Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1977 May 1;73(2):492–504. doi: 10.1083/jcb.73.2.492

Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro

PMCID: PMC2109919  PMID: 558199

Abstract

A post-translational modification of tubulin with potential regulatory significance has been revealed by the discovery of an enzyme (tubulin- tyrosine ligase) in brain extracts which can add a tyrosine residue to the alpha chain, apparently through peptide bond linkage to a C- terminal glutamate. We have investigated whether this modification also occurs in vivo, and whether it alters the extent to which tubulin can assemble in vitro. Cytoplasmic tubulin purified from bovine brain by cycles of assembly was shown to be partially tyrosylated. Carboxypeptidase A digestion of isolated alpha chains liberated about 0.3 equivalent of tyrosine. Brief digestion of native tubulin increased the proportion of alpha chains which could be tyrosylated by ligase, from 25 to 45%. The tubulin assembled to the same extent before and after carboxypeptidase treatment. When tubulin was purified after introducing labeled tyrosine with ligase, the labeled species assembled in the same proportion as unlabeled. Thus tubulin can be incorporated into microbubules in vitro with or without C-terminal tyrosine. An apparent resolution of alpha chain into two components by hydroxylapatite chromatography was shown not to be due to the presence or absence of C-terminal tyrosine. Tubulin-tyrosine ligase was found in extracts of every rat tissue examined, but was not detected in sea urchin eggs before or after fertilization, in Tetrahymena cells or cilia, or in yeast. Cultured neuroblastoma cells fixed tyrosine into tubulin alpha chains under conditions where protein synthesis was inhibited; this in vivo fixation appeared to be into an insoluble moiety of tubulin. Incidental to these studies, a new assay utilizing an enamine substrate for carboxypeptidase was investigated.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. 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]
  2. Deanin G. G., Gordon M. W. The distribution of tyrosyltubulin ligase in brain and other tissues. Biochem Biophys Res Commun. 1976 Jul 26;71(2):676–683. doi: 10.1016/0006-291x(76)90841-x. [DOI] [PubMed] [Google Scholar]
  3. Hendler R. W., Burgess A. H. Fractionation of the electron-transport chain of Escherichia coli. Biochim Biophys Acta. 1974 Aug 23;357(2):215–230. doi: 10.1016/0005-2728(74)90062-0. [DOI] [PubMed] [Google Scholar]
  4. Jacobs M., Bennett P. M., Dickens M. J. Duplex microtubule is a new form of tubulin assembly induced by polycations. Nature. 1975 Oct 23;257(5528):707–709. doi: 10.1038/257707a0. [DOI] [PubMed] [Google Scholar]
  5. Jacobs M., Caplow M. Microtubular protein reaction with nucleotides. Biochem Biophys Res Commun. 1976 Jan 12;68(1):127–135. doi: 10.1016/0006-291x(76)90019-x. [DOI] [PubMed] [Google Scholar]
  6. Kobayashi T., Simizu T. Roles of nucleoside triphosphates in microtubule assembly. J Biochem. 1976 Jun;79(6):1357–1364. doi: 10.1093/oxfordjournals.jbchem.a131190. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Luduena R. F., Woodward D. O. Isolation and partial characterization of alpha and beta-tubulin from outer doublets of sea-urchin sperm and microtubules of chick-embryo brain. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3594–3598. doi: 10.1073/pnas.70.12.3594. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Morgan J. L., Seeds N. W. Tubulin constancy during morphological differentiation of mouse neuroblastoma cells. J Cell Biol. 1975 Oct;67(1):136–145. doi: 10.1083/jcb.67.1.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Moss B., Rosenblum E. N. Hydroxylapatite chromatography of protein-sodium dodecyl sulfate complexes. A new method for the separation of polypeptide subunits. J Biol Chem. 1972 Aug 25;247(16):5194–5198. [PubMed] [Google Scholar]
  11. Murphy D. B., Borisy G. G. Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2696–2700. doi: 10.1073/pnas.72.7.2696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Raff R. A., Greenhouse G., Gross K. W., Gross P. R. Synthesis and storage of microtubule proteins by sea urchin embryos. J Cell Biol. 1971 Aug;50(2):516–527. doi: 10.1083/jcb.50.2.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Rodriguez J. A., Barra H. S., Arce C. A., Hallak M. E., Caputto R. The reciprocal exclusion by L-dopa (L-3,4-dihydroxyphenylalanine) and L-tyrosine of their incorporation as single units into a soluble rat brain protein. Biochem J. 1975 Jul;149(1):115–121. doi: 10.1042/bj1490115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Sloboda R. D., Rudolph S. A., Rosenbaum J. L., Greengard P. Cyclic AMP-dependent endogenous phosphorylation of a microtubule-associated protein. Proc Natl Acad Sci U S A. 1975 Jan;72(1):177–181. doi: 10.1073/pnas.72.1.177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sluder G. Experimental manipulation of the amount of tubulin available for assembly into the spindle of dividing sea urchin eggs. J Cell Biol. 1976 Jul;70(1):75–85. doi: 10.1083/jcb.70.1.75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Weber K., Pringle J. R., Osborn M. Measurement of molecular weights by electrophoresis on SDS-acrylamide gel. Methods Enzymol. 1972;26:3–27. doi: 10.1016/s0076-6879(72)26003-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Weisenberg R. C., Borisy G. G., Taylor E. W. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry. 1968 Dec;7(12):4466–4479. doi: 10.1021/bi00852a043. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES