A nuclear export signal in hnRNP A1: A signal-mediated, temperature-dependent nuclear protein export pathway

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.

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A high-throughput screening system targeting the nuclear export pathway via the third nuclear export signal of influenza A virus nucleoprotein

Virus Research ◽

10.1016/j.virusres.2016.02.007 ◽

2016 ◽

Vol 217 ◽

pp. 23-31 ◽

Cited By ~ 6

Author(s):

Michinori Kakisaka ◽

Takafumi Mano ◽

Yoko Aida

Keyword(s):

Influenza A Virus ◽

High Throughput ◽

Nuclear Export ◽

High Throughput Screening ◽

Influenza A ◽

Nuclear Export Signal ◽

Screening System ◽

The Third ◽

Export Pathway ◽

Export Signal

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A nuclear export signal is essential for the cytosolic localization of the Ran binding protein, RanBP1.

The Journal of Cell Biology ◽

10.1083/jcb.134.5.1157 ◽

1996 ◽

Vol 134 (5) ◽

pp. 1157-1168 ◽

Cited By ~ 100

Author(s):

S A Richards ◽

K M Lounsbury ◽

K L Carey ◽

I G Macara

Keyword(s):

Nuclear Export ◽

Protein Import ◽

Nuclear Protein ◽

Fluorescent Protein ◽

Binding Protein ◽

Nuclear Export Signal ◽

Cyclin B1 ◽

Nuclear Accumulation ◽

Protein Fusion ◽

Export Signal

RanBP1 is a Ran/TC4 binding protein that can promote the interaction between Ran and beta-importin /beta-karyopherin, a component of the docking complex for nuclear protein cargo. This interaction occurs through a Ran binding domain (RBD). Here we show that RanBP1 is primarily cytoplasmic, but the isolated RBD accumulates in the nucleus. A region COOH-terminal to the RBD is responsible for this cytoplasmic localization. This domain acts heterologously, localizing a nuclear cyclin B1 mutant to the cytoplasm. The domain contains a nuclear export signal that is necessary but not sufficient for the nuclear export of a functional RBD In transiently transfected cells, epitope-tagged RanBP1 promotes dexamethasone-dependent nuclear accumulation of a glucocorticoid receptor-green fluorescent protein fusion, but the isolated RBD potently inhibits this accumulation. The cytosolic location of RanBP1 may therefore be important for nuclear protein import. RanBP1 may provide a key link between the nuclear import and export pathways.

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Adenovirus Late Gene Expression Does Not Require a Rev-Like Nuclear RNA Export Pathway

Journal of Virology ◽

10.1128/jvi.74.14.6684-6688.2000 ◽

2000 ◽

Vol 74 (14) ◽

pp. 6684-6688 ◽

Cited By ~ 17

Author(s):

Claudia Rabino ◽

Anders Aspegren ◽

Kara Corbin-Lickfett ◽

Eileen Bridge

Keyword(s):

Nuclear Export ◽

Nuclear Export Signal ◽

Late Gene Expression ◽

Export Pathway ◽

Early Proteins ◽

Immunodeficiency Virus ◽

Rna Export ◽

Nuclear Rna Export ◽

Export Signal

ABSTRACT Adenovirus late mRNA export is facilitated by viral early proteins of 55 and 34 kDa. The 34-kDa protein contains a leucine-rich nuclear export signal (NES) similar to that of the human immunodeficiency virus Rev protein. It was proposed that the 34-kDa protein might facilitate the export of adenovirus late mRNA through a Rev-like NES-mediated export pathway. We have tested the role of NES-mediated RNA export during adenovirus infection, and we find that it is not essential for the expression of adenovirus late genes.

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The HIV-1 Rev Activation Domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs

Cell ◽

10.1016/0092-8674(95)90436-0 ◽

1995 ◽

Vol 82 (3) ◽

pp. 475-483 ◽

Cited By ~ 740

Author(s):

Utz Fischer ◽

Jochen Huber ◽

Wilbert C Boelens ◽

Lain W Mattajt ◽

Reinhard Lührmann

Keyword(s):

Nuclear Export ◽

Nuclear Export Signal ◽

Activation Domain ◽

Export Pathway ◽

Export Signal ◽

Hiv 1

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Characterization of the Interaction between the Interferon-Induced Protein P56 and the Int6 Protein Encoded by a Locus of Insertion of the Mouse Mammary Tumor Virus

Journal of Virology ◽

10.1128/jvi.74.4.1892-1899.2000 ◽

2000 ◽

Vol 74 (4) ◽

pp. 1892-1899 ◽

Cited By ~ 45

Author(s):

Jinjiao Guo ◽

Ganes C. Sen

Keyword(s):

Nuclear Export ◽

Nuclear Protein ◽

Nuclear Export Signal ◽

Structural Basis ◽

Specific Domain ◽

Interacting Protein ◽

Amino Terminal ◽

Tumor Virus ◽

Export Signal ◽

Two Hybrid

ABSTRACT For determining cellular functions of the interferon-inducible human cytoplasmic protein P56, we undertook a Saccharomyces cerevisiae two-hybrid screen that identified Int6 as a P56-interacting protein. That the interaction also occurs in human cells was confirmed by coimmunoprecipitation and the observed cytoplasmic displacement of nuclear Int6 upon coexpression of P56. Because Int6 has been claimed to be both a cytoplasmic and a nuclear protein, we investigated the structural basis of this discrepancy. By mutational analyses, we showed that the Int6 protein contains a bipartite nuclear localization signal and a nuclear export signal at the far end of the amino terminus. The 20 amino-terminal residues of Int6, when they were attached to a different nuclear protein, were sufficient to translocate that protein to the cytoplasm. Within this region, replacement of any of the three leucine residues with alanine destroyed the function of the export signal. The specific domain of P56 that is required for its interaction with Int6 was mapped using the yeast two-hybrid assay and a mammalian coimmunoprecipitation assay. Both assays demonstrated that the C-terminal region of P56 containing three specific tetratricopeptide motifs is required for this interaction. In contrast, removal of an internal domain of P56 enhanced the interaction, as quantified by the two-hybrid assay.

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Nup358/RanBP2 Attaches to the Nuclear Pore Complex via Association with Nup88 and Nup214/CAN and Plays a Supporting Role in CRM1-Mediated Nuclear Protein Export

Molecular and Cellular Biology ◽

10.1128/mcb.24.6.2373-2384.2004 ◽

2004 ◽

Vol 24 (6) ◽

pp. 2373-2384 ◽

Cited By ~ 112

Author(s):

Rafael Bernad ◽

Hella van der Velde ◽

Maarten Fornerod ◽

Helen Pickersgill

Keyword(s):

Nuclear Export ◽

Nuclear Protein ◽

Nuclear Export Signal ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Strong Reduction ◽

Cytoplasmic Face ◽

Pore Complexes ◽

Export Signal

ABSTRACT Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus.

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Nuclear export of ubiquitinated proteins via the UBIN-POST system

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1711017115 ◽

2018 ◽

Vol 115 (18) ◽

pp. E4199-E4208 ◽

Cited By ~ 5

Author(s):

Shoshiro Hirayama ◽

Munechika Sugihara ◽

Daisuke Morito ◽

Shun-ichiro Iemura ◽

Tohru Natsume ◽

...

Keyword(s):

Nuclear Export ◽

Nuclear Protein ◽

Nuclear Export Signal ◽

Proteasome Inhibition ◽

Protein Homeostasis ◽

Dependent Manner ◽

Nuclear Pores ◽

Ubiquitinated Proteins ◽

Uba Domain ◽

Export Signal

Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.

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