title: Novel approaches for the identification of nuclear transport receptor substrates.
abstract: Nucleocytoplasmic transport affects the subcellular localization of a large proportion of cellular proteins. Transported proteins interact with a set of ~20 transport receptors, importins and exportins, which mediate translocation through the nuclear pore complex. Here we describe two novel methods based on quantitative proteome analysis for the identification of cargo proteins that are transported by a specific importin or exportin. The first approach is based on SILAC (stable isotope labeling of amino acids in cells) using cells that have been treated or not with specific reagents, followed by subcellular fractionation. Applying this approach to cells treated with or without the selective CRM1 inhibitor leptomycin B, we identified substrates of CRM1, the major nuclear export receptor. In the second SILAC approach, digitonin-permeabilized cells are incubated with nuclear and cytosolic extracts in the absence or presence of particular import receptors of interest. Proteomic analysis of the permeabilized cells then yields proteins whose nuclear import depends specifically on the added import receptor. Using this system, we identified substrates of two representative import receptors, transportin and importin-α/β.
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kimura
authors.affiliation: Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Göttingen, Göttingen, Germany.
authors.forename: Ketan
authors.initials: K
authors.lastname: Thakar
authors.affiliation: Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Samir
authors.initials: S
authors.lastname: Karaca
authors.affiliation: Cellular Dynamics Laboratory, RIKEN, Wako, Saitama, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Imamoto
authors.affiliation: Department of Molecular Biology, Faculty of Medicine, Georg-August-University of Göttingen, Göttingen, Germany.
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Kehlenbach
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000596:Amino Acids / Q000378:metabolism; D000935:Antifungal Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D004072:Digitonin / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D028884:Karyopherins / Q000037:antagonists & inhibitors; D013058:Mass Spectrometry; D022022:Nuclear Pore / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors; D015398:Signal Transduction; D028901:alpha Karyopherins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism; D000097606:Exportin 1 Protein
chemical_list: D000596:Amino Acids; D000935:Antifungal Agents; D004072:Digitonin; D005231:Fatty Acids, Unsaturated; D028884:Karyopherins; D018160:Receptors, Cytoplasmic and Nuclear; D028901:alpha Karyopherins; D028961:beta Karyopherins; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C038753:leptomycin B
keywords: Digitonin; Exportin; Importin; In vitro import assay; Leptomycin B; Mass spectrometry; SILAC; Semipermeabilized cells