Importin α/β and Ran-GTP Regulate XCTK2 Microtubule Binding through a Bipartite Nuclear Localization Signal

Abstract

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin α and/or importin β to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin α and β (α/β) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin α/β-mediated regulation of XCTK2 microtubule binding. Our data show that importin α/β directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.