Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana

Transport across the nuclear envelope is mediated by transport receptors fr om the Importin beta family. We identified Exportin 1 from Arabidopsis (AtX PO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-ri ch nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its fu nctional homologues from humans and yeasts. We functionally characterised A tXPO1 by its interaction with NESs of animal and plant proteins, which is i nhibited by the cytotoxin leptomycin B (LMB), and also by its interaction w ith the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we d emonstrated the existence of a nuclear export pathway for proteins in plant s. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter prot ein fused to both a nuclear localisation signal (NLS) and an NES motif. Usi ng this in vivo assay we demonstrated that the NES of the heterologous prot ein Rev is also functional in plants and that its export is inhibited by LM B. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protei n, is disrupted by mutating three long chain hydrophobic amino acid residue s to alanine (L(176)A, L(179)A, V(181)A). In BY-2 protoplasts the NES of At RanBP1a is functionally indistinguishable from the Rev NES. Our results dem onstrate that the machinery for the nuclear export of proteins is functiona lly conserved in plants.