Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Apr 15;339(Pt 2):463–471.

Association of tubulin carboxypeptidase with microtubules in living cells.

M A Contin 1, J J Sironi 1, H S Barra 1, C A Arce 1
PMCID: PMC1220178  PMID: 10191280

Abstract

Tubulin carboxypeptidase is the enzyme that releases the C-terminal tyrosine from alpha-tubulin, converting tyrosine-terminated (Tyr) to detyrosinated (Glu) tubulin. The present study demonstrates that this enzyme is associated with microtubules in living cells. We extracted cultured cells (COS-7) with Triton X-100 under microtubule-stabilizing conditions and found tubulin carboxypeptidase activity in the cytoskeleton fraction. We ruled out, by using several control experiments, the possibility that this result was due to contamination of the isolated cytoskeletons by non-associated proteins contained in the detergent fraction or to an artifact in vitro during the extraction procedure. The associated carboxypeptidase activity showed characteristics similar to those of brain tubulin carboxypeptidase and different from those of pancreatic carboxypeptidase A. In comparison with cultures at confluence, those at low cell density contained small (if any) amounts of carboxypeptidase activity associated with microtubules. In addition, the enzyme was shown to be associated only with cold-labile microtubules. The tubulin carboxypeptidase/microtubule association was also demonstrated in Chinese hamster ovary, NIH 3T3 and PC12 cells. Interestingly, this association was not observed in cultured embryonic brain cells. Our results demonstrate that tubulin carboxypeptidase is indeed associated with microtubules in living cells. Furthermore, the findings that this association occurs with a subset of microtubules and that its magnitude depends on the degree of confluence of the cell culture indicate that it could be part of the mechanism that regulates the tyrosination state of microtubules.

Full Text

The Full Text of this article is available as a PDF (240.3 KB).

Selected References

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

  1. Arce C. A., Barra H. S. Association of tubulinyl-tyrosine carboxypeptidase with microtubules. FEBS Lett. 1983 Jun 27;157(1):75–78. doi: 10.1016/0014-5793(83)81119-3. [DOI] [PubMed] [Google Scholar]
  2. Arce C. A., Barra H. S. Release of C-terminal tyrosine from tubulin and microtubules at steady state. Biochem J. 1985 Feb 15;226(1):311–317. doi: 10.1042/bj2260311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arce C. A., Hallak M. E., Rodriguez J. A., Barra H. S., Caputto R. Capability of tubulin and microtubules to incorporate and to release tyrosine and phenylalanine and the effect of the incorporation of these amino acids on tubulin assembly. J Neurochem. 1978 Jul;31(1):205–210. doi: 10.1111/j.1471-4159.1978.tb12449.x. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Barra H. S., Arce C. A., Argaraña C. E. Posttranslational tyrosination/detyrosination of tubulin. Mol Neurobiol. 1988 Summer;2(2):133–153. doi: 10.1007/BF02935343. [DOI] [PubMed] [Google Scholar]
  6. Barra H. S., Arce C. A., Caputto R. Total tubulin and its aminoacylated and non-aminoacylated forms during the development of rat brain. Eur J Biochem. 1980 Aug;109(2):439–446. doi: 10.1111/j.1432-1033.1980.tb04813.x. [DOI] [PubMed] [Google Scholar]
  7. Black M. M., Kurdyla J. T. Microtubule-associated proteins of neurons. J Cell Biol. 1983 Oct;97(4):1020–1028. doi: 10.1083/jcb.97.4.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bré M. H., Kreis T. E., Karsenti E. Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules. J Cell Biol. 1987 Sep;105(3):1283–1296. doi: 10.1083/jcb.105.3.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bulinski J. C., Gundersen G. G. Stabilization of post-translational modification of microtubules during cellular morphogenesis. Bioessays. 1991 Jun;13(6):285–293. doi: 10.1002/bies.950130605. [DOI] [PubMed] [Google Scholar]
  10. Duerr A., Pallas D., Solomon F. Molecular analysis of cytoplasmic microtubules in situ: identification of both widespread and specific proteins. Cell. 1981 Apr;24(1):203–211. doi: 10.1016/0092-8674(81)90516-x. [DOI] [PubMed] [Google Scholar]
  11. Gundersen G. G., Bulinski J. C. Microtubule arrays in differentiated cells contain elevated levels of a post-translationally modified form of tubulin. Eur J Cell Biol. 1986 Dec;42(2):288–294. [PubMed] [Google Scholar]
  12. 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]
  13. Gundersen G. G., Khawaja S., Bulinski J. C. Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules. J Cell Biol. 1987 Jul;105(1):251–264. doi: 10.1083/jcb.105.1.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hallak M. E., Rodriguez J. A., Barra H. S., Caputto R. Release of tyrosine from tyrosinated tubulin. Some common factors that affect this process and the assembly of tubulin. FEBS Lett. 1977 Feb 1;73(2):147–150. doi: 10.1016/0014-5793(77)80968-x. [DOI] [PubMed] [Google Scholar]
  15. Kreis T. E. Microtubules containing detyrosinated tubulin are less dynamic. EMBO J. 1987 Sep;6(9):2597–2606. doi: 10.1002/j.1460-2075.1987.tb02550.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Lopez R. A., Arce C. A., Barra H. S. Effect of polyanions and polycations on detyrosination of tubulin and microtubules at steady state. Biochim Biophys Acta. 1990 Jun 19;1039(2):209–217. doi: 10.1016/0167-4838(90)90188-l. [DOI] [PubMed] [Google Scholar]
  20. Paturle-Lafanechère L., Eddé B., Denoulet P., Van Dorsselaer A., Mazarguil H., Le Caer J. P., Wehland J., Job D. Characterization of a major brain tubulin variant which cannot be tyrosinated. Biochemistry. 1991 Oct 29;30(43):10523–10528. doi: 10.1021/bi00107a022. [DOI] [PubMed] [Google Scholar]
  21. Paturle-Lafanechère L., Manier M., Trigault N., Pirollet F., Mazarguil H., Job D. Accumulation of delta 2-tubulin, a major tubulin variant that cannot be tyrosinated, in neuronal tissues and in stable microtubule assemblies. J Cell Sci. 1994 Jun;107(Pt 6):1529–1543. doi: 10.1242/jcs.107.6.1529. [DOI] [PubMed] [Google Scholar]
  22. Paturle L., Wehland J., Margolis R. L., Job D. Complete separation of tyrosinated, detyrosinated, and nontyrosinatable brain tubulin subpopulations using affinity chromatography. Biochemistry. 1989 Mar 21;28(6):2698–2704. doi: 10.1021/bi00432a050. [DOI] [PubMed] [Google Scholar]
  23. Pillus L., Solomon F. Components of microtubular structures in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2468–2472. doi: 10.1073/pnas.83.8.2468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Saxton W. M., Stemple D. L., Leslie R. J., Salmon E. D., Zavortink M., McIntosh J. R. Tubulin dynamics in cultured mammalian cells. J Cell Biol. 1984 Dec;99(6):2175–2186. doi: 10.1083/jcb.99.6.2175. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schulze E., Kirschner M. Microtubule dynamics in interphase cells. J Cell Biol. 1986 Mar;102(3):1020–1031. doi: 10.1083/jcb.102.3.1020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sironi J. J., Barra H. S., Arce C. A. The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes. Mol Cell Biochem. 1997 May;170(1-2):9–16. doi: 10.1023/a:1006846828547. [DOI] [PubMed] [Google Scholar]
  27. Solomon F. Direct identification of microtubule-associated proteins by selective extraction of cultured cells. Methods Enzymol. 1986;134:139–147. doi: 10.1016/0076-6879(86)34082-5. [DOI] [PubMed] [Google Scholar]
  28. Solomon F., Magendantz M., Salzman A. Identification with cellular microtubules of one of the co-assemlbing microtubule-associated proteins. Cell. 1979 Oct;18(2):431–438. doi: 10.1016/0092-8674(79)90062-x. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. Webster D. R., Modesti N. M., Bulinski J. C. Regulation of cytoplasmic tubulin carboxypeptidase activity during neural and muscle differentiation: characterization using a microtubule-based assay. Biochemistry. 1992 Jun 30;31(25):5849–5856. doi: 10.1021/bi00140a021. [DOI] [PubMed] [Google Scholar]
  31. Webster D. R., Oxford M. G. Regulation of cytoplasmic tubulin carboxypeptidase activity in vitro by cations and sulfhydryl-modifying compounds. J Cell Biochem. 1996 Mar 1;60(3):424–436. doi: 10.1002/(sici)1097-4644(19960301)60:3<424::aid-jcb13>3.0.co;2-k. [DOI] [PubMed] [Google Scholar]
  32. Wehland J., Weber K. Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells. J Cell Sci. 1987 Sep;88(Pt 2):185–203. doi: 10.1242/jcs.88.2.185. [DOI] [PubMed] [Google Scholar]
  33. Wessel D., Flügge U. I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem. 1984 Apr;138(1):141–143. doi: 10.1016/0003-2697(84)90782-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES