γ-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 γ-tubulin small complex (γTuSC; ∼280,000 D) and Drosophila γTuRC (∼2,200,000 D). In addition to γ-tubulin, the γTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The γTuSC is a structural subunit of the γTuRC, a larger complex containing about six additional polypeptides. Like the γTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), the Drosophila γTuRC 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 γTuSC also nucleates microtubules, but much less efficiently than the γTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the γTuSC reveals that γ-tubulin binds preferentially to GDP over GTP, rendering γ-tubulin an unusual member of the tubulin superfamily.
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22 February 1999
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February 22 1999
Characterization of Two Related Drosophila γ-tubulin Complexes that Differ in Their Ability to Nucleate Microtubules
Karen Oegema,
Karen Oegema
*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
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Christiane Wiese,
Christiane Wiese
*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
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Ona C. Martin,
Ona C. Martin
*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
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Ronald A. Milligan,
Ronald A. Milligan
*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
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Akihiro Iwamatsu,
Akihiro Iwamatsu
*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
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Timothy J. Mitchison,
Timothy J. Mitchison
*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
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Yixian Zheng
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
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Karen Oegema
*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
Christiane Wiese
*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
Ona C. Martin
*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
Ronald A. Milligan
*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
Akihiro Iwamatsu
*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
Timothy J. Mitchison
*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
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
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: [email protected] or Yixian Zheng, Department of Embryology, Carnegie Institute of Washington, Baltimore, MD 21210. Tel.: 410-554-1232. Fax: 410-243-6311. E-mail: [email protected]
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.
Received:
September 09 1998
Revision Received:
January 11 1999
Online ISSN: 1540-8140
Print ISSN: 0021-9525
1999
J Cell Biol (1999) 144 (4): 721–733.
Article history
Received:
September 09 1998
Revision Received:
January 11 1999
Citation
Karen Oegema, Christiane Wiese, Ona C. Martin, Ronald A. Milligan, Akihiro Iwamatsu, Timothy J. Mitchison, Yixian Zheng; Characterization of Two Related Drosophila γ-tubulin Complexes that Differ in Their Ability to Nucleate Microtubules . J Cell Biol 22 February 1999; 144 (4): 721–733. doi: https://doi.org/10.1083/jcb.144.4.721
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