NTF2 mediates nuclear import of Ran

Importin beta family transport receptors shuttle between the nucleus and th e cytoplasm and mediate transport of macromolecules through nuclear pore co mplexes (NPCs). The interactions between these receptors and their cargoes are regulated by binding RanGTP; all receptors probably exit the nucleus co mplexed with RanGTP, and so should deplete RanGTP continuously from the nuc leus. We describe here the development of an in vitro system to study how n uclear Ran is replenished. Nuclear import of Ran does not rely on simple di ffusion as Ran's small size would permit, but instead is stimulated by solu ble transport factors. This facilitated import is specific for cytoplasmic RanGDP and employs nuclear transport factor 2 (NTF2) as the actual carrier. NTF2 binds RanGDP initially to NPCs and probably also mediates translocati on of the NTF2-RanGDP complex to the nuclear side of the NPCs, A direct NTF 2-RanGDP interaction is crucial for this process, since point mutations tha t disturb the RanGDP-NTF2 interaction also interfere with Ran import. The s ubsequent nuclear accumulation of Ran also requires GTP, but not GTP hydrol ysis. The release of Ran from NTF2 into the nucleus, and thus the direction ality of Ran import, probably involves nucleotide exchange to generate RanG TP, for which NTF2 has no detectable affinity, followed by binding of the R anGTP to an importin beta family transport receptor.