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J. Cell Biol.,
Volume 139, Number 4, November 17, 1997 841-849
Department of Anatomy and Cell Biology, Osaka University Medical School, Suita, Osaka 565, Japan
A 97-kD component of nuclear pore-targeting complex (the 
-subunit of nuclear pore-targeting
complex [PTAC]/importin/karyopherin) mediates the
import of nuclear localization signal (NLS)-containing proteins by anchoring the NLS receptor protein (the
-subunit of PTAC/importin/karyopherin) to the nuclear pore complex (NPC). The import requires a small
GTPase Ran, which interacts directly with the -subunit. The present study describes an examination of the
behavior of the -subunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically
injected -subunit rapidly migrates into the nucleus.
The use of deletion mutants reveals that nuclear migration of the -subunit requires neither Ran- nor -subunit-binding but only the NPC-binding domain of this molecule, which is also involved in NLS-mediated import. Furthermore, unlike NLS-mediated import, a
dominant-negative Ran, defective in GTP-hydrolysis,
did not inhibit nuclear migration of the -subunit. In
the digitonin-permeabilized cell-free import assay, the
-subunit transits rapidly through the NPC into the nucleus in a saturating manner in the absence of exogenous addition of soluble factors. These results show
that the -subunit undergoes translocation at the NPC
in a Ran-unassisted manner when it does not carry
-subunit/NLS substrate. Therefore, a requirement for
Ran arises only when the -subunit undergoes a translocation reaction together with the -subunit/NLS substrate. The results provide an insight to the yet unsolved question regarding the mechanism by which
proteins are directionally transported through the NPC,
and the role of Ran in this process.
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