Regulation of Microtubule Assembly and Organization in Mitosis by the AAA+ ATPase Pontin

Daniel Ducat,* ${dagger}$ Shin-ichi Kawaguchi,* ${ddagger}$ Hongbin Liu, ${sect}$ || John R. Yates III, ${sect}$ and Yixian Zheng* ${dagger}$

^*Department of Embryology, Carnegie Institution for Science and Howard Hughes Medical Institute, Baltimore, MD 21210; Department of Biology, Johns Hopkins University, Baltimore MD 21218; Department of Cell Biology, The Scripps Research Institute, LA Jolla, CA 92037

Monitoring Editor: Stephen Doxsey

To identify novel proteinsimportant for microtubule assembly in mitosis, we have useda centrosome-based complementation assay to enrich for proteinswith mitotic functions. An RNAi-based screen of these proteinsallowed us to uncover 13 novel mitotic regulators. We carriedout in-depth analyses of one of these proteins, Pontin, whichis known to have a number of functions in interphase, includingchromatin remodeling, DNA repair, and transcription. We showthat reduction of Pontin by RNAi resulted in defects in spindleassembly in Drosophila S2 cells and in a number of mammaliantissue culture cell lines. Further characterization of Pontinin Xenopus egg extracts demonstrates that Pontin interacts withthe ${gamma}$ -tubulin ring complex ( ${gamma}$ TuRC). Because depletion of Pontinleads to defects in the assembly and organization of microtubulearrays in egg extracts, our studies suggest that Pontin hasa mitosis-specific function in regulating microtubule assembly.

Present addresses: Temasek Life Sciences Laboratory, Singapore 117604, Singapore; ^||Protein Analytical Chemistry, Genentech Inc. South San Francisco, CA 94080.

Address correspondence to: Yixian Zheng (zheng{at}ciwemb.edu)