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 approximately 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 alpha/beta-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 alpha, which promotes the formation of an importin alpha/beta complex. These results provide physiological evidence that release of Ran-GTP from importin beta by RanBP1 and importin alpha is critical for the recycling of importin beta to a transport-competent state.
Highlights
Molecular transport between the cytoplasm and the nucleus occurs through nuclear pore complexes (NPCs),1 large supramolecular structures that span the nuclear envelope
We found that a novel Ran mutant with four of its five acidic C-terminal amino acids modified to alanine (RanC4A) has an ϳ20-fold reduced affinity for Ran-binding protein 1 (RanBP1)
Biochemical Characterization of RanC4A—The acidic C terminus of Ran promotes the interaction between Ran and RanBP1 [15, 16, 39], whereas it appears to impede the interaction between Ran and importin  [16, 40]
Summary
Cell Culture—HeLa cells were grown on either plastic dishes or coverslips in Dulbecco’s modified Eagle’s medium or in suspension in Joklik’s modified S-MEM. Aliquots were removed at regular intervals and applied to HPLC analysis to measure the protein-bound GTP and GDP concentration. The kinetic constant kon of the Ran-GST-RanBP1 association was calculated with Kaleidagraph 3.0.5 (Synergy Software) as described previously [15]. For the measurement of the dissociation kinetics (koff), a 1:1 complex between 200 nM Ran-mant-Gpp(NH)p and GST-RanBP1 was generated in 20 mM K2HPO4/KH2PO4, pH 7.4, 5 mM MgCl2, 5 mM dithioerythritol, in a titration experiment at 25 °C. The permeabilized cells were incubated in nuclear import reactions for 30 min at 30 °C as described [34], except the import substrate was a fusion protein between the amino terminus of human SRP␣ and -galactosidase When suspension HeLa cells were used for import assays, the reactions were performed as described in Ref. 36, containing 2.5 mg/ml cytosol, unless otherwise
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