Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal.
The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta 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 alpha and beta (alpha/beta) 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 alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta 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.
D000818:Animals; D002478:Cells, Cultured; D004926:Escherichia coli; D016547:Kinesins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism; D028901:alpha Karyopherins / Q000378:metabolism*; D028961:beta Karyopherins / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000378:metabolism*
C104595:KIFC1 protein, Xenopus; D019913:Nuclear Localization Signals; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D028901:alpha Karyopherins; D028961:beta Karyopherins; D016547:Kinesins; D020931:ran GTP-Binding Protein