Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Jul;9(7):1357–1364. doi: 10.1110/ps.9.7.1357

Autopalmitoylation of tubulin.

J Wolff 1, A M Zambito 1, P J Britto 1, L Knipling 1
PMCID: PMC2144690  PMID: 10933501

Abstract

Pure rat brain tubulin is readily palmitoylated in vitro using [3H]palmitoyl CoA but no added enzymes. A maximum of approximately six palmitic acids are added per dimer in 2-3 h at 36-37 degrees C under native conditions. Both alpha and beta tubulin are labeled, and 63-73% of the label was hydroxylamine-labile, presumed thioesters. Labeling increases with increasing pH and temperature, and with low concentrations of guanidine HCl or KCl (but not with urea) to a maximum of approximately 13 palmitates/dimer. High SDS and guanidine HCl concentrations are inhibitory. At no time could all 20 cysteine residues of the dimer be palmitoylated. Polymerization to microtubules, or use of tubulin S, markedly decreases the accessibility of the palmitoylation sites. Palmitoylation increases the electrophoretic mobility of a portion of alpha tubulin toward the beta band. Palmitoylated tubulin binds a colchicine analogue normally, but during three warm/cold polymerization/depolymerization cycles there is a progressive loss of palmitoylated tubulin, indicating decreased polymerization competence. We postulate that local electrostatic factors are major regulators of reactivity of tubulin cysteine residues toward palmitoyl CoA, and that the negative charges surrounding a number of the cysteines are sensitive to negative charges on palmitoyl CoA.

Full Text

The Full Text of this article is available as a PDF (406.7 KB).

Selected References

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

  1. Alvarez E., Gironès N., Davis R. J. Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid. J Biol Chem. 1990 Sep 25;265(27):16644–16655. [PubMed] [Google Scholar]
  2. Bañ M. C., Jackson C. S., Magee A. I. Pseudo-enzymatic S-acylation of a myristoylated yes protein tyrosine kinase peptide in vitro may reflect non-enzymatic S-acylation in vivo. Biochem J. 1998 Mar 1;330(Pt 2):723–731. doi: 10.1042/bj3300723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bharadwaj M., Bizzozero O. A. Myelin P0 glycoprotein and a synthetic peptide containing the palmitoylation site are both autoacylated. J Neurochem. 1995 Oct;65(4):1805–1815. doi: 10.1046/j.1471-4159.1995.65041805.x. [DOI] [PubMed] [Google Scholar]
  4. Bhattacharyya B., Sackett D. L., Wolff J. Tubulin, hybrid dimers, and tubulin S. Stepwise charge reduction and polymerization. J Biol Chem. 1985 Aug 25;260(18):10208–10216. [PubMed] [Google Scholar]
  5. 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]
  6. Bizzozero O. A., McGarry J. F., Lees M. B. Autoacylation of myelin proteolipid protein with acyl coenzyme A. J Biol Chem. 1987 Oct 5;262(28):13550–13557. [PubMed] [Google Scholar]
  7. Caron J. M. Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies. Mol Biol Cell. 1997 Apr;8(4):621–636. doi: 10.1091/mbc.8.4.621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chapman E. R., Blasi J., An S., Brose N., Johnston P. A., Südhof T. C., Jahn R. Fatty acylation of synaptotagmin in PC12 cells and synaptosomes. Biochem Biophys Res Commun. 1996 Aug 5;225(1):326–332. doi: 10.1006/bbrc.1996.1174. [DOI] [PubMed] [Google Scholar]
  9. Clottes E., Couthon F., Denoroy L., Vial C. Creatine kinase compactness and thiol accessibility during sodium dodecyl sulfate denaturation estimated by resonance energy transfer and 2-nitro-5-thiocyanobenzoic acid cleavage. Biochim Biophys Acta. 1994 Dec 14;1209(2):171–176. doi: 10.1016/0167-4838(94)90181-3. [DOI] [PubMed] [Google Scholar]
  10. Degtyarev M. Y., Spiegel A. M., Jones T. L. The G protein alpha s subunit incorporates [3H]palmitic acid and mutation of cysteine-3 prevents this modification. Biochemistry. 1993 Aug 17;32(32):8057–8061. doi: 10.1021/bi00083a001. [DOI] [PubMed] [Google Scholar]
  11. Duncan J. A., Gilman A. G. Autoacylation of G protein alpha subunits. J Biol Chem. 1996 Sep 20;271(38):23594–23600. doi: 10.1074/jbc.271.38.23594. [DOI] [PubMed] [Google Scholar]
  12. Dunphy J. T., Linder M. E. Signalling functions of protein palmitoylation. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):245–261. doi: 10.1016/s0005-2760(98)00130-1. [DOI] [PubMed] [Google Scholar]
  13. Faulstich H., Tews P., Heintz D. Determination and derivatization of protein thiols by n-octyldithionitrobenzoic acid. Anal Biochem. 1993 Feb 1;208(2):357–362. doi: 10.1006/abio.1993.1061. [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. Ford D. A., Horner C. C., Gross R. W. Protein kinase C acylation by palmitoyl coenzyme A facilitates its translocation to membranes. Biochemistry. 1998 Aug 25;37(34):11953–11961. doi: 10.1021/bi980565w. [DOI] [PubMed] [Google Scholar]
  16. Gundersen C. B., Mastrogiacomo A., Faull K., Umbach J. A. Extensive lipidation of a Torpedo cysteine string protein. J Biol Chem. 1994 Jul 29;269(30):19197–19199. [PubMed] [Google Scholar]
  17. Hartel-Schenk S., Agre P. Mammalian red cell membrane Rh polypeptides are selectively palmitoylated subunits of a macromolecular complex. J Biol Chem. 1992 Mar 15;267(8):5569–5574. [PubMed] [Google Scholar]
  18. Ichihara K., Neely J. R. Recovery of ventricular function in reperfused ischemic rat hearts exposed to fatty acids. Am J Physiol. 1985 Sep;249(3 Pt 2):H492–H497. doi: 10.1152/ajpheart.1985.249.3.H492. [DOI] [PubMed] [Google Scholar]
  19. Ikeda Y., Steiner M. Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding. Biochemistry. 1978 Aug 22;17(17):3454–3459. doi: 10.1021/bi00610a005. [DOI] [PubMed] [Google Scholar]
  20. Khan I. A., Ludueña R. F. Possible regulation of the in vitro assembly of bovine brain tubulin by the bovine thioredoxin system. Biochim Biophys Acta. 1991 Jan 29;1076(2):289–297. doi: 10.1016/0167-4838(91)90280-d. [DOI] [PubMed] [Google Scholar]
  21. Knipling L., Hwang J., Wolff J. Preparation and properties of pure tubulin S. Cell Motil Cytoskeleton. 1999;43(1):63–71. doi: 10.1002/(SICI)1097-0169(1999)43:1<63::AID-CM7>3.0.CO;2-Z. [DOI] [PubMed] [Google Scholar]
  22. Ludueña R. F., Roach M. C. Interaction of tubulin with drugs and alkylating agents. 1. Alkylation of tubulin by iodo[14C]acetamide and N,N'-ethylenebis(iodoacetamide). Biochemistry. 1981 Jul 21;20(15):4437–4444. doi: 10.1021/bi00518a031. [DOI] [PubMed] [Google Scholar]
  23. Magee A. I., Gutierrez L., McKay I. A., Marshall C. J., Hall A. Dynamic fatty acylation of p21N-ras. EMBO J. 1987 Nov;6(11):3353–3357. doi: 10.1002/j.1460-2075.1987.tb02656.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Milligan G., Parenti M., Magee A. I. The dynamic role of palmitoylation in signal transduction. Trends Biochem Sci. 1995 May;20(5):181–187. doi: 10.1016/s0968-0004(00)89004-0. [DOI] [PubMed] [Google Scholar]
  25. Mollner S., Ferreira P., Beck K., Pfeuffer T. Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the alpha-subunit of the stimulatory GTP-binding protein Gs. Eur J Biochem. 1998 Oct 1;257(1):236–241. doi: 10.1046/j.1432-1327.1998.2570236.x. [DOI] [PubMed] [Google Scholar]
  26. O'Brien P. J., St Jules R. S., Reddy T. S., Bazan N. G., Zatz M. Acylation of disc membrane rhodopsin may be nonenzymatic. J Biol Chem. 1987 Apr 15;262(11):5210–5215. [PubMed] [Google Scholar]
  27. Omary M. B., Trowbridge I. S. Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem. 1981 Dec 25;256(24):12888–12892. [PubMed] [Google Scholar]
  28. Parente A., Merrifield B., Geraci G., D'Alessio G. Molecular basis of superreactivity of cysteine residues 31 and 32 of seminal ribonuclease. Biochemistry. 1985 Feb 26;24(5):1098–1104. doi: 10.1021/bi00326a005. [DOI] [PubMed] [Google Scholar]
  29. Phelps K. K., Walker R. A. NEM tubulin inhibits microtubule minus end assembly by a reversible capping mechanism. Biochemistry. 2000 Apr 11;39(14):3877–3885. doi: 10.1021/bi992200x. [DOI] [PubMed] [Google Scholar]
  30. Quesnel S., Silvius J. R. Cysteine-containing peptide sequences exhibit facile uncatalyzed transacylation and acyl-CoA-dependent acylation at the lipid bilayer interface. Biochemistry. 1994 Nov 15;33(45):13340–13348. doi: 10.1021/bi00249a021. [DOI] [PubMed] [Google Scholar]
  31. Resh M. D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta. 1999 Aug 12;1451(1):1–16. doi: 10.1016/s0167-4889(99)00075-0. [DOI] [PubMed] [Google Scholar]
  32. Sackett D. L., Bhattacharyya B., Wolff J. Local unfolding and the stepwise loss of the functional properties of tubulin. Biochemistry. 1994 Nov 1;33(43):12868–12878. doi: 10.1021/bi00209a019. [DOI] [PubMed] [Google Scholar]
  33. Snyder G. H., Cennerazzo M. J., Karalis A. J., Field D. Electrostatic influence of local cysteine environments on disulfide exchange kinetics. Biochemistry. 1981 Nov 10;20(23):6509–6519. doi: 10.1021/bi00526a001. [DOI] [PubMed] [Google Scholar]
  34. Snyder G. H., Reddy M. K., Cennerazzo M. J., Field D. Use of local electrostatic environments of cysteines to enhance formation of a desired species in a reversible disulfide exchange reaction. Biochim Biophys Acta. 1983 Dec 28;749(3):219–226. doi: 10.1016/0167-4838(83)90228-5. [DOI] [PubMed] [Google Scholar]
  35. Stephens R. E. Membrane tubulin. Biol Cell. 1986;57(2):95–109. doi: 10.1111/j.1768-322x.1986.tb00467.x. [DOI] [PubMed] [Google Scholar]
  36. Sudo Y., Valenzuela D., Beck-Sickinger A. G., Fishman M. C., Strittmatter S. M. Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. EMBO J. 1992 Jun;11(6):2095–2102. doi: 10.1002/j.1460-2075.1992.tb05268.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tao N., Wagner S. J., Lublin D. M. CD36 is palmitoylated on both N- and C-terminal cytoplasmic tails. J Biol Chem. 1996 Sep 13;271(37):22315–22320. doi: 10.1074/jbc.271.37.22315. [DOI] [PubMed] [Google Scholar]
  38. Veit M., Sachs K., Heckelmann M., Maretzki D., Hofmann K. P., Schmidt M. F. Palmitoylation of rhodopsin with S-protein acyltransferase: enzyme catalyzed reaction versus autocatalytic acylation. Biochim Biophys Acta. 1998 Oct 2;1394(1):90–98. doi: 10.1016/s0005-2760(98)00097-6. [DOI] [PubMed] [Google Scholar]
  39. Weimbs T., Stoffel W. Proteolipid protein (PLP) of CNS myelin: positions of free, disulfide-bonded, and fatty acid thioester-linked cysteine residues and implications for the membrane topology of PLP. Biochemistry. 1992 Dec 15;31(49):12289–12296. doi: 10.1021/bi00164a002. [DOI] [PubMed] [Google Scholar]
  40. Wilson L. Microtubules as drug receptors: pharmacological properties of microtubule protein. Ann N Y Acad Sci. 1975 Jun 30;253:213–231. doi: 10.1111/j.1749-6632.1975.tb19201.x. [DOI] [PubMed] [Google Scholar]
  41. Wolff J., Knipling L., Sackett D. L. Charge-shielding and the "paradoxical" stimulation of tubulin polymerization by guanidine hydrochloride. Biochemistry. 1996 May 7;35(18):5910–5920. doi: 10.1021/bi9527395. [DOI] [PubMed] [Google Scholar]
  42. Wolff J., Sackett D. L., Knipling L. Cation selective promotion of tubulin polymerization by alkali metal chlorides. Protein Sci. 1996 Oct;5(10):2020–2028. doi: 10.1002/pro.5560051008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yamashita A., Watanabe M., Tonegawa T., Sugiura T., Waku K. Acyl-CoA binding and acylation of UDP-glucuronosyltransferase isoforms of rat liver: their effect on enzyme activity. Biochem J. 1995 Nov 15;312(Pt 1):301–308. doi: 10.1042/bj3120301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zambito A. M., Wolff J. Palmitoylation of tubulin. Biochem Biophys Res Commun. 1997 Oct 29;239(3):650–654. doi: 10.1006/bbrc.1997.7525. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES