Nuclear pore localization and nucleocytoplasmic transport of eIF-5A: evidence for direct interaction with the export receptor CRM1

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine. The exact in vivo function of eIF-5A, however, is to date unknown. The finding that eIF-5A is an essential cofactor of the human immunodeficiency virus type 1 (HIV-1) Rev RNA transport factor suggested that eIF-5A is part of a specific nuclear export pathway. In this study we used indirect immunofluorescence and immunogold electron microscopy to demonstrate that eIF-5A accumulates at nuclear pore-associated intranuclear filaments in mammalian cells and Xenopus oocytes. We are able to show that eIF-5A interacts with the general nuclear export receptor, CRM1. Furthermore, microinjection studies in somatic cells revealed that eIF-5A is transported from the nucleus to the cytoplasm, and that this nuclear export is blocked by leptomycin B. Our data demonstrate that eIF-5A is a nucleocytoplasmic shuttle protein.

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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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The Yeast Nucleoporin Nup53p Specifically Interacts with Nic96p and Is Directly Involved in Nuclear Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3885 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3885-3896 ◽

Cited By ~ 22

Author(s):

Birthe Fahrenkrog ◽

Wolfgang Hübner ◽

Anna Mandinova ◽

Nelly Panté ◽

Walter Keller ◽

...

Keyword(s):

Nuclear Export ◽

Protein Import ◽

Nuclear Protein ◽

Genomic Library ◽

Nuclear Periphery ◽

Nucleocytoplasmic Transport ◽

Immunogold Electron Microscopy ◽

Nuclear Pore ◽

Nuclear Protein Import

The bidirectional nucleocytoplasmic transport of macromolecules is mediated by the nuclear pore complex (NPC) which, in yeast, is composed of ∼30 different proteins (nucleoporins). Pre-embedding immunogold-electron microscopy revealed that Nic96p, an essential yeast nucleoporin, is located about the cytoplasmic and the nuclear periphery of the central channel, and near or at the distal ring of the yeast NPC. Genetic approaches further implicated Nic96p in nuclear protein import. To more specifically explore the potential role of Nic96p in nuclear protein import, we performed a two-hybrid screen withNIC96 as the bait against a yeast genomic library to identify transport factors and/or nucleoporins involved in nuclear protein import interacting with Nic96p. By doing so, we identified the yeast nucleoporin Nup53p, which also exhibits multiple locations within the yeast NPC and colocalizes with Nic96p in all its locations. Whereas Nup53p is directly involved in NLS-mediated protein import by its interaction with the yeast nuclear import receptor Kap95p, it appears not to participate in NES-dependent nuclear export.

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Formation of a Tap/NXF1 Homotypic Complex Is Mediated through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins

Molecular Biology of the Cell ◽

10.1091/mbc.e07-03-0255 ◽

2008 ◽

Vol 19 (1) ◽

pp. 327-338 ◽

Cited By ~ 11

Author(s):

Leah H. Matzat ◽

Stephen Berberoglu ◽

Lyne Lévesque

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Direct Interaction ◽

Nuclear Pore ◽

Amino Terminal ◽

Multimeric Complex ◽

Rna Export ◽

Amino Terminal Domain

Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.

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

Molecular and Cellular Biology ◽

10.1128/mcb.00468-09 ◽

2009 ◽

Vol 29 (19) ◽

pp. 5327-5338 ◽

Cited By ~ 48

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.

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Nup180, a novel nuclear pore complex protein localizing to the cytoplasmic ring and associated fibrils.

The Journal of Cell Biology ◽

10.1083/jcb.123.6.1345 ◽

1993 ◽

Vol 123 (6) ◽

pp. 1345-1354 ◽

Cited By ~ 27

Author(s):

N Wilken ◽

U Kossner ◽

J L Senécal ◽

U Scheer ◽

M C Dabauvalle

Keyword(s):

Nuclear Pore Complex ◽

Mammalian Cells ◽

Protein Transport ◽

Nucleocytoplasmic Transport ◽

Transport Processes ◽

Nuclear Pore ◽

Nuclear Surface ◽

Apparent Lack

Using an autoimmune serum from a patient with overlap connective tissue disease we have identified by biochemical and immunocytochemical approaches an evolutionarily conserved nuclear pore complex (NPC) protein with an estimated molecular mass of 180 kD and an isoelectric point of approximately 6.2 which we have designated as nup180. Extraction of isolated nuclear envelopes with 2 M urea and chromatography of the solubilized proteins on WGA-Sepharose demonstrated that nup180 is a peripheral membrane protein and does not react with WGA. Affinity-purified antibodies yielded a punctate immunofluorescent pattern of the nuclear surface of mammalian cells and stained brightly the nuclear envelope of cryosectioned Xenopus oocytes. Nuclei reconstituted in vitro in Xenopus egg extract were also stained in the characteristic punctate fashion. Immunogold EM localized nup180 exclusively to the cytoplasmic ring of NPCs and short fibers emanating therefrom into the cytoplasm. Antibodies to nup180 did not inhibit nuclear protein transport in vivo nor in vitro. Despite the apparent lack of involvement in NPC assembly or nucleocytoplasmic transport processes, the conservation of nup180 across species and its exclusive association with the NPC cytoplasmic ring suggests an important, though currently undefined function for this novel NPC protein.

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Selective nuclear export of mRNAs is promoted by DRBD18 in Trypanosoma brucei

10.1101/2021.06.14.448361 ◽

2021 ◽

Author(s):

Amartya Mishra ◽

Jan Naseer Kaur ◽

Daniel I. McSkimming ◽

Eva Hegedusova ◽

Ashutosh P. Dubey ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Nuclear Export ◽

Rna Binding ◽

Rna Binding Proteins ◽

Direct Interaction ◽

Nuclear Pore ◽

Control Gene Expression ◽

Export Receptors ◽

Posttranscriptional Level

Kinetoplastids, including Trypanosoma brucei, control gene expression primarily at the posttranscriptional level. Nuclear mRNA export is an important, but understudied, step in this process. The general heterodimeric export factors, Mex67/Mtr2, function in the export of mRNAs and tRNAs in T. brucei, but RNA binding proteins (RBPs) that regulate export processes by controlling the dynamics of Mex67/Mtr2 ribonucleoprotein formation or transport have not been identified. Here, we report that DRBD18, an essential and abundant T. brucei RBP, associates with Mex67/Mtr2 in vivo, likely through its direct interaction with Mtr2. DRBD18 downregulation results in partial accumulation of poly(A)+ mRNA in the nucleus, but has no effect on localization of intron-containing or mature tRNAs. Comprehensive analysis of transcriptomes from whole cell and cytosol in DRBD18 knockdown parasites demonstrates that depletion of DRBD18 leads to impairment of nuclear export of a subset of mRNAs. CLIP experiments reveal association of DRBD18 with several of these mRNAs. Moreover, DRBD18 knockdown leads to a partial accumulation of the Mex67/Mtr2 export receptors in the nucleus. Taken together, the current study supports a model in which DRBD18 regulates the selective nuclear export of mRNAs by promoting the mobilization of export competent mRNPs to the cytosol through the nuclear pore complex.

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Localization matters: nuclear-trapped Survivin sensitizes glioblastoma cells to temozolomide by elevating cellular senescence and impairing homologous recombination

Cellular and Molecular Life Sciences ◽

10.1007/s00018-021-03864-0 ◽

2021 ◽

Author(s):

Thomas R. Reich ◽

Christian Schwarzenbach ◽

Juliana Brandstetter Vilar ◽

Sven Unger ◽

Fabian Mühlhäusler ◽

...

Keyword(s):

Homologous Recombination ◽

Nuclear Export ◽

Cell Model ◽

Chromosome Instability ◽

Direct Interaction ◽

High Grade Glioma ◽

Strand Break ◽

Γh2ax Foci

AbstractTo clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

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SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex

The Journal of Cell Biology ◽

10.1083/jcb.140.3.499 ◽

1998 ◽

Vol 140 (3) ◽

pp. 499-509 ◽

Cited By ~ 316

Author(s):

Michael J. Matunis ◽

Jian Wu ◽

Günter Blobel

Keyword(s):

Nuclear Localization ◽

Nuclear Localization Signal ◽

Nuclear Export ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Gtpase Activating Protein ◽

Terminal Domain ◽

Localization Signal

RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm.

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Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

Molecular Biology of the Cell ◽

10.1091/mbc.e15-08-0601 ◽

2016 ◽

Vol 27 (1) ◽

pp. 167-176 ◽

Cited By ~ 6

Author(s):

Ogheneochukome Lolodi ◽

Hiroya Yamazaki ◽

Shotaro Otsuka ◽

Masahiro Kumeta ◽

Shige H. Yoshimura

Keyword(s):

Steady State ◽

Molecular Transport ◽

Nucleocytoplasmic Transport ◽

Live Cells ◽

Release Mechanism ◽

Nuclear Pore ◽

Catch And Release ◽

Import And Export ◽

Nucleocytoplasmic Distribution

Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP.

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Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽

10.7554/elife.10785 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 44

Author(s):

Andrei Vovk ◽

Chad Gu ◽

Michael G Opferman ◽

Larisa E Kapinos ◽

Roderick YH Lim ◽

...

Keyword(s):

Spatial Organization ◽

Intrinsically Disordered Proteins ◽

Nucleocytoplasmic Transport ◽

Transport Proteins ◽

Disordered Proteins ◽

Biophysical Model ◽

Nuclear Pore ◽

Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

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