J. Cell Biol., Volume 144, Number 4, February 22, 1999 721-733

Characterization of Two Related Drosophila -tubulin Complexes that Differ in Their Ability to Nucleate Microtubules

Karen Oegema,^* Christiane Wiese, Ona C. Martin, Ronald A. Milligan,^§ Akihiro Iwamatsu, Timothy J. Mitchison,^* and Yixian Zheng

^* Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115;  Department of Embryology, Carnegie Institute of Washington, Baltimore, Maryland 21210; ^§ Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037; and  Central Laboratories for Key Technology, Kirin Brewery Company, Ltd., Yokohama 236, Japan

-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name -tubulin small complex (TuSC; ~280,000 D) and Drosophila TuRC (~2,200,000 D). In addition to -tubulin, the TuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The TuSC is a structural subunit of the TuRC, a larger complex containing about six additional polypeptides. Like the TuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila TuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with ~13 radially arranged structural repeats. The TuSC also nucleates microtubules, but much less efficiently than the TuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the TuSC reveals that -tubulin binds preferentially to GDP over GTP, rendering -tubulin an unusual member of the tubulin superfamily.

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