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

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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.

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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

K D :

Equilibrium dissociation constant

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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.

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Author notes

  1. Iraia García-Santisteban

    Present address: Division of Cell Biology I, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Authors and Affiliations

  1. Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain

    Iraia García-Santisteban & Jose A. Rodríguez

  2. Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain

    Igor Arregi, Marián Alonso-Mariño, María A. Urbaneja & Sonia Bañuelos

  3. Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands

    Juan J. Garcia-Vallejo

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  1. Iraia García-Santisteban
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  2. Igor Arregi
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Corresponding authors

Correspondence to Sonia Bañuelos or Jose A. Rodríguez.

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A patent application on the SRV100 biosensor has been submitted by the University of the Basque Country UPV/EHU.

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García-Santisteban, I., Arregi, I., Alonso-Mariño, M. et al. A cellular reporter to evaluate CRM1 nuclear export activity: functional analysis of the cancer-related mutant E571K. Cell. Mol. Life Sci. 73, 4685–4699 (2016). https://doi.org/10.1007/s00018-016-2292-0

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