Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1

Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ra n and Ran-binding protein 1 (RanBP1), The acidic C terminus of Ran is requi red for high affinity interaction between Ran and RanBP1. We found that a n ovel Ran mutant with four of its five acidic C-terminal amino acids modifie d to alanine (RanC4A) has an similar to 20-fold reduced affinity for RanBP1 , We investigated the effects of RanC4A on nuclear import and export in per meabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly st imulates nuclear export of GFP-NFAT, Since RanC4A exhibits an elevated affi nity for CRM1 and other nuclear transport receptors, this suggests that for mation of the export complex containing CRM1, Ran-GTP, and substrate is a r ate-limiting step in export, not release from Nup214. Conversely, importin alpha/beta -dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A Inh ibition can be reversed by additional importin alpha, which promotes the fo rmation of an importin alpha/beta complex. These results provide physiologi cal evidence that release of Ran-GTP from importin beta by RanBP1 and impor tin alpha is critical for the recycling of importin beta to a transport-com petent state.