Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Aug 1;365(Pt 3):889–895. doi: 10.1042/BJ20020175

Association of brain gamma-tubulins with alpha beta-tubulin dimers.

Vadym Sulimenko 1, Tetyana Sulimenko 1, Slobodan Poznanovic 1, Volodymyr Nechiporuk-Zloy 1, Konrad J Böhm 1, Libor Macurek 1, Eberhard Unger 1, Pavel Dráber 1
PMCID: PMC1222706  PMID: 11939909

Abstract

gamma-Tubulin is necessary for nucleation and polar orientation of microtubules in vivo. The molecular mechanism of microtubule nucleation by gamma-tubulin and the regulation of this process are not fully understood. Here we show that there are two gamma-tubulin forms in the brain that are present in complexes of various sizes. Large complexes tend to dissociate in the presence of a high salt concentration. Both gamma-tubulins co-polymerized with tubulin dimers, and multiple gamma-tubulin bands were identified in microtubule protein preparations under conditions of non-denaturing electrophoresis. Immunoprecipitation experiments with monoclonal antibodies against gamma-tubulin and alpha-tubulin revealed interactions of both gamma-tubulin forms with tubulin dimers, irrespective of the size of complexes. We suggest that, besides small and large gamma-tubulin complexes, other molecular gamma-tubulin form(s) exist in brain extracts. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin in both brain extracts and microtubule protein preparations. Post-translational modification(s) of gamma-tubulins might therefore have an important role in the regulation of microtubule nucleation in neuronal cells.

Full Text

The Full Text of this article is available as a PDF (198.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Best D., Warr P. J., Gull K. Influence of the composition of commercial sodium dodecyl sulfate preparations on the separation of alpha- and beta-tubulin during polyacrylamide gel electrophoresis. Anal Biochem. 1981 Jul 1;114(2):281–284. doi: 10.1016/0003-2697(81)90481-4. [DOI] [PubMed] [Google Scholar]
  2. Caudron N., Valiron O., Usson Y., Valiron P., Job D. A reassessment of the factors affecting microtubule assembly and disassembly in vitro. J Mol Biol. 2000 Mar 17;297(1):211–220. doi: 10.1006/jmbi.2000.3554. [DOI] [PubMed] [Google Scholar]
  3. Correia J. J., Williams R. C., Jr Characterization of oligomers of tubulin by two-dimensional native electrophoresis. Arch Biochem Biophys. 1985 May 15;239(1):120–129. doi: 10.1016/0003-9861(85)90818-5. [DOI] [PubMed] [Google Scholar]
  4. Detraves C., Mazarguil H., Lajoie-Mazenc I., Julian M., Raynaud-Messina B., Wright M. Protein complexes containing gamma-tubulin are present in mammalian brain microtubule protein preparations. Cell Motil Cytoskeleton. 1997;36(2):179–189. doi: 10.1002/(SICI)1097-0169(1997)36:2<179::AID-CM7>3.0.CO;2-4. [DOI] [PubMed] [Google Scholar]
  5. Dráber P., Dráberová E., Linhartová I., Viklický V. Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies. J Cell Sci. 1989 Mar;92(Pt 3):519–528. doi: 10.1242/jcs.92.3.519. [DOI] [PubMed] [Google Scholar]
  6. Dráber P., Lagunowich L. A., Dráberová E., Viklický V., Damjanov I. Heterogeneity of tubulin epitopes in mouse fetal tissues. Histochemistry. 1988;89(5):485–492. doi: 10.1007/BF00492606. [DOI] [PubMed] [Google Scholar]
  7. Dráberová E., Dráber P., Havlícek F., Viklický V. A common antigenic determinant of vimentin and desmin defined by monoclonal antibody. Folia Biol (Praha) 1986;32(5):295–303. [PubMed] [Google Scholar]
  8. Dráberová L., Dráberová E., Surviladze Z., Dráber P., Dráber P. Protein tyrosine kinase p53/p56(lyn) forms complexes with gamma-tubulin in rat basophilic leukemia cells. Int Immunol. 1999 Nov;11(11):1829–1839. doi: 10.1093/intimm/11.11.1829. [DOI] [PubMed] [Google Scholar]
  9. Feng Y., Hodge D. R., Palmieri G., Chase D. L., Longo D. L., Ferris D. K. Association of polo-like kinase with alpha-, beta- and gamma-tubulins in a stable complex. Biochem J. 1999 Apr 15;339(Pt 2):435–442. [PMC free article] [PubMed] [Google Scholar]
  10. George H. J., Misra L., Field D. J., Lee J. C. Polymorphism of brain tubulin. Biochemistry. 1981 Apr 28;20(9):2402–2409. doi: 10.1021/bi00512a006. [DOI] [PubMed] [Google Scholar]
  11. Görg A., Obermaier C., Boguth G., Weiss W. Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis. 1999 Apr-May;20(4-5):712–717. doi: 10.1002/(SICI)1522-2683(19990101)20:4/5<712::AID-ELPS712>3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
  12. Inclán Y. F., Nogales E. Structural models for the self-assembly and microtubule interactions of gamma-, delta- and epsilon-tubulin. J Cell Sci. 2001 Jan;114(Pt 2):413–422. doi: 10.1242/jcs.114.2.413. [DOI] [PubMed] [Google Scholar]
  13. Joshi H. C., Palacios M. J., McNamara L., Cleveland D. W. Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature. 1992 Mar 5;356(6364):80–83. doi: 10.1038/356080a0. [DOI] [PubMed] [Google Scholar]
  14. Kellogg D. R., Moritz M., Alberts B. M. The centrosome and cellular organization. Annu Rev Biochem. 1994;63:639–674. doi: 10.1146/annurev.bi.63.070194.003231. [DOI] [PubMed] [Google Scholar]
  15. Knop M., Schiebel E. Spc98p and Spc97p of the yeast gamma-tubulin complex mediate binding to the spindle pole body via their interaction with Spc110p. EMBO J. 1997 Dec 1;16(23):6985–6995. doi: 10.1093/emboj/16.23.6985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  17. Lajoie-Mazenc I., Détraves C., Rotaru V., Garès M., Tollon Y., Jean C., Julian M., Wright M., Raynaud-Messina B. A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres. J Cell Sci. 1996 Oct;109(Pt 10):2483–2492. doi: 10.1242/jcs.109.10.2483. [DOI] [PubMed] [Google Scholar]
  18. Leguy R., Melki R., Pantaloni D., Carlier M. F. Monomeric gamma -tubulin nucleates microtubules. J Biol Chem. 2000 Jul 21;275(29):21975–21980. doi: 10.1074/jbc.M000688200. [DOI] [PubMed] [Google Scholar]
  19. Lessman C. A., Kim H. Soluble tubulin complexes in oocytes of the common leopard frog, Rana pipiens, contain gamma-tubulin. Mol Reprod Dev. 2001 Sep;60(1):128–136. doi: 10.1002/mrd.1069. [DOI] [PubMed] [Google Scholar]
  20. Li Q., Joshi H. C. gamma-tubulin is a minus end-specific microtubule binding protein. J Cell Biol. 1995 Oct;131(1):207–214. doi: 10.1083/jcb.131.1.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Linhartová I., Dráber P., Dráberová E., Viklický V. Immunological discrimination of beta-tubulin isoforms in developing mouse brain. Post-translational modification of non-class-III beta-tubulins. Biochem J. 1992 Dec 15;288(Pt 3):919–924. doi: 10.1042/bj2880919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Liu B., Marc J., Joshi H. C., Palevitz B. A. A gamma-tubulin-related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner. J Cell Sci. 1993 Apr;104(Pt 4):1217–1228. doi: 10.1242/jcs.104.4.1217. [DOI] [PubMed] [Google Scholar]
  23. Llamazares S., Tavosanis G., Gonzalez C. Cytological characterisation of the mutant phenotypes produced during early embryogenesis by null and loss-of-function alleles of the gammaTub37C gene in Drosophila. J Cell Sci. 1999 Mar;112(Pt 5):659–667. doi: 10.1242/jcs.112.5.659. [DOI] [PubMed] [Google Scholar]
  24. Llanos R., Chevrier V., Ronjat M., Meurer-Grob P., Martinez P., Frank R., Bornens M., Wade R. H., Wehland J., Job D. Tubulin binding sites on gamma-tubulin: identification and molecular characterization. Biochemistry. 1999 Nov 30;38(48):15712–15720. doi: 10.1021/bi990895w. [DOI] [PubMed] [Google Scholar]
  25. Ludueña R. F. Multiple forms of tubulin: different gene products and covalent modifications. Int Rev Cytol. 1998;178:207–275. doi: 10.1016/s0074-7696(08)62138-5. [DOI] [PubMed] [Google Scholar]
  26. Mandelkow E. M., Herrmann M., Rühl U. Tubulin domains probed by limited proteolysis and subunit-specific antibodies. J Mol Biol. 1985 Sep 20;185(2):311–327. doi: 10.1016/0022-2836(85)90406-1. [DOI] [PubMed] [Google Scholar]
  27. Meads T., Schroer T. A. Polarity and nucleation of microtubules in polarized epithelial cells. Cell Motil Cytoskeleton. 1995;32(4):273–288. doi: 10.1002/cm.970320404. [DOI] [PubMed] [Google Scholar]
  28. Moritz M., Braunfeld M. B., Fung J. C., Sedat J. W., Alberts B. M., Agard D. A. Three-dimensional structural characterization of centrosomes from early Drosophila embryos. J Cell Biol. 1995 Sep;130(5):1149–1159. doi: 10.1083/jcb.130.5.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Moritz M., Zheng Y., Alberts B. M., Oegema K. Recruitment of the gamma-tubulin ring complex to Drosophila salt-stripped centrosome scaffolds. J Cell Biol. 1998 Aug 10;142(3):775–786. doi: 10.1083/jcb.142.3.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moudjou M., Bordes N., Paintrand M., Bornens M. gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms. J Cell Sci. 1996 Apr;109(Pt 4):875–887. doi: 10.1242/jcs.109.4.875. [DOI] [PubMed] [Google Scholar]
  31. Murphy S. M., Preble A. M., Patel U. K., O'Connell K. L., Dias D. P., Moritz M., Agard D., Stults J. T., Stearns T. GCP5 and GCP6: two new members of the human gamma-tubulin complex. Mol Biol Cell. 2001 Nov;12(11):3340–3352. doi: 10.1091/mbc.12.11.3340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Murphy S. M., Urbani L., Stearns T. The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p. J Cell Biol. 1998 May 4;141(3):663–674. doi: 10.1083/jcb.141.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Nováková M., Dráberová E., Schürmann W., Czihak G., Viklický V., Dr-aber P. gamma-Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies. Cell Motil Cytoskeleton. 1996;33(1):38–51. doi: 10.1002/(SICI)1097-0169(1996)33:1<38::AID-CM5>3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  34. Oakley B. R., Oakley C. E., Yoon Y., Jung M. K. Gamma-tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell. 1990 Jun 29;61(7):1289–1301. doi: 10.1016/0092-8674(90)90693-9. [DOI] [PubMed] [Google Scholar]
  35. Oakley C. E., Oakley B. R. Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature. 1989 Apr 20;338(6217):662–664. doi: 10.1038/338662a0. [DOI] [PubMed] [Google Scholar]
  36. Oegema K., Wiese C., Martin O. C., Milligan R. A., Iwamatsu A., Mitchison T. J., Zheng Y. Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules. J Cell Biol. 1999 Feb 22;144(4):721–733. doi: 10.1083/jcb.144.4.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Raynaud-Messina B., Debec A., Tollon Y., Garès M., Wright M. Differential properties of the two Drosophila gamma-tubulin isotypes. Eur J Cell Biol. 2001 Oct;80(10):643–649. doi: 10.1078/0171-9335-00195. [DOI] [PubMed] [Google Scholar]
  38. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vassilev A., Kimble M., Silflow C. D., LaVoie M., Kuriyama R. Identification of intrinsic dimer and overexpressed monomeric forms of gamma-tubulin in Sf9 cells infected with baculovirus containing the Chlamydomonas gamma-tubulin sequence. J Cell Sci. 1995 Mar;108(Pt 3):1083–1092. doi: 10.1242/jcs.108.3.1083. [DOI] [PubMed] [Google Scholar]
  40. Viklický V., Dráber P., Hasek J., Bártek J. Production and characterization of a monoclonal antitubulin antibody. Cell Biol Int Rep. 1982 Aug;6(8):725–731. doi: 10.1016/0309-1651(82)90164-3. [DOI] [PubMed] [Google Scholar]
  41. Vogel J., Drapkin B., Oomen J., Beach D., Bloom K., Snyder M. Phosphorylation of gamma-tubulin regulates microtubule organization in budding yeast. Dev Cell. 2001 Nov;1(5):621–631. doi: 10.1016/s1534-5807(01)00073-9. [DOI] [PubMed] [Google Scholar]
  42. Weingarten M. D., Lockwood A. H., Hwo S. Y., Kirschner M. W. A protein factor essential for microtubule assembly. Proc Natl Acad Sci U S A. 1975 May;72(5):1858–1862. doi: 10.1073/pnas.72.5.1858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wiese C., Zheng Y. A new function for the gamma-tubulin ring complex as a microtubule minus-end cap. Nat Cell Biol. 2000 Jun;2(6):358–364. doi: 10.1038/35014051. [DOI] [PubMed] [Google Scholar]
  44. Wiese C., Zheng Y. Gamma-tubulin complexes and their interaction with microtubule-organizing centers. Curr Opin Struct Biol. 1999 Apr;9(2):250–259. doi: 10.1016/S0959-440X(99)80035-9. [DOI] [PubMed] [Google Scholar]
  45. Wilson P. G., Zheng Y., Oakley C. E., Oakley B. R., Borisy G. G., Fuller M. T. Differential expression of two gamma-tubulin isoforms during gametogenesis and development in Drosophila. Dev Biol. 1997 Apr 15;184(2):207–221. doi: 10.1006/dbio.1997.8545. [DOI] [PubMed] [Google Scholar]
  46. Wise D. O., Krahe R., Oakley B. R. The gamma-tubulin gene family in humans. Genomics. 2000 Jul 15;67(2):164–170. doi: 10.1006/geno.2000.6247. [DOI] [PubMed] [Google Scholar]
  47. Wolff A., Denoulet P., Jeantet C. High level of tubulin microheterogeneity in the mouse brain. Neurosci Lett. 1982 Aug 31;31(3):323–328. doi: 10.1016/0304-3940(82)90041-6. [DOI] [PubMed] [Google Scholar]
  48. Zheng Y., Wong M. L., Alberts B., Mitchison T. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature. 1995 Dec 7;378(6557):578–583. doi: 10.1038/378578a0. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES