RanGTP-Regulated Interactions of CRM1 with Nucleoporins and a Shuttling DEAD-Box Helicase

Abstract

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

ACKNOWLEDGMENTS

We thank Ursula Bodendorf and Nathalie Salome (TMV, Heidelberg, Germany), Anne Uv and Anna Wickberg (University of Umeå, Umeå, Sweden), and Karsten Weis (University of California, Berkeley) for communicating unpublished results; Gert-Jan Arts and Scott Kuersten for sharing materials; and members of the Mattaj lab and Judith Boer for valuable discussions and comments on the manuscript. The Resource Center of the German Human Genome Project is acknowledged for the screening of Xenopus cDNA libraries.

P.A., M.F., and M.O. were supported by EMBL short-term, EMBL long-term, and Deutsche Forschungsgemeinschaft fellowships, respectively.