Dissecting the interactions between NTF2, RanGDP, and the nucleoporin XFXFG repeats

We have used a range of complementary biochemical and biophysical methods t o investigate the interactions between nuclear transport factor 2 (NTF2), t he Ras family GTPase Ran, and XFXFG nucleoporin repeats that are crucial fo r nuclear trafficking. Microcalorimetry, microtiter plate binding, and fluo rescence quenching in solution are all consistent with the binding constant for the NTF2-RanGDP interaction being in the 100 nM range, whereas the int eraction between NTF2 and XFXFG repeat-containing nucleoporins such as Nsp1 p is in the 1 mu M range. Although the accumulation of NTF2 at the nuclear envelope is enhanced by RanGDP, we show that Ran binding does not alter the affinity of NTF2 for nucleoporins nor does the binding of nucleoporins alt er the affinity of NTF2 for RanGDP, These results indicate that, instead, R an increases the binding of NTF2 to nucleoporins by another mechanism, most probably by Ran itself binding to nucleoporins and NTF2 binding to this nu clear pore-associated Ran.