutational analysis demonstrated that Nup98 physically and functionally interacts with Crm1 in a RanGTP-dependent manner through its N-terminal phenylalanine-glycine (FG) repeat region. Moreover, the activity of the Nup98-Crm1 complex was modulated by RanBP3, a known cofactor for Crm1-mediated nuclear export. Finally, cytoplasmic microinjection of anti-Nup98 inhibited the Crm1-dependent nuclear export of proteins, concomitant with the accumulation of anti-Nup98 in the nucleus. These results clearly demonstrate that Nup98 functions as a novel shuttling cofactor for Crm1-mediated nuclear export in conjunction with RanBP3.

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CRM1 promotes capsid disassembly and nuclear envelope translocation of adenovirus independently of its export function

Journal of Virology ◽

10.1128/jvi.01273-21 ◽

2021 ◽

Author(s):

Floriane Lagadec ◽

Irene Carlon-Andres ◽

Jessica Ragues ◽

Sarah Port ◽

Harald Wodrich ◽

...

Keyword(s):

Nuclear Export ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Microtubule Organizing Center ◽

Leptomycin B ◽

Pore Complexes ◽

Nuclear Export Receptor ◽

Mitotic Cells ◽

Capsid Disassembly

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly, both in interphase and in mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export-competent but deficient in viral capsid disassembly, both in interphase and in mitotic cells.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.12.4295-4308.2000 ◽

2000 ◽

Vol 20 (12) ◽

pp. 4295-4308 ◽

Cited By ~ 44

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.

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The deubiquitylase Ubp15 couples transcription to mRNA export

eLife ◽

10.7554/elife.61264 ◽

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.

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Porcine Reproductive and Respiratory Syndrome Virus Nonstructural Protein 1 Beta Interacts with Nucleoporin 62 To Promote Viral Replication and Immune Evasion

Journal of Virology ◽

10.1128/jvi.00469-19 ◽

2019 ◽

Vol 93 (14) ◽

Cited By ~ 4

Author(s):

Hanzhong Ke ◽

Mingyuan Han ◽

Jineui Kim ◽

Kurt E. Gustin ◽

Dongwan Yoo

Keyword(s):

Immune Evasion ◽

Nuclear Pore Complex ◽

Nuclear Export ◽

Protein Production ◽

Nonstructural Protein ◽

Respiratory Syndrome Virus ◽

Nuclear Pore ◽

Wild Type ◽

Antiviral Protein ◽

Nonstructural Protein 1

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) blocks host mRNA nuclear export to the cytoplasm, and nonstructural protein 1 beta (nsp1β) of PRRSV has been identified as the protein that disintegrates the nuclear pore complex. In the present study, the molecular basis for the inhibition of host mRNA nuclear export was investigated. Nucleoporin 62 (Nup62) was found to bind to nsp1β, and the region representing the C-terminal residues 328 to 522 of Nup62 was determined to be the binding domain for nsp1β. The nsp1β L126A mutant in the SAP domain did not bind to Nup62, and in L126A-expressing cells, host mRNA nuclear export occurred normally. The vL126A mutant PRRSV generated by reverse genetics replicated at a lower rate, and the titer was lower than for wild-type virus. In nsp1β-overexpressing cells or small interfering RNA (siRNA)-mediated Nup62 knockdown cells, viral protein synthesis increased. Notably, the production of type I interferons (IFN-α/β), IFN-stimulated genes (PKR, OAS, Mx1, and ISG15 genes), IFN-induced proteins with tetratricopeptide repeats (IFITs) 1 and 2, and IFN regulatory factor 3 decreased in these cells. As a consequence, the growth of vL126A mutant PRRSV was rescued to the level of wild-type PRRSV. These findings are attributed to nuclear pore complex (NPC) disintegration by nsp1β, resulting in increased viral protein production and decreased host protein production, including antiviral proteins in the cytoplasm. Our study reveals a new strategy of PRRSV for immune evasion and enhanced replication during infection.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS and is known to effectively suppress host innate immunity. The PRRSV nsp1β protein blocks host mRNA nuclear export, which has been shown to be one of the viral mechanisms for inhibition of antiviral protein production. nsp1β binds to the cellular protein nucleoporin 62 (Nup62), and as a consequence, the nuclear pore complex (NPC) is disintegrated and the nucleocytoplasmic trafficking of host mRNAs and host proteins is blocked. We show the dual benefits of Nup62 and nsp1β binding for PRRSV replication: the inhibition of host antiviral protein expression and the exclusive use of host translation machinery by the virus. Our study unveils a novel strategy of PRRSV for immune evasion and enhanced replication during infection.

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The Deubiquitylase Ubp15 Couples Transcription to mRNA Export

10.1101/2020.08.20.258822 ◽

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.

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Role of the Ndc1 interaction network in yeast nuclear pore complex assembly and maintenance

The Journal of Cell Biology ◽

10.1083/jcb.200810030 ◽

2009 ◽

Vol 185 (3) ◽

pp. 475-491 ◽

Cited By ~ 97

Author(s):

Evgeny Onischenko ◽

Leslie H. Stanton ◽

Alexis S. Madrid ◽

Thomas Kieselbach ◽

Karsten Weis

Keyword(s):

Nuclear Pore Complex ◽

Structural Integrity ◽

Fluorescent Protein ◽

Interaction Network ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Binding Partners ◽

Interaction Partners ◽

Redundant Functions

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

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The single transmembrane segment of gp210 is sufficient for sorting to the pore membrane domain of the nuclear envelope.

The Journal of Cell Biology ◽

10.1083/jcb.119.6.1441 ◽

1992 ◽

Vol 119 (6) ◽

pp. 1441-1449 ◽

Cited By ~ 75

Author(s):

R W Wozniak ◽

G Blobel

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Immunofluorescence Microscopy ◽

Nuclear Pore ◽

Transmembrane Helices ◽

Wild Type ◽

Transmembrane Segment ◽

Membrane Domain ◽

3T3 Cells ◽

Type Mutant

The glycoprotein gp210 is located in the "pore membrane," a specialized domain of the nuclear envelope to which the nuclear pore complex (NPC) is anchored. gp210 contains a large cisternal domain, a single transmembrane segment (TM), and a COOH-terminal, 58-amino acid residue cytoplasmic tail (CT) (Wozniak, R. W., E. Bartnik, and G. Blobel. 1989. J. Cell Biol. 108:2083-2092; Greber, U. F., A. Senior, and L. Gerace. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:1495-1502). To locate determinants for sorting of gp210 to the pore membrane, we constructed various cDNAs coding for wild-type, mutant, and chimeric gp210, and monitored localization of the expressed protein in 3T3 cells by immunofluorescence microscopy using appropriate antibodies. The large cisternal domain of gp210 (95% of its mass) did not reveal any sorting determinants. Surprisingly, the TM of gp210 is sufficient for sorting to the pore membrane. The CT also contains a sorting determinant, but it is weaker than that of the TM. We propose specific lateral association of the transmembrane helices of two proteins to yield either a gp210 homodimer or a heterodimer of gp210 and another protein. The cytoplasmically oriented tails of these dimers may bind cooperatively to the adjacent NPCs. In addition, we demonstrate that gp210 co-localizes with cytoplasmically dispersed nucleoporins, suggesting a cytoplasmic association of these components.

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

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1571-1582.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1571-1582 ◽

Cited By ~ 49

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.

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