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© The Rockefeller University Press, 0021-9525/1999//721 $5.00
The Journal of Cell Biology, Volume 144, Number 4, , 1999 721-733

Regular Articles

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



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

* Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; {ddagger} 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

{gamma}-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 {gamma}-tubulin small complex ({gamma}TuSC; ~280,000 D) and Drosophila {gamma}TuRC (~2,200,000 D). In addition to {gamma}-tubulin, the {gamma}TuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The {gamma}TuSC is a structural subunit of the {gamma}TuRC, a larger complex containing about six additional polypeptides. Like the {gamma}TuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), the Drosophila {gamma}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 {gamma}TuSC also nucleates microtubules, but much less efficiently than the {gamma}TuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the {gamma}TuSC reveals that {gamma}-tubulin binds preferentially to GDP over GTP, rendering {gamma}-tubulin an unusual member of the tubulin superfamily.

Key Words: centrosome • cytoskeleton • nucleation • GTP • GDP

Abbreviations used in this paper: Dgrip, Drosophila gamma ring protein; {gamma}TuRC, {gamma}-tubulin ring complex; {gamma}TuSC, {gamma}-tubulin small complex; MT, microtubule; PCM, pericentriolar material; PEG, polyethylene glycol; Sc {gamma}TuSC, Saccharomyces cerevisiae 6 S complex.

Address correspondence to Karen Oegema, European Molecular Biology Laboratory, Cell Biology Program, Meyerhofstrasse 1, D-69117 Heidelberg, Germany. Tel.: 49-6221-387-337. Fax: 49-6221-387-512-306. E-mail: Karen.Oegema{at}EMBL-Heidelberg.DE or Yixian Zheng, Department of Embryology, Carnegie Institute of Washington, Baltimore, MD 21210. Tel.: 410-554-1232. Fax: 410-243-6311. E-mail: zheng{at}mail1.ciwemb.edu

We dedicate this work to Christine Mirzayan. The authors thank Mike Sepanski and Mei Lie Wong for the negative stain EM. We also thank Arshad Desai for much help during the course of this work and Arshad Desai and Doug Kellogg for critical reading of the manuscript.


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