Different structural and kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2 and importin-beta

The cytoplasmic disassembly of Ran.GTP.importin and Ran.GTP.exportin.cargo complexes is an essential step in the corresponding nuclear import and expo rt cycles. It has previously been shown that such disassembly can be mediat ed by RanBP1 in the presence of RanGAP. The nuclear pore complex protein Ra nBP2 (Nup358) contains four Ran-binding domains (RanBDi) that might functio n like RanBP1. We used biophysical assays based on fluorescence-labeled pro bes and on surface plasmon resonance to investigate the dynamic interplay o f Ran in its GDP- and GTP-complexed states with RanBDis and with importin-b eta. We show that RanBP1 and the four RanBDis from RanBP2 have comparable a ffinities for]Ran GTP (10(8)-10(9) M-l). Deletion of Ran' s C-terminal (DED DDL216)-D-211 sequence weakens the interaction of Ran GTP with RanBPis appr oximately 2000-fold, but accelerates the association of Ran GTP with import in-beta 10-fold. Importin-beta binds Ran GTP with a moderate rate, but atta ins a high affinity for Ran (K-D = 140 pM) via an extremely low dissociatio n rate of 10(-5) s(-1). Association with Ran is accelerated 3-fold in the p resence of RanBPI, which presumably prevents steric hindrance caused by the Ran C-terminus. In addition, we show that the RanBDis of RanBP2 are full e quivalents of RanBP1 in that they also costimulate RanGAP-catalyzed GTP hyd rolysis in Ran and relieve the GTPase block in a Ran GTP.transportin comple x. Our data suggest that the C-terminus of Ran functions like a loose tethe r in Ran GTP complexes of importins or exportins that exit the nucleus. Thi s flag is then recognized by the multiple RanBDis at or near the nuclear po re complex, allowing efficient disassembly of these Ran GTP complexes.