Stimulation of Nuclear Export and Inhibition of Nuclear Import by a Ran Mutant Deficient in Binding to Ran-binding Protein 1

Abstract

Receptor-mediated nucleocytoplasmic transport is dependent on the GTPase Ran and Ran-binding protein 1 (RanBP1). The acidic C terminus of Ran is required for high affinity interaction between Ran and RanBP1. We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an ∼20-fold reduced affinity for RanBP1. We investigated the effects of RanC4A on nuclear import and export in permeabilized HeLa cells. Although RanC4A promotes accumulation of the nuclear export receptor CRM1 at the cytoplasmic nucleoporin Nup214, it strongly stimulates nuclear export of GFP-NFAT. Since RanC4A exhibits an elevated affinity for CRM1 and other nuclear transport receptors, this suggests that formation of the export complex containing CRM1, Ran-GTP, and substrate is a rate-limiting step in export, not release from Nup214. Conversely, importin α/β-dependent nuclear import of bovine serum albumin, coupled to a classical nuclear localization sequence is strongly inhibited by RanC4A. Inhibition can be reversed by additional importin α, which promotes the formation of an importin α/β complex. These results provide physiological evidence that release of Ran-GTP from importin β by RanBP1 and importin α is critical for the recycling of importin 13 to a transport-competent state.