Structural basis for the nuclear export activity of Importin13

Grünwald, Marlene; Lazzaretti, D.; Bono, Fulvia

doi:10.1038/emboj.2013.29

Cited by 38 publications

(72 citation statements)

References 73 publications

(140 reference statements)

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“…The closest paralog of Tnpo3 is Importin 13 (Imp13), with which it shares 21.6% amino acid sequence identity. The overall structures of isolated Tnpo3 and of its complex with RanGTP are predictably similar to the corresponding Imp13 structures reported previously (37)(38)(39). Akin to Imp13 and some other β-karyopherins, Tnpo3 is composed of 20 HEAT repeats and adopts an open circular/toroidal shape ( Fig.…”

Section: Resultssupporting

confidence: 53%

Structural basis for nuclear import of splicing factors by human Transportin 3

Maertens

Cook

Wang

et al. 2014

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

128

167

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Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginineserine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran-and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.T he transport of macromolecules between cytoplasm and nucleus is orchestrated by a family of nuclear import and export receptors (1). Referred to as importins and exportins, these proteins bind their specific cargoes and translocate them across the nuclear pore complex. The process is regulated by the small GTPase Ran that partitions between cytoplasm and nucleus in the predominantly GDP-and GTP-bound form, respectively. Importins associate with their cargoes in the cytoplasm, and the competitive binding of RanGTP induces them to release their cargoes in the nucleus (2). Most nuclear import/export receptors belong to the β-karyopherin family of proteins, with 22 members encoded in the human genome (3). The majority of β-karyopherins bind their cargoes directly, recognizing a linear nuclear localization or export signal and/or a specific tertiary/quaternary structural feature (4, 5).A fundamentally important type of a nuclear localization signal (NLS), comprising sequences rich in Arg-Ser and/or ArgAsp/Glu/Gly dipeptides (referred to as RS or RS-like domains), belongs to the family of Ser/Arg-rich (SR) proteins. These nuclear proteins also contain RNA recognition motif (RRM) domains and play essential roles in pre-mRNA splicing and 3′ processing and participate in transcription regulation, mRNA transport, translation, and nonsense-mediated mRNA decay (6). The splicing factors alternative splicing factor/splicing factor 2 (ASF/SF2) and SC35 along with the cleavage and polyadenylation specificity factor 6 (CPSF6, also known as CF-Im-68) are among the best-characterized metazoan SR proteins (7-10). The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12-16). Phosphorylation of SR proteins is thought to...

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Section: Resultssupporting

confidence: 53%

Structural basis for nuclear import of splicing factors by human Transportin 3

Maertens

Cook

Wang

et al. 2014

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

128

167

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“…Upon overexpression of importin 13, however, the protein was found in the cytoplasm. Essentially the same observation was made for overexpressed, GFP-tagged eIF1A, as reported previously (18). Likewise, overexpressed GFP-GST-Ubc9 and GFP-GST-Mago were cytoplasmic in control cells, but nuclear in importin 13-overexpressing cells.…”

Section: Resultssupporting

confidence: 75%

“…Different Importin 13 Interaction Modes of Individual Cargoes-As a bi-directional transporter, importin 13 uses different regions for interaction with import or export cargoes (17,18). Curiously, however, also import cargoes bind differently FIG.…”

Section: Fig 5 Analysis Of Importin 13 Cargo Candidates Enriched Wimentioning

confidence: 99%

Extensive Identification and In-depth Validation of Importin 13 Cargoes

Baade

Spillner

Schmitt

et al. 2018

Molecular & Cellular Proteomics

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Importin 13 is a member of the importin β family of transport receptors. Unlike most family members, importin 13 mediates both, nuclear protein import and export. To search for novel importin 13 cargoes, we used stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Using stringent criteria, we identified 255 importin 13 substrates, including the known cargoes Ubc9, Mago and eIF1A, and validate many of them as transport cargoes by extensive biochemical and cell biological characterization. Several novel cargoes can also be transported by the export receptor CRM1, demonstrating a clear redundancy in receptor choice. Using importin 13 mutants, we show that many of the novel substrates contact regions on the transport receptor that are not used by Ubc9, Mago or eIF1A. Together, this study significantly expands the repertoire of importin 13 cargoes and sets the basis for a more detailed characterization of this extremely versatile transport receptor.

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“…3A and B) contacts Asp426, Leu361 and Thr362 of Imp13, and that Ubc9-R17E mutation disrupts in its interaction with Imp13 [13]. Further, GFPtagged Ubc9-R17E mutant exhibits a striking decrease in its nuclear accumulation compared to GFP-tagged Ubc9-WT [14]. Moreover, we noticed that the nuclear/cytoplasmic ratios of GFP-Ubc9-R17E mutant [14] were much lower than those of GFPUbc9-WT in cells with RNAi-depletion of Imp13 (Fig.…”

Section: The N-terminal Ubc9 Mutations That Disrupt Its Interaction Wmentioning

confidence: 79%