The C-Terminal Extension of the Small GTPase Ran is Essential for Defining the GDP-Bound Form

Abstract

The small GTPase Ran controls cellular processes by interacting with members of the importin β family that bind specifically to the GTP-bound form of Ran, and this regulates the interaction between importin β-like proteins and cellular factors. The structures of RanGDP and RanGTP are markedly different, and major structural changes are found in the switch I and switch II regions and in the C-terminal extension of Ran. Here, we show that a deletion mutant of Ran, lacking the entire C-terminal extension, termed Ran Core, can bind to importin β in its GDP-bound form with high affinity. The ability of Ran CoreGDP to dissociate cargo from importin β results in an import block in digitonin-permeabilized cells and leads to microtubule aster formation in mitotic Xenopus egg extract. As for importin β, also transportin, importin 7 and exportin-t can no longer discriminate efficiently between the two nucleotide-bound forms of Ran Core. In contrast, a significant reduction in affinity of the RanGDP-binding protein NTF2 for Ran CoreGDP is observed, indicating that the switch regions have changed conformation in the Ran Core mutant. Our results demonstrate that the C terminus of Ran is a major determinant of the state of Ran, and that removal of this allows the GDP-bound form to adopt a GTP-like conformation, thereby creating a constitutively active protein.