scholarly journals Nuclear export of ubiquitinated proteins via the UBIN-POST system

2018 ◽  
Vol 115 (18) ◽  
pp. E4199-E4208 ◽  
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.

scholarly journals Reconstitution of nuclear protein export in isolated nuclear envelopes

2002 ◽  
Vol 158 (5) ◽  
pp. 849-854 ◽  
Author(s):  
Jan Peter Siebrasse ◽  
Elias Coutavas ◽  
Reiner Peters
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Xenopus Oocytes ◽  
Nuclear Protein ◽  
Nuclear Export Signal ◽  
Protein Export ◽  
Leptomycin B ◽  
Permeabilized Cells ◽  
Export Signal ◽  
Kinase A

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.

scholarly journals RanGTP-Binding Protein NXT1 Facilitates Nuclear Export of Different Classes of RNA In Vitro

2000 ◽  
Vol 20 (13) ◽  
pp. 4562-4571 ◽  
Author(s):  
Batool Ossareh-Nazari ◽  
Christèle Maison ◽  
Ben E. Black ◽  
Lyne Lévesque ◽  
Bryce M. Paschal ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Vitro Assay ◽  
U1 Snrna ◽  
Rna Export ◽  
Gtpase Ran ◽  
Export Signal

ABSTRACT To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopusextract-dependent manner. U1 snRNA export requires a 5′ monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.

scholarly journals The microtubule plus-end tracking protein Bik1 is required for chromosome congression

2021 ◽  
Author(s):  
Alexander Julner ◽  
Marjan Abbasi ◽  
Victoria Menendez Benito
Keyword(s):  
Cell Cycle ◽  
Nuclear Export ◽  
Cell Cycle Progression ◽  
Nuclear Export Signal ◽  
Microtubule Dynamics ◽  
Dependent Manner ◽  
Chromosome Congression ◽  
A Cell ◽  
Cycle Dependent ◽  
Export Signal

During mitosis, sister chromatids congress on either side of the spindle equator to facilitate the correct partitioning of the genomic material. Chromosome congression requires a finely tuned control of microtubule dynamics by the kinesin motor proteins. In Saccharomyces cerevisiae, the kinesin proteins Cin8, Kip1, and Kip3 have pivotal roles in chromosome congression. It has been hypothesized that additional proteins that modulate microtubule dynamics are also involved. Here, we show that the microtubule plus-end tracking protein Bik1 (the budding yeast ortholog of CLIP-170) is essential for chromosome congression. We find that nuclear Bik1 localizes to the kinetochores in a cell-cycle-dependent manner. Disrupting the nuclear pool of Bik1 with a nuclear export signal (Bik1-NES) leads to a slower cell cycle progression characterized by a delayed metaphase-anaphase transition. Bik1-NES cells have mispositioned kinetochores along the spindle in metaphase. Furthermore, using proximity-dependent methods, we identify Cin8 as an interaction partner of Bik1. Deleting CIN8 reduces the amount of Bik1 at the spindle. In contrast, Cin8 retains its typical bilobed distribution in Bik1-NES and does not localize to the unclustered kinetochores characteristic of Bik1-NES cells. Thus, we propose that Bik1 functions together with Cin8 to regulate kinetochore-microtubule dynamics for correct kinetochore positioning and chromosome congression.

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

1996 ◽  
Vol 134 (5) ◽  
pp. 1157-1168 ◽  
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.

scholarly journals 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

2000 ◽  
Vol 74 (4) ◽  
pp. 1892-1899 ◽  
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.

scholarly journals CRM1/Ran-Mediated Nuclear Export of p27Kip1Involves a Nuclear Export Signal and Links p27 Export and Proteolysis

2003 ◽  
Vol 14 (1) ◽  
pp. 201-213 ◽  
Author(s):  
Michael K. Connor ◽  
Rouslan Kotchetkov ◽  
Sandrine Cariou ◽  
Ansgar Resch ◽  
Rafaella Lupetti ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Cyclin E ◽  
Nuclear Export Signal ◽  
Proteasome Inhibition ◽  
S Phase ◽  
Leptomycin B ◽  
Nuclear Export Sequence ◽  
Export Signal ◽  
Hiv 1

We show that p27 localization is cell cycle regulated and we suggest that active CRM1/RanGTP-mediated nuclear export of p27 may be linked to cytoplasmic p27 proteolysis in early G1. p27 is nuclear in G0 and early G1 and appears transiently in the cytoplasm at the G1/S transition. Association of p27 with the exportin CRM1 was minimal in G0 and increased markedly during G1-to-S phase progression. Proteasome inhibition in mid-G1 did not impair nuclear import of p27, but led to accumulation of p27 in the cytoplasm, suggesting that export precedes degradation for at least part of the cellular p27 pool. p27-CRM1 binding and nuclear export were inhibited by S10A mutation but not by T187A mutation. A putative nuclear export sequence in p27 is identified whose mutation reduced p27-CRM1 interaction, nuclear export, and p27 degradation. Leptomycin B (LMB) did not inhibit p27-CRM1 binding, nor did it prevent p27 export in vitro or in heterokaryon assays. Prebinding of CRM1 to the HIV-1 Rev nuclear export sequence did not inhibit p27-CRM1 interaction, suggesting that p27 binds CRM1 at a non-LMB-sensitive motif. LMB increased total cellular p27 and may do so indirectly, through effects on other p27 regulatory proteins. These data suggest a model in which p27 undergoes active, CRM1-dependent nuclear export and cytoplasmic degradation in early G1. This would permit the incremental activation of cyclin E-Cdk2 leading to cyclin E-Cdk2-mediated T187 phosphorylation and p27 proteolysis in late G1 and S phase.

scholarly journals 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

2004 ◽  
Vol 24 (6) ◽  
pp. 2373-2384 ◽  
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.

MALT1 contains nuclear export signals and regulates cytoplasmic localization of BCL10

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4210-4216 ◽  
Author(s):  
Masao Nakagawa ◽  
Yoshitaka Hosokawa ◽  
Masakatsu Yonezumi ◽  
Koh Izumiyama ◽  
Ritsuro Suzuki ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Specific Inhibitor ◽  
B Cell Lymphoma ◽  
Nuclear Factor Κb ◽  
Dependent Manner ◽  
Nucleocytoplasmic Shuttling ◽  
Leptomycin B ◽  
Apoptosis Inhibitor ◽  
Export Signal

MALT1, BCL10 (B-cell lymphoma 10), and API2 (apoptosis inhibitor 2)-MALT1 are key molecules in mucosa-associated lymphoid tissue (MALT) lymphomagenesis. We previously reported that MALT1 and API2-MALT1 were localized only in cytoplasm, where we suggested that both molecules were likely to be active. In the study presented here, we further examined the localization-determining region by generating various mutants and were able to demonstrate that there were nuclear export signal (NES)-containing domains in the MALT1 C-terminal region. The use of leptomycin B, an NES-specific inhibitor, demonstrated that both MALT1 and API2-MALT1 were predominantly retained in the nuclei, indicating that these molecules were shuttling between nucleus and cytoplasm in an NES-dependent manner. It was also found that MALT1 was involved in the nuclear export of BCL10, which is originally localized in both nucleus and cytoplasm. These results correlate well with the nuclear BCL10 expression pattern in both t(1;14) and t(11;18) MALT lymphomas. The nucleocytoplasmic shuttling of MALT1 and BCL10 complex may indicate that these molecules are involved not only in the nuclear factor κB (NF-κB) pathway but also in other biologic functions in lymphocytes.

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