Skip to main content
Biochemical Journal logoLink to Biochemical Journal
. 1996 Feb 1;313(Pt 3):717–720. doi: 10.1042/bj3130717

Chemical inhibition of myristoylation of the G-protein Gi1 alpha by 2-hydroxymyristate does not interfere with its palmitoylation or membrane association. Evidence that palmitoylation, but not myristoylation, regulates membrane attachment.

F Galbiati 1, F Guzzi 1, A I Magee 1, G Milligan 1, M Parenti 1
PMCID: PMC1216969  PMID: 8611146

Abstract

The alpha-subunit of the G-protein Gi1 alpha is normally dually acylated at its N-terminus with the saturated fatty acids myristate and palmitate. Inhibition of protein myristoylation by treatment with 2-hydroxymyristate prevented neither the incorporation of [3H]palmitate nor the membrane association of this protein when expressed in the COS cells. Construction of a mutant of Gi1 alpha in which serine-6 was replaced by aspartic acid prevented both myristoylation and palmitoylation, and the expressed protein was found primarily in the cytoplasmic fraction. These data indicate the myristoylation is not an absolute requirement for palmitoylation of Gi1 alpha and that palmitoylation, but not myristoylation, plays a key role in membrane association of this G-protein alpha-subunit.

Full Text

The Full Text of this article is available as a PDF (332.6 KB).

Selected References

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

  1. Bonner W. M., Laskey R. A. A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem. 1974 Jul 1;46(1):83–88. doi: 10.1111/j.1432-1033.1974.tb03599.x. [DOI] [PubMed] [Google Scholar]
  2. Casey P. J. Protein lipidation in cell signaling. Science. 1995 Apr 14;268(5208):221–225. doi: 10.1126/science.7716512. [DOI] [PubMed] [Google Scholar]
  3. Degtyarev M. Y., Spiegel A. M., Jones T. L. Palmitoylation of a G protein alpha i subunit requires membrane localization not myristoylation. J Biol Chem. 1994 Dec 9;269(49):30898–30903. [PubMed] [Google Scholar]
  4. Galbiati F., Guzzi F., Magee A. I., Milligan G., Parenti M. N-terminal fatty acylation of the alpha-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation. Biochem J. 1994 Nov 1;303(Pt 3):697–700. doi: 10.1042/bj3030697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grassie M. A., McCallum J. F., Guzzi F., Magee A. I., Milligan G., Parenti M. The palmitoylation status of the G-protein G(o)1 alpha regulates its activity of interaction with the plasma membrane. Biochem J. 1994 Sep 15;302(Pt 3):913–920. doi: 10.1042/bj3020913. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Green A., Johnson J. L., Milligan G. Down-regulation of Gi sub-types by prolonged incubation of adipocytes with an A1 adenosine receptor agonist. J Biol Chem. 1990 Mar 25;265(9):5206–5210. [PubMed] [Google Scholar]
  7. Hallak H., Brass L. F., Manning D. R. Failure to myristoylate the alpha subunit of Gz is correlated with an inhibition of palmitoylation and membrane attachment, but has no affect on phosphorylation by protein kinase C. J Biol Chem. 1994 Feb 11;269(6):4571–4576. [PubMed] [Google Scholar]
  8. Jones T. L., Simonds W. F., Merendino J. J., Jr, Brann M. R., Spiegel A. M. Myristoylation of an inhibitory GTP-binding protein alpha subunit is essential for its membrane attachment. Proc Natl Acad Sci U S A. 1990 Jan;87(2):568–572. doi: 10.1073/pnas.87.2.568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Koegl M., Zlatkine P., Ley S. C., Courtneidge S. A., Magee A. I. Palmitoylation of multiple Src-family kinases at a homologous N-terminal motif. Biochem J. 1994 Nov 1;303(Pt 3):749–753. doi: 10.1042/bj3030749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Mumby S. M., Heukeroth R. O., Gordon J. I., Gilman A. G. G-protein alpha-subunit expression, myristoylation, and membrane association in COS cells. Proc Natl Acad Sci U S A. 1990 Jan;87(2):728–732. doi: 10.1073/pnas.87.2.728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Mumby S. M., Kleuss C., Gilman A. G. Receptor regulation of G-protein palmitoylation. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2800–2804. doi: 10.1073/pnas.91.7.2800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nadler M. J., Harrison M. L., Ashendel C. L., Cassady J. M., Geahlen R. L. Treatment of T cells with 2-hydroxymyristic acid inhibits the myristoylation and alters the stability of p56lck. Biochemistry. 1993 Sep 7;32(35):9250–9255. doi: 10.1021/bi00086a034. [DOI] [PubMed] [Google Scholar]
  15. Neer E. J., Pulsifer L., Wolf L. G. The amino terminus of G protein alpha subunits is required for interaction with beta gamma. J Biol Chem. 1988 Jun 25;263(18):8996–8970. [PubMed] [Google Scholar]
  16. Paige L. A., Zheng G. Q., DeFrees S. A., Cassady J. M., Geahlen R. L. Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase. Biochemistry. 1990 Nov 20;29(46):10566–10573. doi: 10.1021/bi00498a021. [DOI] [PubMed] [Google Scholar]
  17. Peitzsch R. M., McLaughlin S. Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins. Biochemistry. 1993 Oct 5;32(39):10436–10443. doi: 10.1021/bi00090a020. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Rodgers W., Crise B., Rose J. K. Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction. Mol Cell Biol. 1994 Aug;14(8):5384–5391. doi: 10.1128/mcb.14.8.5384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Shenoy-Scaria A. M., Dietzen D. J., Kwong J., Link D. C., Lublin D. M. Cysteine3 of Src family protein tyrosine kinase determines palmitoylation and localization in caveolae. J Cell Biol. 1994 Jul;126(2):353–363. doi: 10.1083/jcb.126.2.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Silvius J. R., l'Heureux F. Fluorimetric evaluation of the affinities of isoprenylated peptides for lipid bilayers. Biochemistry. 1994 Mar 15;33(10):3014–3022. doi: 10.1021/bi00176a034. [DOI] [PubMed] [Google Scholar]
  22. Spector A. A., Hoak J. C. An improved method for the addition of long-chain free fatty acid to protein solutions. Anal Biochem. 1969 Nov;32(2):297–302. doi: 10.1016/0003-2697(69)90089-x. [DOI] [PubMed] [Google Scholar]
  23. Spector A. A. Structure and lipid binding properties of serum albumin. Methods Enzymol. 1986;128:320–339. doi: 10.1016/0076-6879(86)28077-5. [DOI] [PubMed] [Google Scholar]
  24. Spiegel A. M., Backlund P. S., Jr, Butrynski J. E., Jones T. L., Simonds W. F. The G protein connection: molecular basis of membrane association. Trends Biochem Sci. 1991 Sep;16(9):338–341. doi: 10.1016/0968-0004(91)90139-m. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Towler D. A., Adams S. P., Eubanks S. R., Towery D. S., Jackson-Machelski E., Glaser L., Gordon J. I. Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase. Proc Natl Acad Sci U S A. 1987 May;84(9):2708–2712. doi: 10.1073/pnas.84.9.2708. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wedegaertner P. B., Wilson P. T., Bourne H. R. Lipid modifications of trimeric G proteins. J Biol Chem. 1995 Jan 13;270(2):503–506. doi: 10.1074/jbc.270.2.503. [DOI] [PubMed] [Google Scholar]
  28. van der Neut R., Pantaloni C., Nebout I., Bockaert J., Audigier Y. Mutagenesis of the amino-terminal glycine to alanine in Gs alpha subunit alters beta gamma-dependent properties and decreases adenylylcyclase activation. J Biol Chem. 1993 Jan 5;268(1):436–441. [PubMed] [Google Scholar]

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

RESOURCES