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Cellular and Molecular Life Sciences

, Volume 73, Issue 24, pp 4685–4699 | Cite as

A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K

  • Iraia García-Santisteban
  • Igor Arregi
  • Marián Alonso-Mariño
  • María A. Urbaneja
  • Juan J. Garcia-Vallejo
  • Sonia Bañuelos
  • Jose A. Rodríguez
Original Article

Abstract

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.

Keywords

NES XPO1 Recurrent mutation Chronic lymphocytic leukemia Cellular assay 

Abbreviations

AML

Acute myeloid leukemia

CLL

Chronic lymphocytic leukemia

CTE motif

C-terminal export motif

LR-NES

Leucine rich NES

NES

Nuclear export signal

NLS

Nuclear localization signal

NPC

Nuclear pore complex

KD

Equilibrium dissociation constant

Notes

Acknowledgments

We thank Dr. Fernando Moro for helping with the analysis of binding curves and Dr. René Medema for his support. We thank the staff from the High Resolution Microscopy Facility (SGIker-UPV/EHU) for technical support. This work is funded by the Spanish Ministry of Economy (Grant SAF2014-57743-R to SB and JAR), and by the University of the Basque Country (UFI 11/20). IG-S is a recipient of a postdoctoral fellowship from the Department of Education of the Basque Country Government.

Supplementary material

18_2016_2292_MOESM1_ESM.pptx (86 kb)
Supplementary material 1 (PPTX 85 kb)
18_2016_2292_MOESM2_ESM.docx (21 kb)
Supplementary material 2 (DOCX 21 kb)
18_2016_2292_MOESM3_ESM.pdf (88 kb)
Supplementary material 3 (PDF 87 kb)

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Copyright information

© Springer International Publishing 2016

Authors and Affiliations

  • Iraia García-Santisteban
    • 1
    • 4
  • Igor Arregi
    • 2
  • Marián Alonso-Mariño
    • 2
  • María A. Urbaneja
    • 2
  • Juan J. Garcia-Vallejo
    • 3
  • Sonia Bañuelos
    • 2
  • Jose A. Rodríguez
    • 1
  1. 1.Department of Genetics, Physical Anthropology and Animal PhysiologyUniversity of the Basque Country (UPV/EHU)LeioaSpain
  2. 2.Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC)University of the Basque CountryLeioaSpain
  3. 3.Department of Molecular Cell Biology and ImmunologyVU University Medical CenterAmsterdamThe Netherlands
  4. 4.Division of Cell Biology IThe Netherlands Cancer InstituteAmsterdamThe Netherlands

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