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Vol. 18, Issue 12, 4992-5003, December 2007

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Cooperative Assembly of CYK-4/MgcRacGAP and ZEN-4/MKLP1 to Form the Centralspindlin Complex

Visnja Pavicic-Kaltenbrunner^*, Masanori Mishima, and Michael Glotzer^*

*Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637; and Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge CB2 1QN, United Kingdom

Submitted May 18, 2007; Revised September 21, 2007; Accepted October 5, 2007
Monitoring Editor: Kerry Bloom

Cytokinesis in metazoan cells requires a set of antiparallel microtubules that become bundled upon anaphase onset to form a structure known as the central spindle. Bundling of these microtubules requires a protein complex, centralspindlin, that consists of the CYK-4/MgcRacGAP Rho-family GTPase-activating protein and the ZEN-4/MKLP1 kinesin-6 motor protein. Centralspindlin, but not its individual subunits, is sufficient to bundle microtubules in vitro. Here, we present a biochemical and genetic dissection of centralspindlin. We show that each of the two subunits of centralspindlin dimerize via a parallel coiled coil. The two homodimers assemble into a high-affinity heterotetrameric complex by virtue of two low-affinity interactions. Conditional mutations in the regions that mediate complex assembly can be readily suppressed by numerous second site mutations in the interacting regions. This unexpected plasticity explains the lack of primary sequence conservation of the regions critical for this essential protein–protein interaction.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Michael Glotzer (mglotzer{at}uchicago.edu).

Abbreviations used: GAP, GTPase-activating protein; ITC, isothermal titration calorimetry; MAP, microtubule-associated protein; MT, microtubule; SPR, surface plasmon resonance; wt, wild type.