A role for RanBP1 in the release of CRM1 from the nuclear pore complex in a terminal step of nuclear export

We recently developed an assay in which nuclear export of the shuttling tra nscription factor NFAT (nuclear factor of activated T cells) can be reconst ituted in permeabilized cells with the GTPase Ran and the nuclear export re ceptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot h ydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alon e do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cy toplasmic periphery of the nuclear pore complex (NPC) in association with t he p62 complex and Can/Nup214, RanGTP-dependent association of CRM1 with th ese nucleoporins was reconstituted in vitro. By biochemical fractionation a nd reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC, Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that R anBP1 and probably RanBP2 are involved in the dissociation of nuclear expor t complexes from the NPC in a terminal step of transport.