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

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.

[1]  B. Alberts,et al.  Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. , 1998 .

[2]  B. Alberts,et al.  Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds , 1998, The Journal of cell biology.

[3]  M. Knop,et al.  Receptors determine the cellular localization of a γ‐tubulin complex and thereby the site of microtubule formation , 1998, The EMBO journal.

[4]  Stephen S. Taylor,et al.  The Human Homologue of Bub3 Is Required for Kinetochore Localization of Bub1 and a Mad3/Bub1-related Protein Kinase , 1998, The Journal of cell biology.

[5]  T. Stearns,et al.  The Mammalian γ-Tubulin Complex Contains Homologues of the Yeast Spindle Pole Body Components Spc97p and Spc98p , 1998, The Journal of cell biology.

[6]  A. Iwamatsu,et al.  Xgrip109: A γ Tubulin–Associated Protein with an Essential Role in γ Tubulin Ring Complex (γTuRC) Assembly and Centrosome Function , 1998, The Journal of cell biology.

[7]  M. Bornens,et al.  Characterization of the Human Homologue of the Yeast Spc98p and Its Association with γ-Tubulin , 1998, The Journal of cell biology.

[8]  K. Oegema,et al.  [41] Purification of cytoskeletal proteins using peptide antibodies , 1998 .

[9]  K. Oegema,et al.  Purification of cytoskeletal proteins using peptide antibodies. , 1998, Methods in enzymology.

[10]  M. Knop,et al.  Spc98p and Spc97p of the yeast γ‐tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p , 1997, The EMBO journal.

[11]  T. Stearns,et al.  Centrosomes Isolated from Spisula solidissima Oocytes Contain Rings and an Unusual Stoichiometric Ratio of α/β Tubulin , 1997, The Journal of cell biology.

[12]  M. Knop,et al.  The spindle pole body component Spc97p interacts with the γ‐tubulin of Saccharomyces cerevisiae and functions in microtubule organization and spindle pole body duplication , 1997, The EMBO journal.

[13]  M. Martin,et al.  The role of γ-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans , 1997 .

[14]  E. Schiebel,et al.  Centrosome-microtubule nucleation. , 1997, Journal of cell science.

[15]  H. Mazarguil,et al.  Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations. , 1997, Cell motility and the cytoskeleton.

[16]  T. Mitchison,et al.  Microtubule polymerization dynamics. , 1997, Annual review of cell and developmental biology.

[17]  H. Erickson,et al.  Protofilaments and rings, two conformations of the tubulin family conserved from bacterial FtsZ to alpha/beta and gamma tubulin , 1996, The Journal of cell biology.

[18]  J. Kilmartin,et al.  The spindle pole body component Spc98p interacts with the gamma‐tubulin‐like Tub4p of Saccharomyces cerevisiae at the sites of microtubule attachment. , 1996, The EMBO journal.

[19]  J. Mulholland,et al.  Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function , 1996, The Journal of cell biology.

[20]  E. Schiebel,et al.  gamma-Tubulin-like Tub4p of Saccharomyces cerevisiae is associated with the spindle pole body substructures that organize microtubules and is required for mitotic spindle formation , 1996, The Journal of cell biology.

[21]  H. Erickson,et al.  Protofilaments and Rings, Two Conformations of the Tubulin Family Conserved from Bacterial FtsZ to od13 and /Tubulin , 1996 .

[22]  B. Alberts,et al.  Microtubule nucleation by γ-tubulin-containing rings in the centrosome , 1995, Nature.

[23]  Yixian Zheng,et al.  Nucleation of microtubule assembly by a γ-tubulin-containing ring complex , 1995, Nature.

[24]  M. Snyder,et al.  A highly divergent gamma-tubulin gene is essential for cell growth and proper microtubule organization in Saccharomyces cerevisiae , 1995, The Journal of cell biology.

[25]  D A Agard,et al.  Three-dimensional structural characterization of centrosomes from early Drosophila embryos , 1995, The Journal of cell biology.

[26]  R. Burns.  Analysis of the gamma-tubulin sequences: implications for the functional properties of gamma-tubulin. , 1995, Journal of cell science.

[27]  Flyvbjerg,et al.  Spontaneous nucleation of microtubules. , 1995, Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics.

[28]  P. Peluso,et al.  The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound. , 1995, Molecular biology of the cell.

[29]  C. Sunkel,et al.  Gamma‐tubulin is required for the structure and function of the microtubule organizing centre in Drosophila neuroblasts. , 1995, The EMBO journal.

[30]  T. Schroer,et al.  Polarity and nucleation of microtubules in polarized epithelial cells. , 1995, Cell motility and the cytoskeleton.

[31]  R. Burns.  Analysis of the γ-tubulin sequences : implications for the functional properties of γ-tubulin , 1995 .

[32]  M. Kirschner,et al.  In vitro reconstitution of centrosome assembly and function: The central role of γ-tubulin , 1994, Cell.

[33]  H. Joshi.  Microtubule organizing centers and γ-tubulin , 1994 .

[34]  M. Félix,et al.  Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation , 1994, The Journal of cell biology.

[35]  W. Theurkauf.  Immunofluorescence analysis of the cytoskeleton during oogenesis and early embryogenesis. , 1994, Methods in cell biology.

[36]  R. Wade,et al.  Cryoelectron microscopy of microtubules. , 1993, Journal of structural biology.

[37]  A. Hyman,et al.  Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. , 1992, Molecular biology of the cell.

[38]  H. Joshi,et al.  γ-Tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation , 1992, Nature.

[39]  B. Alberts,et al.  Purification of a multiprotein complex containing centrosomal proteins from the Drosophila embryo by chromatography with low-affinity polyclonal antibodies. , 1992, Molecular biology of the cell.

[40]  M. Yanagida,et al.  The fission yeast gamma-tubulin is essential for mitosis and is localized at microtubule organizing centers. , 1991, Journal of cell science.

[41]  B. Oakley,et al.  γ-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans , 1990, Cell.

[42]  B. Oakley,et al.  Identification of γ-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans , 1989, Nature.

[43]  H. Erickson,et al.  The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. , 1984, The Journal of biological chemistry.

[44]  H. Ris,et al.  Centriole distribution during tripolar mitosis in Chinese hamster ovary cells , 1984, The Journal of cell biology.

[45]  J. Davies,et al.  Molecular Biology of the Cell , 1983, Bristol Medico-Chirurgical Journal.

[46]  B. Zeeberg,et al.  Determination of free and bound microtubular protein and guanine nucleotide under equilibrium conditions. , 1979, Biochemistry.

[47]  R. Weisenberg,et al.  Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules. , 1976, Biochemistry.

[48]  F. Frankel.  Organization and energy-dependent growth of microtubules in cells. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[49]  K. Weber,et al.  Cytoplasmic microtubules in tissue culture cells appear to grow from an organizing structure towards the plasma membrane. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[50]  K. J. Monty,et al.  Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases. , 1966, Biochimica et biophysica acta.