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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.

SUBMITTER: Thakar K

PROVIDER: S-EPMC3591659 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fluorescent protein biosensors are powerful cellular systems biology tools for dissecting the complexity of cellular processes with high spatial and temporal resolution. As regulated nucleo-cytoplasmic transport is crucial for the modulation of numerous (patho)physiological cellular responses, a detailed understanding of its molecular mechanism would open up novel options for a rational manipulation of the cell. In contrast to genetic approaches, we here established and employed high-content cellular translocation biosensors applicable for dissecting nuclear export by chemicogenomics. A431 cell lines, stably expressing a translocation biosensor composed of glutathione S-transferase, GFP and a rational combination of nuclear import and export signals, were engineered by antibiotic selection and flow cytometry sorting. Using an optimized nuclear translocation algorithm, the translocation response could be robustly quantified on the Cellomics Arrayscan(®) VTI platform. Subsequent to assay optimization, the assay was developed into a higher density 384-well format high-content assay and employed for the screening of the 17K ChemBioNet compound collection. This library was selected on the basis of a genetic algorithm used to identify maximum common chemical substructures in a database of annotated bioactive molecules and hence, is well-placed in the chemical space covered by bioactive compounds. Automated multiparameter data analysis combined with visual inspection allowed us to identify and to rationally discriminate true export inhibitors from false positives, which included fluorescent compounds or cytotoxic substances that dramatically affected the cellular morphology. A total of 120 potential hit compounds were selected for Cellomics Arrayscan(®) VTI based rescreening. The export inhibitory activity of 20 compounds effective at concentrations < 25 ?M were confirmed by fluorescence microscopy in several cell lines. Interestingly, kinetic analysis allowed the identification of inhibitors capable to interfere with the export receptor CRM1-mediated nuclear export not only in an irreversible, but also in a reversible fashion. In sum, exploitation of biosensor based screening allows the identification of chemicogenomic tools applicable for dissecting nucleo-cytoplasmic transport in living cells.

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Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry.

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