Fig. 7. hNMD3 can engage into a complex with CRM1/RanGTP. (A) RanGTP stimulates the direct binding of CRM1 to NMD3. Recombinant 2z-hNMD3 FL (lanes 2 to 4) or 2z-hNMD3C27 (lanes 5 to 7) were incubated with recombinant CRM1 in the absence or presence of RanQ69L(GTP). 2z-hNMD3 associated factors were retrieved from the reaction mixtures by IgG-Sepharose. Bound proteins were separated by SDS-PAGE and detected by Coomassie blue staining. The amount of unspecific binding of CRM1 in the presence of RanGTP to IgG-Sepharose was also determined (lane 1). (B) 2z-hNMD3 forces CRM1 into a complex with RanGTP. Apparent dissociation constants of complexes between RanGTP and CRM1 were estimated in the presence of either 2 µM 2z-hNMD3, 2z-hNMD3C27 or 6His-hNMD3 using the RanGTPase assay. Note that only 2z-hNMD3 and only to some extent 6His-hNMD3, but not 2z-hNMD3C27, are able to force CRM1 into complex with Ran[-³²P]GTP. The complex formation of RanGTP and CRM1 protects the GTP on Ran from RanGAP-induced GTP hydrolysis.