A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K.
The exportin CRM1 binds nuclear export signals (NESs), and mediates active transport of NES-bearing proteins from the nucleus to the cytoplasm. Structural and biochemical analyses have uncovered the molecular mechanisms underlying CRM1/NES interaction. CRM1 binds NESs through a hydrophobic cleft, whose open or closed conformation facilitates NES binding and release. Several cofactors allosterically modulate the conformation of the NES-binding cleft through intramolecular interactions involving an acidic loop and a C-terminal helix in CRM1. This current model of CRM1-mediated nuclear export has not yet been evaluated in a cellular setting. Here, we describe SRV100, a cellular reporter to interrogate CRM1 nuclear export activity. Using this novel tool, we provide evidence further validating the model of NES binding and release by CRM1. Furthermore, using both SRV100-based cellular assays and in vitro biochemical analyses, we investigate the functional consequences of a recurrent cancer-related mutation, which targets a residue near CRM1 NES-binding cleft. Our data indicate that this mutation does not necessarily abrogate the nuclear export activity of CRM1, but may increase its affinity for NES sequences bearing a more negatively charged C-terminal end.
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain.|Division of Cell Biology I, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Iraia
I
García-Santisteban
Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain.
Igor
I
Arregi
Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain.
Marián
M
Alonso-Mariño
Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain.
María A
MA
Urbaneja
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.
Juan J
JJ
Garcia-Vallejo
Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain. sonia.banuelos@ehu.es.
Sonia
S
Bañuelos
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain. josean.rodriguez@ehu.es.
Jose A
JA
Rodríguez
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D017930:Genes, Reporter; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D028884:Karyopherins / Q000737:chemistry / Q000378:metabolism*; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000378:metabolism* / Q000473:pathology; D049790:Nuclear Export Signals; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000378:metabolism*; D000097606:Exportin 1 Protein
D028884:Karyopherins; D050505:Mutant Proteins; D049790:Nuclear Export Signals; D018160:Receptors, Cytoplasmic and Nuclear; D000097606:Exportin 1 Protein
Cellular assay; Chronic lymphocytic leukemia; NES; Recurrent mutation; XPO1