title: Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry.
abstract: Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions.
authors.affiliation: Department of Biochemistry I, Faculty of Medicine, Georg-August-University of Göttingen, Humboldtallee 23, Göttingen, Germany.
authors.forename: Ketan
authors.initials: K
authors.lastname: Thakar
authors.forename: Samir
authors.initials: S
authors.lastname: Karaca
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Port
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Kehlenbach
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D049790:Nuclear Export Signals / Q000235:genetics; D011485:Protein Binding; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D000071456:Sequestosome-1 Protein; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D000097606:Exportin 1 Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002268:Carboxypeptidases; D005231:Fatty Acids, Unsaturated; D019204:GTP-Binding Proteins; D028884:Karyopherins; D008164:Luminescent Proteins; D049790:Nuclear Export Signals; D054648:Protein Phosphatase 2; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D000071456:Sequestosome-1 Protein; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; D000097606:Exportin 1 Protein; C099718:SQSTM1 protein, human; C583027:AGTPBP1 protein, human; C038753:leptomycin B