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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Active Transport, Cell Nucleus / drug effects
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Adaptor Proteins, Signal Transducing / genetics
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Adaptor Proteins, Signal Transducing / metabolism
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Carboxypeptidases / genetics
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Carboxypeptidases / metabolism
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Cell Nucleus / metabolism*
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Exportin 1 Protein
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Fatty Acids, Unsaturated / pharmacology
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GTP-Binding Proteins / genetics
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GTP-Binding Proteins / metabolism
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HeLa Cells
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Humans
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Immunoblotting
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Karyopherins / genetics
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Karyopherins / metabolism*
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Luminescent Proteins / genetics
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Luminescent Proteins / metabolism
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Mass Spectrometry / methods*
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Microscopy, Confocal
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Microscopy, Fluorescence
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Nuclear Export Signals / genetics
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Protein Binding
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Protein Phosphatase 2 / genetics
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Protein Phosphatase 2 / metabolism
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Receptors, Cytoplasmic and Nuclear / genetics
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Receptors, Cytoplasmic and Nuclear / metabolism*
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
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Sequestosome-1 Protein
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Serine-Type D-Ala-D-Ala Carboxypeptidase
Substances
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Adaptor Proteins, Signal Transducing
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Fatty Acids, Unsaturated
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Karyopherins
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Luminescent Proteins
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Nuclear Export Signals
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Receptors, Cytoplasmic and Nuclear
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Recombinant Fusion Proteins
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SQSTM1 protein, human
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Sequestosome-1 Protein
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Protein Phosphatase 2
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Carboxypeptidases
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AGTPBP1 protein, human
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Serine-Type D-Ala-D-Ala Carboxypeptidase
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GTP-Binding Proteins
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leptomycin B