Nuclear import of Ran is mediated by the transport factor NTF2

A concentration gradient of the GTP-bound form of the GTPase Ran across nuc lear pores is essential for the transport of many proteins and nucleic acid s between the nuclear and cytoplasmic compartments of eukaryotic cells [1-4 ]. The mechanisms responsible for the dynamics and maintenance of this Ran gradient have been unclear. We now show that Ran shuttles between the nucle osol and cytosol, and that cytosolic Ran accumulates rapidly in the nucleus in a saturable manner that is dependent on. temperature and on the guanine -nucleotide exchange factor RCC1. Nuclear import in digitonin-permeabilized cells in the absence of added factors was minimal. The addition of energy and nuclear transport factor 2 (NTF2) [5] was sufficient for the accumulati on of Ran in the nucleus. An NTF2 mutant that cannot bind Ran [6] was unabl e to facilitate Ran import. A GTP-bound form of a Ran mutant that cannot bi nd NTF2 was not a substrate for import. A dominant-negative importin-beta m utant inhibited nuclear import of Ran, whereas addition of transportin, whi ch accumulates in the nucleus, enhanced NTF2-dependent Ran import. We concl ude that NTF2 functions as a transport receptor for Ran, permitting rapid e ntry into the nucleus where GTP-GDP exchange mediated by RCC1 [7] converts Ran into its GTP-bound state. The Ran-GTP can associate with nuclear Ran-bi nding proteins, thereby creating a Ran gradient across nuclear pores.