Ran-unassisted Nuclear Migration of a 97-kD Component of Nuclear Pore–targeting Complex

Abstract

A 97-kD component of nuclear pore-targeting complex (the β-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 α-subunit of PTAC/importin/karyopherin) to the nuclear pore complex (NPC). The import requires a small GTPase Ran, which interacts directly with the β-subunit. The present study describes an examination of the behavior of the β-subunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically injected β-subunit rapidly migrates into the nucleus. The use of deletion mutants reveals that nuclear migration of the β-subunit requires neither Ran- nor α-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 β-subunit. In the digitonin-permeabilized cell-free import assay, the β-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 β-subunit undergoes translocation at the NPC in a Ran-unassisted manner when it does not carry α-subunit/NLS substrate. Therefore, a requirement for Ran arises only when the β-subunit undergoes a translocation reaction together with the α-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.

β-Catenin Shows an Overlapping Sequence Requirement but Distinct Molecular Interactions for Its Bidirectional Passage through Nuclear Pores

“…7). The importin ␤-RanGTP complex, which has a weaker affinity for FG-nucleoporins (51), exited the nucleus but did not enter the nucleus (22,31). Our results provide an important indication that if the same FG-nucleoporins provide crucial binding sites for both import and export, import through the NPC could be achieved through stronger binding, whereas export can be achieved by much weaker binding by the same FG-nucleoporins.…”

“…A comparison of the crystal structure of cargo-free importin ␤ and ␤-catenin revealed a remarkable conformational resemblance between the HEAT repeats 4, 5, 6, 7, and 8 of importin ␤ and the ARM repeats 9, 10, 11, and 12 of ␤-catenin (34). The HEAT repeats 4, 5, 6, 7, and 8 of importin ␤ comprise the region that is necessary and sufficient for its NPC translocation (22,35). This evidence prompted us to focus on the latter one-third of the ␤-catenin for its nuclear migrating activity.…”

“…Although nuclear transport in vivo is an energy-consuming process, a number of in vitro studies have demonstrated that the NPC translocation step of transport does not require any metabolic energy (22)(23)(24)(25)(26). In the importin/exportin pathways, metabolic energy is supplied by the Ran GTPase cycle (27), which operates on both sides of the NPC, to assure the directionality of cargo transport.…”

“…The NPC translocation process itself does not require any metabolic energy (22)(23)(24)(25)(26)18), and the importance of specific interactions between various transport receptors with FGnucleoporins has been well characterized (46, 47; for review, see Refs. 1 and 4).…”

Importin 7 and Importin α/Importin β Are Nuclear Import Receptors for the Glucocorticoid Receptor

“…Plasmid pGEX-2t-GFP-NLS was generously provided by Mary S. Moore (Baylor College of Medicine, Houston, TX). For nuclear import assays, pQt Ran (pKW356), pQtRanQ69L (pKW592; Nachury and Weis, 1999), and GST-PTAC97 (importin ␤; Kose et al, 1997) (Picard and Yamamoto, 1987;Freeman and Yamamoto, 2001), ϳ16 h before transfection. Cells were transfected with Lipfectamine 2000 (Invitrogen).…”