Abstract
Ran-GTP interacts strongly with importin-beta, and this interaction promotes the release of the importin-alpha-nuclear localization signal cargo from importin-beta. Ran-GDP also interacts with importin-beta, but this interaction is 4 orders of magnitude weaker than the Ran-GTP.importin-beta interaction. Here we use the yeast complement of nuclear import proteins to show that the interaction between Ran-GDP and importin-beta promotes the dissociation of GDP from Ran. The release of GDP from the Ran-GDP-importin-beta complex stabilizes the complex, which cannot be dissociated by importin-alpha. Although Ran has a higher affinity for GDP compared with GTP, Ran in complex with importin-beta has a higher affinity for GTP. This feature is responsible for the generation of Ran-GTP from Ran-GDP by importin-beta. Ran-binding protein-1 (RanBP1) activates this reaction by forming a trimeric complex with Ran-GDP and importin-beta. Importin-alpha inhibits the GDP exchange reaction by sequestering importin-beta, whereas RanBP1 restores the GDP nucleotide exchange by importin-beta by forming a tetrameric complex with importin-beta, Ran, and importin-alpha. The exchange is also inhibited by nuclear-transport factor-2 (NTF2). We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results.
Highlights
Release and GTP Binding to Gsp1p (Ran) (Gsp1p in yeast) is a Ras-like GTPase that regulates diverse cellular processes, including nuclear transport, mitotic spindle assembly, and post-mitotic nuclear assembly [1, 2]
We suggest a mechanism for nuclear import, additional to the established RCC1 (Ran-guanine exchange factor)-dependent pathway that incorporates these results
Importin- and importin-␣ are able to function in a new cycle of transport, whereas Ran-GDP is transported into the nucleus by NTF2 [14]
Summary
Chi et al [15] have demonstrated that RanGDP forms a stable complex with RanBP1 and importin-; they suggested a role for Ran-GDP in the association of the importin-1⁄7importin-␣1⁄7NLS complex with the nuclear pore and speculated that the importin-1⁄7importin-␣1⁄7NLS1⁄7Ran-GDP1⁄7RanBP1 pentameric complex was the actual translocation complex that moved through the pore This model has been adopted by others (16 –18) who have proposed that a stable Ran-GDP-containing complex was created on nucleoporin Nup358 ( called RanBP2) and that upon displacement of the importin1⁄7importin-␣1⁄7Ran-GDP complex from the RBH (domain homologous to RanBP1) domains of Nup358 by RanBP1, binding of NTF2 triggered translocation to the nucleus. We suggest a mechanism of nuclear import additional to the RCC1-dependent pathway that incorporates our observations
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