Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry
Ketan Thakar1, Samir Karaca, Sarah A Port
1Department of Biochemistry I, Faculty of Medicine, Georg-August-University of Göttingen, Humboldtallee 23, Göttingen, Germany.
Molecular & Cellular Proteomics : MCP
|December 18, 2012
Summary
We developed a mass spectrometry method to identify proteins exported from the nucleus by CRM1. This approach identified over 100 CRM1-dependent proteins, including novel substrates like CCP1.
Area of Science:
- Cell Biology
- Molecular Biology
- Proteomics
Background:
- Chromosome region maintenance 1 (CRM1) is the primary receptor for nuclear protein export.
- CRM1 recognizes nuclear export signals (NESs), but prediction is challenging due to its flexible binding.
- Protein export can also occur via NES-containing adaptor proteins.
Purpose of the Study:
- To develop and apply a quantitative mass spectrometry approach for identifying CRM1-dependent nuclear export substrates.
- To discover novel proteins regulated by the CRM1 export pathway.
Main Methods:
- Utilized quantitative mass spectrometry with stable isotope labeling (SILAC) in HeLa cells.
- Analyzed protein depletion from cytosolic fractions and enrichment in nuclear fractions upon CRM1 inhibition with leptomycin B.
Main Results:
- Identified over 100 proteins affected by CRM1 inhibition.
- Validated novel CRM1 substrates including sequestosome 1, cancerous inhibitor of protein phosphatase 2A, guanine nucleotide-binding protein-like 3-like protein, programmed cell death protein 2-like protein, and cytosolic carboxypeptidase 1 (CCP1).
- Discovered a functional NES in CCP1 mediating direct CRM1 binding.
Conclusions:
- The developed mass spectrometry method effectively identifies CRM1 pathway targets.
- This approach expands the known substrates of CRM1-mediated nuclear export.
- The method is adaptable for studying other nucleocytoplasmic transport pathways and shuttling proteins.
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