Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Jan 1;265(1):87–93. doi: 10.1042/bj2650087

Purification and assembly in vitro of tubulin from Trypanosoma brucei brucei.

T H MacRae 1, K Gull 1
PMCID: PMC1136617  PMID: 2302174

Abstract

Trypanosome tubulin was purified to near homogeneity by chromatography on DEAE-Sephadex, Amicon filtration and assembly-disassembly in vitro. Polymerization of the tubulin in vitro yielded long, structurally normal, microtubules and some sheet structures on addition of GTP and incubation at 37 degrees C, in either the presence or the absence of Mg2+. Tubulin assembly was disrupted by glycerol and a selection of microtubule-reactive drugs. Immunological analysis of the purified tubulin revealed tyrosinated and acetylated alpha-tubulin, in addition to defining the migration characteristics of the alpha- and beta-tubulin on one-dimensional SDS/polyacrylamide gels. This is the first isolation of trypanosome tubulin with the ability to form structurally normal microtubules independent of the addition of taxol or nucleating microtubule fragments. The development of the purification procedure thus provides an important step for subsequent study of microtubule-associated protein-tubulin and plasma-membrane-microtubule cytoskeleton interactions of trypanosomes, and increases the potential for development of tubulin-based anti-trypanosome drugs.

Full text

PDF
87

Images in this article

89Fig. 1.
on p.89
89Fig. 2.
on p.89
90Fig. 3.
on p.90
91Fig. 4.
on p.91

Selected References

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

  1. Alcina A., Hargreaves A. J., Avila J., Hesketh J. E., Fresno M. The detection of a spectrin-like protein in Trypanosoma cruzi with a polyclonal antibody. Cell Biol Int Rep. 1988 Nov;12(11):979–985. doi: 10.1016/0309-1651(88)90166-x. [DOI] [PubMed] [Google Scholar]
  2. Angelopoulos E. Pellicular microtubules in the family Trypanosomatidae. J Protozool. 1970 Feb;17(1):39–51. doi: 10.1111/j.1550-7408.1970.tb05157.x. [DOI] [PubMed] [Google Scholar]
  3. Birkett C. R., Foster K. E., Johnson L., Gull K. Use of monoclonal antibodies to analyse the expression of a multi-tubulin family. FEBS Lett. 1985 Aug 5;187(2):211–218. doi: 10.1016/0014-5793(85)81244-8. [DOI] [PubMed] [Google Scholar]
  4. Blose S. H., Meltzer D. I., Feramisco J. R. 10-nm filaments are induced to collapse in living cells microinjected with monoclonal and polyclonal antibodies against tubulin. J Cell Biol. 1984 Mar;98(3):847–858. doi: 10.1083/jcb.98.3.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bramblett G. T., Chang S. L., Flavin M. Periodic crosslinking of microtubules by cytoplasmic microtubule-associated and microtubule-corset proteins from a trypanosomatid. Proc Natl Acad Sci U S A. 1987 May;84(10):3259–3263. doi: 10.1073/pnas.84.10.3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brun R., Schönenberger Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979 Sep;36(3):289–292. [PubMed] [Google Scholar]
  7. Campbell E. J., MacKinlay S. A., MacRae T. H. Cross-linking of microtubules by microtubule-associated proteins (MAPs) from the brine shrimp, Artemia. J Cell Sci. 1989 May;93(Pt 1):29–39. doi: 10.1242/jcs.93.1.29. [DOI] [PubMed] [Google Scholar]
  8. Chang S. L., Flavin M. Tubulin tyrosination in Crithidia: modifying enzymes and modification states of tubulin. Cell Motil Cytoskeleton. 1988;10(3):400–409. doi: 10.1002/cm.970100307. [DOI] [PubMed] [Google Scholar]
  9. Clayton L., Quinlan R. A., Roobol A., Pogson C. I., Gull K. A comparison of tubulins from mammalian brain and Physarum polycephalum using SDS-polyacrylamide gel electrophorsis and peptide mapping. FEBS Lett. 1980 Jun 30;115(2):301–305. doi: 10.1016/0014-5793(80)81192-6. [DOI] [PubMed] [Google Scholar]
  10. Dolan M. T., Reid C. G., Voorheis H. P. Calcium ions initiate the selective depolymerization of the pellicular microtubules in bloodstream forms of Trypanosoma brucei. J Cell Sci. 1986 Feb;80:123–140. doi: 10.1242/jcs.80.1.123. [DOI] [PubMed] [Google Scholar]
  11. Gallo J. M., Anderton B. H. A subpopulation of trypanosome microtubules recognized by a monoclonal antibody to tubulin. EMBO J. 1983;2(4):479–483. doi: 10.1002/j.1460-2075.1983.tb01450.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gallo J. M., Precigout E. Tubulin expression in trypanosomes. Biol Cell. 1988;64(2):137–143. doi: 10.1016/0248-4900(88)90073-1. [DOI] [PubMed] [Google Scholar]
  13. Gallo J. M., Précigout E., Schrével J. Subcellular sequestration of an antigenically unique beta-tubulin. Cell Motil Cytoskeleton. 1988;9(2):175–183. doi: 10.1002/cm.970090209. [DOI] [PubMed] [Google Scholar]
  14. Gallo J. M., Schrével J. Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody. Eur J Cell Biol. 1985 Mar;36(2):163–168. [PubMed] [Google Scholar]
  15. Gull K., Hussey P. J., Sasse R., Schneider A., Seebeck T., Sherwin T. Tubulin isotypes: generation of diversity in cells and microtubular organelles. J Cell Sci Suppl. 1986;5:243–255. doi: 10.1242/jcs.1986.supplement_5.16. [DOI] [PubMed] [Google Scholar]
  16. Kilmartin J. V., Wright B., Milstein C. Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line. J Cell Biol. 1982 Jun;93(3):576–582. doi: 10.1083/jcb.93.3.576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Macrae T. H., Ludueña R. F. Developmental and comparative aspects of brine shrimp tubulin. Biochem J. 1984 Apr 1;219(1):137–148. doi: 10.1042/bj2190137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ormerod W. E. Human and animal trypanosomiases as world public health problems. Pharmacol Ther. 1979;6(1):1–40. doi: 10.1016/0163-7258(79)90055-x. [DOI] [PubMed] [Google Scholar]
  20. Piperno G., Fuller M. T. Monoclonal antibodies specific for an acetylated form of alpha-tubulin recognize the antigen in cilia and flagella from a variety of organisms. J Cell Biol. 1985 Dec;101(6):2085–2094. doi: 10.1083/jcb.101.6.2085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Russell D. G., Miller D., Gull K. Tubulin heterogeneity in the trypanosome Crithidia fasciculata. Mol Cell Biol. 1984 Apr;4(4):779–790. doi: 10.1128/mcb.4.4.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Russell D. G., Newsam R. J., Palmer G. C., Gull K. Structural and biochemical characterisation of the paraflagellar rod of Crithidia fasciculata. Eur J Cell Biol. 1983 Mar;30(1):137–143. [PubMed] [Google Scholar]
  23. Saborio J. L., Manuel Hernandez J., Narayanswami S., Wrightsman R., Palmer E., Manning J. Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi. J Biol Chem. 1989 Mar 5;264(7):4071–4075. [PubMed] [Google Scholar]
  24. Sasse R., Gull K. Tubulin post-translational modifications and the construction of microtubular organelles in Trypanosoma brucei. J Cell Sci. 1988 Aug;90(Pt 4):577–589. doi: 10.1242/jcs.90.4.577. [DOI] [PubMed] [Google Scholar]
  25. Schneider A., Eichenberger W., Seebeck T. A microtubule-binding protein of Trypanosoma brucei which contains covalently bound fatty acid. J Biol Chem. 1988 May 15;263(14):6472–6475. [PubMed] [Google Scholar]
  26. Schneider A., Hemphill A., Wyler T., Seebeck T. Large microtubule-associated protein of T. brucei has tandemly repeated, near-identical sequences. Science. 1988 Jul 22;241(4864):459–462. doi: 10.1126/science.3393912. [DOI] [PubMed] [Google Scholar]
  27. Schneider A., Lutz H. U., Marugg R., Gehr P., Seebeck T. Spectrin-like proteins in the paraflagellar rod structure of Trypanosoma brucei. J Cell Sci. 1988 Jun;90(Pt 2):307–315. doi: 10.1242/jcs.90.2.307. [DOI] [PubMed] [Google Scholar]
  28. Schneider A., Sherwin T., Sasse R., Russell D. G., Gull K., Seebeck T. Subpellicular and flagellar microtubules of Trypanosoma brucei brucei contain the same alpha-tubulin isoforms. J Cell Biol. 1987 Mar;104(3):431–438. doi: 10.1083/jcb.104.3.431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Seebeck T., Küng V., Wyler T., Müller M. A 60-kDa cytoskeletal protein from Trypanosoma brucei brucei can interact with membranes and with microtubules. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1101–1104. doi: 10.1073/pnas.85.4.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sherwin T., Schneider A., Sasse R., Seebeck T., Gull K. Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei. J Cell Biol. 1987 Mar;104(3):439–446. doi: 10.1083/jcb.104.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stieger J., Wyler T., Seebeck T. Partial purification and characterization of microtubular protein from Trypanosoma brucei. J Biol Chem. 1984 Apr 10;259(7):4596–4602. [PubMed] [Google Scholar]
  32. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vallee R. B. A taxol-dependent procedure for the isolation of microtubules and microtubule-associated proteins (MAPs). J Cell Biol. 1982 Feb;92(2):435–442. doi: 10.1083/jcb.92.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Vickerman K. Developmental cycles and biology of pathogenic trypanosomes. Br Med Bull. 1985 Apr;41(2):105–114. doi: 10.1093/oxfordjournals.bmb.a072036. [DOI] [PubMed] [Google Scholar]
  35. Woods A., Sherwin T., Sasse R., MacRae T. H., Baines A. J., Gull K. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci. 1989 Jul;93(Pt 3):491–500. doi: 10.1242/jcs.93.3.491. [DOI] [PubMed] [Google Scholar]

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

RESOURCES