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.
Highlights
The transport of macromolecules across the nuclear envelope occurs through large proteinaceous structures called nuclear pore complexes (NPCs).1 NPCs are composed of ϳ30
We set out to devise a method to systematically search for Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1)-substrates that would be applicable for different cell lines under different growth conditions
In the absence of leptomycin B (LMB), Rev(48 –116)-GFP2-M9 mostly localized to the nucleus, but it was detected in the cytoplasm to some extent
Summary
The transport of macromolecules across the nuclear envelope occurs through large proteinaceous structures called nuclear pore complexes (NPCs).1 NPCs are composed of ϳ30. We conclude from these results that the changes we observed in total cell extracts upon LMB treatment cannot account for most of the putative CRM1 substrates as found in the subcellular fractions.
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