Protein nuclear import factors are not, in general, believed to function in
the nuclear export of macromolecules and their reutilization therefore req
uires their recycling from the nucleus to the cytoplasm. Two possible mecha
nisms for recycling have been proposed. On the one hand, protein import fac
tors such as importin beta and transportin (Trn) could continuously shuttle
between cytoplasm and nucleoplasm. On the other hand, these proteins could
penetrate into the nucleus only as far as the inner surface of the nuclear
pore complex and then directly return to the cytoplasm. In this manuscript
, we have used microinjection analysis in human cells, and in vitro nuclear
assays, to demonstrate that importin beta, transportin and importin alpha
are all nucleocytoplasmic shuttle proteins that efficiently enter and exit
the cell nucleoplasm. In the case of transportin, we have mapped sequences
required for nucleocytoplasmic shuttling to the carboxy-terminal 270 amino
acids of this 890 amino acid import factor, thus demonstrating that nuclear
export is independent of the amino-terminal Ran-binding domain of Trn. We
further show that Trn shuttling is independent of nuclear RNA transcription
, Overall, these data suggest that nucleocytoplasmic shuttling is likely to
be a general attribute of protein nuclear import factors.