The small GTPase Ran is essential for virtually all nucleocytoplasmic trans
port events. It is hypothesized that Ran drives vectorial transport of macr
omolecules into and out of the nucleus via the establishment of a Ran gradi
ent between the cytoplasm and nucleoplasm. Although Ran shuttles between th
e nucleus and cytoplasm, it is concentrated in the nucleus at steady state.
We show that nuclear transport factor 2 (NTF2) is required to concentrate
Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analy
ze the mechanism of Ran import into the nucleus by NTF2, we use mutants in
a variety of nuclear transport factors along with biochemical analyses of N
TF2 complexes. We find that Ran remains concentrated in the nucleus when im
portin-mediated protein import is disrupted and demonstrate that NTF2 does
not form a stable complex with the transport receptor, importin-beta. Consi
stent with a critical role for NTF2 in establishing and maintaining the Ran
gradient, we show that NTP2 is required for early embryogenesis in Caenorh
abditis elegans. Our data distinguish between two possible mechanisms for R
an import by NTF2 and demonstrate that Ran import is independent from impor
tin-beta-mediated protein import.