RAN-UNASSISTED NUCLEAR MIGRATION OF A 97-KD COMPONENT OF NUCLEAR PORE-TARGETING COMPLEX

A 97-kD component of nuclear pore-targeting complex (the beta-subunit of nuclear pore-targeting complex [PT4C]/importin/karyopherin) mediate s the import of nuclear localization signal (NLS)-containing proteins by anchoring the NLS receptor protein (the alpha-subunit of PTAC/impor t/karyopherin) to the nuclear pore complex (NPC). The import requires a small GTPase Ran, which interacts directly with the beta-subunit. Th e present study describes an examination of the behavior of the beta-s ubunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically injected beta-subunit rapidly migrates into th e nucleus. The use of deletion mutants reveals that nuclear migration of the beta-subunit requires neither Ran- nor alpha-subunit-binding bu t only the NPC-binding domain of this molecule, which is also involved in NLS-mediated import. Furthermore, unlike NLS-mediated import, a do minant-negative Ran, defective in GTP-hydrolysis, did not inhibit nucl ear migration of the beta-subunit. In the digitonin-permeabilized cell -free import assay, the beta-subunit transits rapidly through the NPC into the nucleus in a saturating manner in the absence of exogenous ad dition of soluble factors. These results show that the beta-subunit un dergoes translocation at the NPC in a Ran-unassisted manner when it do es not carry alpha-subunit/NLS substrate. Therefore, a requirement for Ran arises only when the beta-subunit undergoes a translocation react ion together with the alpha-subunit/NLS substrate. The results provide an insight to the yet unsolved question regarding the mechanism by wh ich proteins are directionally transported through the NPC, and the ro le of Ran in this process.