Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358.
Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358.
Department of Tumor Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Dieuwke
D
Engelsma
Rafael
R
Bernad
Jero
J
Calafat
Maarten
M
Fornerod
D021581:Active Transport, Cell Nucleus; D001943:Breast Neoplasms / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D028884:Karyopherins / Q000235:genetics / Q000378:metabolism*; D007930:Leucine; D016350:Leucine Zippers; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D019151:Peptide Library; D034622:RNA Interference; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
D028884:Karyopherins; D007930:Leucine; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; D010446:Peptide Fragments; D019151:Peptide Library; D018160:Receptors, Cytoplasmic and Nuclear; D020931:ran GTP-Binding Protein; D000097606:Exportin 1 Protein; C094493:ran-binding protein 2