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Cell Mol Life Sci. 2016 Dec;73(24):4685-4699. Epub 2016 Jun 16.

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

Author information

1
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain.
2
Division of Cell Biology I, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
3
Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain.
4
Department of Molecular Cell Biology and Immunology, VU University Medical Center, Amsterdam, The Netherlands.
5
Department of Biochemistry and Molecular Biology, Biofisika Institute (UPV/EHU, CSIC), University of the Basque Country, Leioa, Spain. sonia.banuelos@ehu.es.
6
Department of Genetics, Physical Anthropology and Animal Physiology, University of the Basque Country (UPV/EHU), Leioa, Spain. josean.rodriguez@ehu.es.

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:

Cellular assay; Chronic lymphocytic leukemia; NES; Recurrent mutation; XPO1

PMID:
27312238
DOI:
10.1007/s00018-016-2292-0
[Indexed for MEDLINE]

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