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. 1982 Jul 1;94(1):159–164. doi: 10.1083/jcb.94.1.159

Posttranslational processing of alpha-tubulin during axoplasmic transport in CNS axons

PMCID: PMC2112196  PMID: 6181079

Abstract

Tubulin proteins in mouse retinal ganglion cell (RGC) neurons were analyzed to determine whether they undergo posttranslational processing during axoplasmic transport. Alpha- and beta-tubulin comprised heterogeneous proteins in the primary optic pathway (optic nerve and optic tract) when examined by two-dimensional (2D) PAGE. In addition, however, alpha-tubulin exhibited regional heterogeneity when consecutive 1.1-mm segments of the optic pathway were analyzed separately. In proximal segments, alpha-tubulin consisted of two predominant proteins separable by isoelectric point and several less abundant species. In more distal segments, these predominant proteins decreased progressively and the alpha-tubulin region of the gel was represented by less abundant multiple forms only; beta-tubulin region of the gel was represented by less abundant multiple forms only; beta- tubulin was the same in all segments. After intravitreal injection of [3H]proline to mice, radiolabeled alpha- and beta-tubulin heteroproteins were conveyed together at a rate of 0.1-0.2 mm/d in the slowest phase of axoplasmic transport. At 45 d postinjection, the distribution of radiolabeled heterogeneous forms a alpha- and beta- tubulin in consecutive segments of optic pathway resembled the distribution of unlabeled proteins by 2D PAGE, indicating that regional heterogeneity of tubulin arises during axonal transport. Peptide mapping studies demonstrated that the progressive alteration of alpha- tubulin revealed by PAGE analysis cannot be explained by contamination of the alpha-tubulin region by other proteins on gels. The results are consistent with the posttranslational processing of alpha-tubulin during axoplasmic transport. These observations, along with the accompanying report (J. Cell Biol., 1982, 94:150-158), provide additional evidence that CNS axons may be regionally specialized.

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Selected References

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  1. Arce C. A., Rodriguez J. A., Barra H. S., Caputo R. Incorporation of L-tyrosine, L-phenylalanine and L-3,4-dihydroxyphenylalanine as single units into rat brain tubulin. Eur J Biochem. 1975 Nov 1;59(1):145–149. doi: 10.1111/j.1432-1033.1975.tb02435.x. [DOI] [PubMed] [Google Scholar]
  2. Berkowitz S. A., Katagiri J., Binder H. K., Williams R. C., Jr Separation and characterization of microtubule proteins from calf brain. Biochemistry. 1977 Dec 13;16(25):5610–5617. doi: 10.1021/bi00644a035. [DOI] [PubMed] [Google Scholar]
  3. Bhattacharyya B., Volff J. Membrane-bound tubulin in brain and thyroid tissue. J Biol Chem. 1975 Oct 10;250(19):7639–7646. [PubMed] [Google Scholar]
  4. Brown B. A., Nixon R. A., Strocchi P., Marotta C. A. Characterization and comparison of neurofilament proteins from rat and mouse CNS. J Neurochem. 1981 Jan;36(1):143–153. doi: 10.1111/j.1471-4159.1981.tb02389.x. [DOI] [PubMed] [Google Scholar]
  5. Bryan J. Biochemical properties of microtubules. Fed Proc. 1974 Feb;33(2):152–157. [PubMed] [Google Scholar]
  6. Bryan R. N., Cutter G. A., Hayashi M. Separate mRNAs code for tubulin subunits. Nature. 1978 Mar 2;272(5648):81–83. doi: 10.1038/272081a0. [DOI] [PubMed] [Google Scholar]
  7. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  8. Cleveland D. W., Hughes S. H., Stubblefield E., Kirschner M. W., Varmus H. E. Multiple alpha and beta tubulin genes represent unlinked and dispersed gene families. J Biol Chem. 1981 Mar 25;256(6):3130–3134. [PubMed] [Google Scholar]
  9. Cleveland D. W., Kirschner M. W., Cowan N. J. Isolation of separate mRNAs for alpha- and beta-tubulin and characterization of the corresponding in vitro translation products. Cell. 1978 Nov;15(3):1021–1031. doi: 10.1016/0092-8674(78)90286-6. [DOI] [PubMed] [Google Scholar]
  10. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  11. Dahl D. R., Redburn D. A., Samson F. E., Jr Regional distribution of colchicine-binding (microtubular) protein in the rat brain. J Neurochem. 1970 Aug;17(8):1215–1219. doi: 10.1111/j.1471-4159.1970.tb03371.x. [DOI] [PubMed] [Google Scholar]
  12. Eipper B. A. Rat brain tubulin and protein kinase activity. J Biol Chem. 1974 Mar 10;249(5):1398–1406. [PubMed] [Google Scholar]
  13. Estridge M. Polypeptides similar to the alpha and beta subunits of tubulin are exposed on the neuronal surface. Nature. 1977 Jul 7;268(5615):60–63. doi: 10.1038/268060a0. [DOI] [PubMed] [Google Scholar]
  14. Feit H., Barondes S. H. Colchicine-binding activity in particulate fractions of mouse brain. J Neurochem. 1970 Sep;17(9):1355–1364. doi: 10.1111/j.1471-4159.1970.tb06870.x. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. Feit H., Slusarek L., Shelanski M. L. Heterogeneity of tubulin subunits. Proc Natl Acad Sci U S A. 1971 Sep;68(9):2028–2031. doi: 10.1073/pnas.68.9.2028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Forgue S. T., Dahl J. L. Rat brain tubulin: subunit heterogeneity and phosphorylation. J Neurochem. 1979 Mar;32(3):1015–1025. doi: 10.1111/j.1471-4159.1979.tb04588.x. [DOI] [PubMed] [Google Scholar]
  18. Gainer H., Sarne Y., Brownstein M. J. Neurophysin biosynthesis: conversion of a putative precursor during axonal transport. Science. 1977 Mar 25;195(4284):1354–1356. doi: 10.1126/science.65791. [DOI] [PubMed] [Google Scholar]
  19. Gilbert J. M., Strocchi P., Brown B. A., Marotta C. A. Tubulin synthesis in rat forebrain: studies with free and membrane-bound polysomes. J Neurochem. 1981 Mar;36(3):839–846. doi: 10.1111/j.1471-4159.1981.tb01670.x. [DOI] [PubMed] [Google Scholar]
  20. Gozes I., Littauer U. Z. The alpha-subunit of tubulin is preferentially associated with brain presynaptic membrnae. FEBS Lett. 1979 Mar 1;99(1):86–90. doi: 10.1016/0014-5793(79)80255-0. [DOI] [PubMed] [Google Scholar]
  21. Gozes I., Littauer U. Z. Tubulin microheterogeneity increases with rat brain maturation. Nature. 1978 Nov 23;276(5686):411–413. doi: 10.1038/276411a0. [DOI] [PubMed] [Google Scholar]
  22. Herzog W., Weber K. In vitro assembly of pure tubulin into microtubules in the absence of microtubule-associated proteins and glycerol. Proc Natl Acad Sci U S A. 1977 May;74(5):1860–1864. doi: 10.1073/pnas.74.5.1860. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kelly P. T., Cotman C. W. Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide. J Cell Biol. 1978 Oct;79(1):173–183. doi: 10.1083/jcb.79.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kornguth S. E., Sunderland E. Isolation and partial characterization of a tubulin-like protein from human and swine synaptosomal membranes. Biochim Biophys Acta. 1975 May 30;393(1):100–114. doi: 10.1016/0005-2795(75)90220-2. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. 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]
  27. Lagnado J. R., Lyons C., Wickremasinghe G. The subcellular distribution of colchicine-binding protein ('microtubule protein') in rat brain. FEBS Lett. 1971 Jun 24;15(3):254–258. doi: 10.1016/0014-5793(71)80324-1. [DOI] [PubMed] [Google Scholar]
  28. Lu R. C., Elzinga M. Chromatographic resolution of the subunits of calf brain tubulin. Anal Biochem. 1977 Jan;77(1):243–250. doi: 10.1016/0003-2697(77)90310-4. [DOI] [PubMed] [Google Scholar]
  29. Marotta C. A., Harris J. L., Gilbert J. M. Characterization of multiple forms of brain tubulin subunits. J Neurochem. 1978 Jun;30(6):1431–1440. doi: 10.1111/j.1471-4159.1978.tb10475.x. [DOI] [PubMed] [Google Scholar]
  30. Marotta C. A., Strocchi P., Gilbert J. M. Biosynthesis of heterogeneous forms of mammalian brain tubulin subunits by multiple messenger RNAs. J Neurochem. 1979 Jul;33(1):231–246. doi: 10.1111/j.1471-4159.1979.tb11725.x. [DOI] [PubMed] [Google Scholar]
  31. Marotta C. A., Strocchi P., Gilbert J. M. Microheterogeneity of brain cytoplasmic and synaptoplasmic actins. J Neurochem. 1978 Jun;30(6):1441–1451. doi: 10.1111/j.1471-4159.1978.tb10476.x. [DOI] [PubMed] [Google Scholar]
  32. Marotta C. A., Strocchi P., Gilbert J. M. Sununit structure of synaptosomal tubulin. Brain Res. 1979 May 5;167(1):93–106. doi: 10.1016/0006-8993(79)90265-8. [DOI] [PubMed] [Google Scholar]
  33. Matus A., Bernhardt R., Hugh-Jones T. High molecular weight microtubule-associated proteins are preferentially associated with dendritic microtubules in brain. Proc Natl Acad Sci U S A. 1981 May;78(5):3010–3014. doi: 10.1073/pnas.78.5.3010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nath J., Flavin M. A structural difference between cytoplasmic and membrane-bound tubulin of brain. FEBS Lett. 1978 Nov 15;95(2):335–338. doi: 10.1016/0014-5793(78)81024-2. [DOI] [PubMed] [Google Scholar]
  35. Nixon R. A., Brown B. A., Marotta C. A. Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons. J Cell Biol. 1982 Jul;94(1):150–158. doi: 10.1083/jcb.94.1.150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nixon R. A. Increased axonal proteolysis in myelin-deficient mutant mice. Science. 1982 Feb 19;215(4535):999–1001. doi: 10.1126/science.7156980. [DOI] [PubMed] [Google Scholar]
  37. Nixon R. A. Protein degradation in the mouse visual system. I. Degradation of axonally transported and retinal proteins. Brain Res. 1980 Oct 27;200(1):69–83. doi: 10.1016/0006-8993(80)91095-1. [DOI] [PubMed] [Google Scholar]
  38. Olmsted J. B., Borisy G. G. Microtubules. Annu Rev Biochem. 1973;42:507–540. doi: 10.1146/annurev.bi.42.070173.002451. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Portier M. M., Jeantet C., Gros F. Partial purification of the alpha-tubulin and beta-tubulin messenger RNAs from rat brain. Eur J Biochem. 1980 Dec;112(3):601–609. doi: 10.1111/j.1432-1033.1980.tb06125.x. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Raybin D., Flavin M. Modification of tubulin by tyrosylation in cells and extracts and its effect on assembly in vitro. J Cell Biol. 1977 May;73(2):492–504. doi: 10.1083/jcb.73.2.492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Reddington M., Tan L. P., Lagnado J. R. The phosphorylation of brain microtubular proteins in situ and in vitro. J Neurochem. 1976 Nov;27(5):1229–1236. doi: 10.1111/j.1471-4159.1976.tb00332.x. [DOI] [PubMed] [Google Scholar]
  44. Shelanski M. L., Gaskin F., Cantor C. R. Microtubule assembly in the absence of added nucleotides. Proc Natl Acad Sci U S A. 1973 Mar;70(3):765–768. doi: 10.1073/pnas.70.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Soifer D., Czosnek H. Association of newly synthesized tubulin with brain microsomal membranes. J Neurochem. 1980 Nov;35(5):1128–1136. doi: 10.1111/j.1471-4159.1980.tb07868.x. [DOI] [PubMed] [Google Scholar]
  47. Soifer D. Enzymatic acitivity in tubulin preparations: cyclic-AMP dependent protein kinase activity of brain microtubule protein. J Neurochem. 1975 Jan;24(1):21–33. doi: 10.1111/j.1471-4159.1975.tb07623.x. [DOI] [PubMed] [Google Scholar]
  48. Wang Y. J., Mahler H. R. Topography of the synaptosomal membrane. J Cell Biol. 1976 Nov;71(2):639–658. doi: 10.1083/jcb.71.2.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wilson L. Properties of colchicine binding protein from chick embryo brain. Interactions with vinca alkaloids and podophyllotoxin. Biochemistry. 1970 Dec 8;9(25):4999–5007. doi: 10.1021/bi00827a026. [DOI] [PubMed] [Google Scholar]
  50. Zisapel N., Levi M., Gozes I. Tubulin: an integral protein of mammalian synaptic vesicle membranes. J Neurochem. 1980 Jan;34(1):26–32. doi: 10.1111/j.1471-4159.1980.tb04617.x. [DOI] [PubMed] [Google Scholar]

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