Nuclear transport factor p10/NTF2 functions as a RAN-GDP dissociation inhibitor (RAN-GDI)

Abstract

The cytosolic nuclear transport factor p10/NTF2 is required for the translocation of karyophilic molecules through nuclear pores, and the small GTPase Ran is a key regulator of protein transport between the nucleus and cytoplasm. It has been reported that p10/NTF2 interacts directly and specifically with Ran-GDP but not with Ran-GTP. The precise role(s) of p10/NTF2 in the Ran GTP/GDP cycle are thus far unclear, however. In this study, we show that mammalian p10/NTF2 dramatically inhibits the dissociation of [3H]GDP from Ran and the binding of [35S]GTPγS to Ran following the dissociation of nonradioactive GDP by RCC1, the only known mammalian guanine nucleotide exchange factor for Ran (Ran-GEF). In contrast, the dissociation of [35S]GTPγS from Ran, which was also catalyzed by RCC1, was not affected by p10/NTF2. Furthermore, the activities of wild-type p10/NTF2 and the mutant forms M84T and D92G in an assay of nuclear protein import in a digitonin-permeabilized cell-free system correlated with their level of inhibition of the dissociation of nucleotide from Ran-GDP. These results suggest that p10/NTF2 acts as a GDP dissociation inhibitor for Ran (Ran-GDI), thereby coordinating the Ran-dependent reactions that underlie nuclear protein import.