Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1985 Jan;4(1):51–56. doi: 10.1002/j.1460-2075.1985.tb02316.x

Structural differences between brain beta 1- and beta 2-tubulins: implications for microtubule assembly and colchicine binding.

M Little, R F Ludueña
PMCID: PMC554150  PMID: 4018027

Abstract

Brain beta 1- and beta 2-tubulins are the major and minor beta-tubulin components of chordate brain tissue, respectively. Two cysteines of beta 1, but not beta 2, can be specifically cross-linked with the bifunctional sulfhydryl reagent N,N'-ethylenebis(iodoacetamide) (EBI). They are in positions 239 and 354. Although separated by 115 amino acid residues along the beta 1-chain, the two sulfur atoms are maximally 9 A apart in the beta 1 tertiary structure. The failure of beta 2 to form a similar cross-bridge is due to the absence of a cysteine in position 239. At least 10 other sequence differences are also present between beta 1 and beta 2. Positions 239 and 354 of beta 1 probably occupy a key part of the tubulin molecule. The microtubule assembly inhibitors colchicine and podophyllotoxin appear to bind on or near this site and EBI is a potent inhibitor of microtubule assembly. Furthermore, the beta 1-cysteine in position 239 appears to be the most reactive in brain tubulin under the given conditions. The marked difference between beta 1 and beta 2 in this critical region suggests that they may have different functions in brain tissue.

Full text

PDF
51

Images in this article

52Fig. 1.
on p.52

Selected References

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

  1. Alexandraki D., Ruderman J. V. Evolution of alpha q- and beta-tubulin genes as inferred by the nucleotide sequences of sea urchin cDNA clones. J Mol Evol. 1983;19(6):397–410. doi: 10.1007/BF02102315. [DOI] [PubMed] [Google Scholar]
  2. Brauer A. W., Margolies M. N., Haber E. The application of 0.1 M quadrol to the microsequence of proteins and the sequence of tryptic peptides. Biochemistry. 1975 Jul;14(13):3029–3035. doi: 10.1021/bi00684a036. [DOI] [PubMed] [Google Scholar]
  3. CRESTFIELD A. M., MOORE S., STEIN W. H. The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J Biol Chem. 1963 Feb;238:622–627. [PubMed] [Google Scholar]
  4. Cortese F., Bhattacharyya B., Wolff J. Podophyllotoxin as a probe for the colchicine binding site of tubulin. J Biol Chem. 1977 Feb 25;252(4):1134–1140. [PubMed] [Google Scholar]
  5. Davidse L. C., Flach W. Differential binding of methyl benzimidazol-2-yl carbamate to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J Cell Biol. 1977 Jan;72(1):174–193. doi: 10.1083/jcb.72.1.174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fellous A., Francon J., Lennon A. M., Nunez J. Microtubule assembly in vitro. Purification of assembly-promoting factors. Eur J Biochem. 1977 Aug 15;78(1):167–174. doi: 10.1111/j.1432-1033.1977.tb11726.x. [DOI] [PubMed] [Google Scholar]
  7. Hoebeke J., Van Nijen G., De Brabander M. Interaction of oncodazole (R 17934), a new antitumoral drug, with rat brain tubulin. Biochem Biophys Res Commun. 1976 Mar 22;69(2):319–324. doi: 10.1016/0006-291x(76)90524-6. [DOI] [PubMed] [Google Scholar]
  8. Jany K. D., Keil W., Meyer H., Kiltz H. H. Preparation of a highly purified bovine trypsin for use in protein sequence analysis. Biochim Biophys Acta. 1976 Nov 26;453(1):62–66. doi: 10.1016/0005-2795(76)90250-6. [DOI] [PubMed] [Google Scholar]
  9. Keates R. A., Sarangi F., Ling V. Structural and functional alterations in microtubule protein from Chinese hamster ovary cell mutants. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5638–5642. doi: 10.1073/pnas.78.9.5638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kilmartin J. V. Purification of yeast tubulin by self-assembly in vitro. Biochemistry. 1981 Jun 9;20(12):3629–3633. doi: 10.1021/bi00515a050. [DOI] [PubMed] [Google Scholar]
  11. Krauhs E., Little M., Kempf T., Hofer-Warbinek R., Ade W., Ponstingl H. Complete amino acid sequence of beta-tubulin from porcine brain. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4156–4160. doi: 10.1073/pnas.78.7.4156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuriyama R., Sakai H. Role of tubulin-SH groups in polymerization to microtubules. Functional-SH groups in tubulin for polymerization. J Biochem. 1974 Sep;76(3):651–654. doi: 10.1093/oxfordjournals.jbchem.a130609. [DOI] [PubMed] [Google Scholar]
  13. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  14. 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]
  15. Ling V., Aubin J. E., Chase A., Sarangi F. Mutants of Chinese hamster ovary (CHO) cells with altered colcemid-binding affinity. Cell. 1979 Oct;18(2):423–430. doi: 10.1016/0092-8674(79)90061-8. [DOI] [PubMed] [Google Scholar]
  16. Little M. Identification of a second beta chain in pig brain tubulin. FEBS Lett. 1979 Dec 1;108(1):283–286. doi: 10.1016/0014-5793(79)81229-6. [DOI] [PubMed] [Google Scholar]
  17. Ludueña R. F., Roach M. C. Interaction of tubulin with drugs and alkylating agents. 1. Alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide). Biochemistry. 1981 Jul 21;20(15):4437–4444. doi: 10.1021/bi00518a031. [DOI] [PubMed] [Google Scholar]
  18. Ludueña R. F., Roach M. C. Interaction of tubulin with drugs and alkylating agents. 2. Effects of colchicine, podophyllotoxin, and vinblastine on the alkylation of tubulin. Biochemistry. 1981 Jul 21;20(15):4444–4450. doi: 10.1021/bi00518a032. [DOI] [PubMed] [Google Scholar]
  19. Ludueña R. F., Roach M. C., Trcka P. P., Little M., Palanivelu P., Binkley P., Prasad V. beta 2-Tubulin, a form of chordate brain tubulin with lesser reactivity toward an assembly-inhibiting sulfhydryl-directed cross-linking reagent. Biochemistry. 1982 Sep 14;21(19):4787–4794. doi: 10.1021/bi00262a041. [DOI] [PubMed] [Google Scholar]
  20. Mann K., Giesel M., Fasold H., Haase W. Isolation of native microtubules from porcine brain and characterization of SH groups essential for polymerization at the GTP binding sites. FEBS Lett. 1978 Aug 1;92(1):45–48. doi: 10.1016/0014-5793(78)80718-2. [DOI] [PubMed] [Google Scholar]
  21. Mellon M. G., Rebhun L. I. Sulfhydryls and the in vitro polymerization of tubulin. J Cell Biol. 1976 Jul;70(1):226–238. doi: 10.1083/jcb.70.1.226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Neff N. F., Thomas J. H., Grisafi P., Botstein D. Isolation of the beta-tubulin gene from yeast and demonstration of its essential function in vivo. Cell. 1983 May;33(1):211–219. doi: 10.1016/0092-8674(83)90350-1. [DOI] [PubMed] [Google Scholar]
  23. Ponstingl H., Krauhs E., Little M., Kempf T. Complete amino acid sequence of alpha-tubulin from porcine brain. Proc Natl Acad Sci U S A. 1981 May;78(5):2757–2761. doi: 10.1073/pnas.78.5.2757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmitt H., Atlas D. Specific affinity labelling of tubulin with bromocolchicine. J Mol Biol. 1976 Apr 25;102(4):743–758. doi: 10.1016/0022-2836(76)90289-8. [DOI] [PubMed] [Google Scholar]
  25. Serrano L., Avila J., Maccioni R. B. Limited proteolysis of tubulin and the localization of the binding site for colchicine. J Biol Chem. 1984 May 25;259(10):6607–6611. [PubMed] [Google Scholar]
  26. Sheir-Neiss G., Lai M. H., Morris N. R. Identification of a gene for beta-tubulin in Aspergillus nidulans. Cell. 1978 Oct;15(2):639–647. doi: 10.1016/0092-8674(78)90032-6. [DOI] [PubMed] [Google Scholar]
  27. Valenzuela P., Quiroga M., Zaldivar J., Rutter W. J., Kirschner M. W., Cleveland D. W. Nucleotide and corresponding amino acid sequences encoded by alpha and beta tubulin mRNAs. Nature. 1981 Feb 19;289(5799):650–655. doi: 10.1038/289650a0. [DOI] [PubMed] [Google Scholar]
  28. Wehland J., Henkart M., Klausner R., Sandoval I. V. Role of microtubules in the distribution of the Golgi apparatus: effect of taxol and microinjected anti-alpha-tubulin antibodies. Proc Natl Acad Sci U S A. 1983 Jul;80(14):4286–4290. doi: 10.1073/pnas.80.14.4286. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES