Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

Engelsma, Dieuwke; Bernad, Rafael; Calafat, Jero; Fornerod, Maarten

doi:10.1038/sj.emboj.7600370

Cited by 88 publications

(119 citation statements)

References 59 publications

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“…1, B and C). We have previously shown that depletion of Nup358 causes a small reduction in export of a Rev(1.4)-GFP-NES reporter protein (43), which is targeted to the cytoplasm and sensitive to leptomycin B (44,49). In addition to an NLS, the Rev(1.4) protein also provides nuclear retention activity which permits a more stringent assessment of nuclear export.…”

Section: Resultsmentioning

confidence: 99%

“…In vitro, certain recombinant versions of NMD3 possess a very high affinity for CRM1 (40), ϳ100-fold higher than regular NESs. To test whether the export defect due to depletion of Nup214-Nup88 was specific for high affinity NESs, we determined the nuclear export driven by an NES of a similar affinity, the supraphysiological S1 NES (44). Depletion of Nup214-Nup88 did not induce nuclear accumulation of this reporter protein, indicating that the effects were not related to high affinity CRM1 binding of NMD3 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Plasmid Construction-pSUPER-358 (43), pSUPER-214, Rev-S1-GFP (44), and HA-Nup153 (45) were described previously. The NLS-eGFP-NES insert containing the SV40 nuclear localization signal (NLS) and protein kinase A inhibitor NES was subcloned from pBSSK (46) into the pcDNA3 vector (Stratagene) using the HindIII-NotI restriction sites.…”

Section: Methodsmentioning

confidence: 99%

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Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Bernad¹,

Engelsma

Sanderson³

et al. 2006

Journal of Biological Chemistry

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The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG-repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/ hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Bernad¹,

Engelsma

Sanderson³

et al. 2006

Journal of Biological Chemistry

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“…However, the fact that both CSR and stability require maintenance of key hydrophobic positions in the NES and show a similar dependence on the identity of the interdigitating hydrophilic residues suggests that they might both depend on the quality of the AID-exportin interaction. Changes in the hydrophilic interdigitating amino acids in an NES have been shown in other systems to affect the quality of exportin-binding and kinetics of intracellular transport (20,21). The dependence of CSR and protein stability on the nature of the NES could therefore simply reflect a requirement for particular kinetics of nucleocytoplasmic shuttling.…”

Section: Discussionmentioning

confidence: 99%

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Geisberger

Rada²,

Neuberger³

2009

Proc. Natl. Acad. Sci. U.S.A.

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The carboxyterminal region of activation-induced deaminase (AID) is required for its function in Ig class switch recombination (CSR) and also contains a nuclear-export sequence (NES). Here, based on an extensive fine-structure mutation analysis of the AID NES, as well as from AID chimeras bearing heterologous NESs, we show that while a functional NES is indeed essential for CSR, it is not sufficient. The precise nature of the NES is critical both for AID stabilization and CSR function: minor changes in the NES can perturb stabilization and CSR without jeopardizing nuclear export. The results indicate that the AID NES fulfills a function beyond simply providing a signal for nuclear export and suggest the possibility that the quality of exportin-binding may be critical to the stabilization of AID and its activity in CSR.activation-induced deaminase ͉ antibody diversification ͉ immunoglobulin class switching ͉ exportin ͉ nuclear transport A ctivation-induced deaminase (AID) plays a central role in antibody gene-diversification, triggering both somatic hypermutation (SHM) and class-switch recombination (CSR) by deaminating deoxycytidine residues within the Ig locus to yield deoxyuridine (1, 2).AID is largely found in the cytoplasm of activated B cells but shuttles between cytoplasm and nucleus (3-6). The C-terminal amino acids of AID comprise a nuclear-export sequence (NES): removal of this NES causes AID to be restricted to the nucleus, whereas its addition to a heterologous nuclear protein drives export of the fusion protein into the cytoplasm (3-5). The sequence of the AID C-terminus conforms well to the consensus established for substrates of the Crm1-dependent export machinery: in keeping with this, export mediated by the AID NES is sensitive to leptomycin B.Analysis of both clinical and contrived mutations in AID have revealed that the AID C-terminal region is also essential for its function in CSR, although not in SHM (7-9). The C-terminal mutations that interfere with AID's ability to potentiate CSR do not compromise its ability to deaminate deoxycytidine in biochemical or bacterial genetic assays. Thus, as previously suggested by others, either the nuclear/cytoplasmic shuttling of AID must be specifically required for CSR or, alternatively, factors specific for CSR must interact with the C-terminal region of AID in an area overlapping the NES (4, 5, 7-9).It was with a view to discriminating these possibilities that we embarked on a refined mutagenic dissection of the AID NES and investigated the ability of heterologous NESs to substitute for the AID NES. Our results suggest that although nuclear export is likely necessary for CSR, this is not the sole function of the AID C-terminal region, with the data raising the possibility that successful CSR depends on the precise quality of AID binding to exportin or a related protein. Results NES Function and CSR Correlate in Alanine-Scanning Mutagenesis.To ascertain whether there was a correlation between export activity and CSR, we performed an alanine-scanning mut...

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“…However, Engelsma et al (29) identified NES-like peptides from a pool of randomly generated synthetic peptides, that exhibited a marked nuclear envelope accumulation in permeabilized HeLa cells. These synthetic NESs showed unusually strong interaction with Crm1, leading to their description as "supraphysiological" (29). These results led us to consider that the NPC localization of the Nmd3 mutant proteins may similarly depend on increased binding to Crm1.…”

Section: Accumulation Of Mutant Nmd3mentioning

confidence: 99%

Novel Interaction of the 60S Ribosomal Subunit Export Adapter Nmd3 at the Nuclear Pore Complex

West¹,

Hedges²,

et al. 2007

Journal of Biological Chemistry

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Nuclear export of the large (60S) ribosomal subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.

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Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358

Cited by 88 publications

References 59 publications

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

The stability of AID and its function in class-switching are critically sensitive to the identity of its nuclear-export sequence

Novel Interaction of the 60S Ribosomal Subunit Export Adapter Nmd3 at the Nuclear Pore Complex

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