Abstract
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.
Highlights
Selective transport of macromolecules between the cytoplasm and the nucleus occurs through nuclear pore complexes (NPCs)1, which are built of ϳ30 different nucleoporins and serve as the only gateway across the nuclear envelope [1]
To identify novel binding partners of the transport receptor, we set up proteomic screens using immobilized importin 13 fused to an HZZ-tag as bait and HeLa cell extracts as a source of potential substrates
In a first version of the screen (“SILAC 1”), which served as a feasibility test toward the more sophisticated screens (“SILAC 2– 4”), we either immobilized the HZZ-importin 13 fusion protein or the HZZ-tag alone and compared binding of cytosolic proteins to the two different matrices and to importin 13 in the presence or absence of RanGTP
Summary
Selective transport of macromolecules between the cytoplasm and the nucleus occurs through nuclear pore complexes (NPCs)1, which are built of ϳ30 different nucleoporins and serve as the only gateway across the nuclear envelope [1]. Ubc9 as a Competitor for Cargo Binding to Importin 13—To get a better idea about specific importin 13 binding partners and, ideally, to discriminate between import and export cargoes, we first treated the cell lysates with phenyl-Sepharose to deplete endogenous transport receptors, which are known to interact with RanGTP and might affect binding of potential substrates to importin 13 [40].
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