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. 1987 Mar 1;104(3):439–446. doi: 10.1083/jcb.104.3.439

Distinct localization and cell cycle dependence of COOH terminally tyrosinolated alpha-tubulin in the microtubules of Trypanosoma brucei brucei

PMCID: PMC2114556  PMID: 3546334

Abstract

alpha-Tubulin can be posttranslationally modified in that its COOH- terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of alpha-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both alpha- tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated alpha-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated alpha-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated alpha-tubulin is a marker for newly formed microtubules.

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Selected References

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  1. 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]
  2. Argarana C. E., Barra H. S., Caputto R. Tubulinyl-tyrosine carboxypeptidase from chicken brain: properties and partial purification. J Neurochem. 1980 Jan;34(1):114–118. doi: 10.1111/j.1471-4159.1980.tb04628.x. [DOI] [PubMed] [Google Scholar]
  3. Barra H. S., Rodriguez J. A., Arce C. A., Caputto R. A soluble preparation from rat brain that incorporates into its own proteins ( 14 C)arginine by a ribonuclease-sensitive system and ( 14 C)tyrosine by a ribonuclease-insensitive system. J Neurochem. 1973 Jan;20(1):97–108. doi: 10.1111/j.1471-4159.1973.tb12108.x. [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. Cleveland D. W., Sullivan K. F. Molecular biology and genetics of tubulin. Annu Rev Biochem. 1985;54:331–365. doi: 10.1146/annurev.bi.54.070185.001555. [DOI] [PubMed] [Google Scholar]
  6. Cumming R., Burgoyne R. D., Lytton N. A. Immunocytochemical demonstration of alpha-tubulin modification during axonal maturation in the cerebellar cortex. J Cell Biol. 1984 Jan;98(1):347–351. doi: 10.1083/jcb.98.1.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cumming R., Williamson J. Differential labelling of trypanosome microtubules using tubulin subunit monoclonal antibodies. Cell Biol Int Rep. 1984 Jan;8(1):2–2. doi: 10.1016/0309-1651(84)90174-7. [DOI] [PubMed] [Google Scholar]
  8. Deanin G. G., Preston S. F., Gordon M. W. Carboxyl terminal tyrosine metabolism of alpha tubulin and changes in cell shape: Chinese hamster ovary cells. Biochem Biophys Res Commun. 1981 Jun;100(4):1642–1650. doi: 10.1016/0006-291x(81)90707-5. [DOI] [PubMed] [Google Scholar]
  9. Eipper B. A. Rat brain tubulin and protein kinase activity. J Biol Chem. 1974 Mar 10;249(5):1398–1406. [PubMed] [Google Scholar]
  10. Feit H., Shelanski M. L. Is tubulin a glycoprotein? Biochem Biophys Res Commun. 1975 Oct 6;66(3):920–927. doi: 10.1016/0006-291x(75)90728-7. [DOI] [PubMed] [Google Scholar]
  11. Flavin M., Murofushi H. Tyrosine incorporation in tubulin. Methods Enzymol. 1984;106:223–237. doi: 10.1016/0076-6879(84)06024-9. [DOI] [PubMed] [Google Scholar]
  12. Forrest G. L., Klevecz R. R. Tyrosyltubulin ligase and colchicine binding activity in synchronized Chinese hamster cells. J Cell Biol. 1978 Aug;78(2):441–450. doi: 10.1083/jcb.78.2.441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gabius H. J., Graupner G., Cramer F. Activity patterns of aminoacyl-tRNA synthetases, tRNA methylases, arginyltransferase and tubulin: tyrosine ligase during development and ageing of Caenorhabditis elegans. Eur J Biochem. 1983 Mar 1;131(1):231–234. doi: 10.1111/j.1432-1033.1983.tb07254.x. [DOI] [PubMed] [Google Scholar]
  14. Gundersen G. G., Kalnoski M. H., Bulinski J. C. Distinct populations of microtubules: tyrosinated and nontyrosinated alpha tubulin are distributed differently in vivo. Cell. 1984 Oct;38(3):779–789. doi: 10.1016/0092-8674(84)90273-3. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Kimmel B. E., Samson S., Wu J., Hirschberg R., Yarbrough L. R. Tubulin genes of the African trypanosome Trypanosoma brucei rhodesiense:nucleotide sequence of a 3.7-kb fragment containing genes for alpha and beta tubulins. Gene. 1985;35(3):237–248. doi: 10.1016/0378-1119(85)90002-2. [DOI] [PubMed] [Google Scholar]
  17. Kumar N., Flavin M. Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin. J Biol Chem. 1981 Jul 25;256(14):7678–7686. [PubMed] [Google Scholar]
  18. L'Hernault S. W., Rosenbaum J. L. Chlamydomonas alpha-tubulin is posttranslationally modified in the flagella during flagellar assembly. J Cell Biol. 1983 Jul;97(1):258–263. doi: 10.1083/jcb.97.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Martensen T. M. Preparation of brain tyrosinotubulin carboxypeptidase. Methods Cell Biol. 1982;24:265–269. doi: 10.1016/s0091-679x(08)60660-3. [DOI] [PubMed] [Google Scholar]
  20. Matsumoto G., Ichikawa M., Tasaki A. Axonal microtubules necessary for generation of sodium current in squid giant axons: II. Effect of colchicine upon asymmetrical displacement current. J Membr Biol. 1984;77(2):93–99. doi: 10.1007/BF01925859. [DOI] [PubMed] [Google Scholar]
  21. Matsumoto G., Ichikawa M., Tasaki A., Murofushi H., Sakai H. Axonal microtubules necessary for generation of sodium current in squid giant axons: I. Pharmacological study on sodium current and restoration of sodium current by microtubule proteins and 260K protein. J Membr Biol. 1984;77(2):77–91. doi: 10.1007/BF01925858. [DOI] [PubMed] [Google Scholar]
  22. Nath J., Flavin M., Schiffmann E. Stimulation of tubulin tyrosinolation in rabbit leukocytes evoked by the chemoattractant formyl-methionyl-leucyl-phenylalanine. J Cell Biol. 1981 Oct;91(1):232–239. doi: 10.1083/jcb.91.1.232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nath J., Flavin M. Tubulin tyrosylation in vivo and changes accompanying differentiation of cultured neuroblastoma-glioma hybrid cells. J Biol Chem. 1979 Nov 25;254(22):11505–11510. [PubMed] [Google Scholar]
  24. Nath J., Gallin J. I. Modulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMM). Kroc Found Ser. 1984;16:95–110. [PubMed] [Google Scholar]
  25. Ponstingl H., Krauhs E., Little M., Kempf T., Hofer-Warbinek R., Ade W. Amino acid sequence of alpha- and beta-tubulins from pig brain: heterogeneity and regional similarity to muscle proteins. Cold Spring Harb Symp Quant Biol. 1982;46(Pt 1):191–197. doi: 10.1101/sqb.1982.046.01.022. [DOI] [PubMed] [Google Scholar]
  26. Preston S. F., Deanin G. G., Hanson R. K., Gordon M. W. The phylogenetic distribution of tubulin:tyrosine ligase. J Mol Evol. 1979 Oct;13(3):233–244. doi: 10.1007/BF01739482. [DOI] [PubMed] [Google Scholar]
  27. Raybin D., Flavin M. An enzyme tyrosylating alpha-tubulin and its role in microtubule assembly. Biochem Biophys Res Commun. 1975 Aug 4;65(3):1088–1095. doi: 10.1016/s0006-291x(75)80497-9. [DOI] [PubMed] [Google Scholar]
  28. Raybin D., Flavin M. Enzyme which specifically adds tyrosine to the alpha chain of tubulin. Biochemistry. 1977 May 17;16(10):2189–2194. doi: 10.1021/bi00629a023. [DOI] [PubMed] [Google Scholar]
  29. Russell D. G., Gull K. Flagellar regeneration of the trypanosome Crithidia fasciculata involves post-translational modification of cytoplasmic alpha tubulin. Mol Cell Biol. 1984 Jun;4(6):1182–1185. doi: 10.1128/mcb.4.6.1182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Sandoval I. V., Cuatrecasas P. Protein kinase associated with tubulin: affinity chromatography and properties. Biochemistry. 1976 Aug 10;15(16):3424–3432. doi: 10.1021/bi00661a005. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Schröder H. C., Wehland J., Weber K. Purification of brain tubulin-tyrosine ligase by biochemical and immunological methods. J Cell Biol. 1985 Jan;100(1):276–281. doi: 10.1083/jcb.100.1.276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Thompson W. C., Deanin G. G., Gordon M. W. Intact microtubules are required for rapid turnover of carboxyl-terminal tyrosine of alpha-tubulin in cell cultures. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1318–1322. doi: 10.1073/pnas.76.3.1318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Thompson W. C. The cyclic tyrosination/detyrosination of alpha tubulin. Methods Cell Biol. 1982;24:235–255. doi: 10.1016/s0091-679x(08)60658-5. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Wehland J., Schröder H. C., Weber K. Amino acid sequence requirements in the epitope recognized by the alpha-tubulin-specific rat monoclonal antibody YL 1/2. EMBO J. 1984 Jun;3(6):1295–1300. doi: 10.1002/j.1460-2075.1984.tb01965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Wehland J., Willingham M. C., Sandoval I. V. A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo. J Cell Biol. 1983 Nov;97(5 Pt 1):1467–1475. doi: 10.1083/jcb.97.5.1467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Williamson D. H., Fennell D. J. The use of fluorescent DNA-binding agent for detecting and separating yeast mitochondrial DNA. Methods Cell Biol. 1975;12:335–351. doi: 10.1016/s0091-679x(08)60963-2. [DOI] [PubMed] [Google Scholar]

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