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. 1994 Nov 1;303(Pt 3):697–700. doi: 10.1042/bj3030697

N-terminal fatty acylation of the alpha-subunit of the G-protein Gi1: only the myristoylated protein is a substrate for palmitoylation.

F Galbiati 1, F Guzzi 1, A I Magee 1, G Milligan 1, M Parenti 1
PMCID: PMC1137601  PMID: 7980434

Abstract

The alpha-subunit of the G-protein Gi1 carries two fatty acyl moieties covalently bound to its N-terminal region: myristic acid is linked to glycine-2 and palmitic acid is linked to cysteine-3. Using site-directed mutagenesis on a cDNA construct of alpha i1 we have generated an alpha i1-G2A mutant, carrying alanine instead of glycine at position 2, and alpha i1-C3S mutant, in which serine replaced cysteine-3 and a double mutant with both substitutions (alpha i1-G2A/C3S). These constructs were individually expressed by transfection in Cos-7 cells, and incorporation of fatty acids into the various mutants was compared with wild-type alpha i1 monitoring metabolic labelling with [3H]palmitate or [3H]myristate. The disruption of the palmitoylation site in alpha i1-C3S did not influence myristoylation, whereas prevention of myristoylation in alpha i1-G2A also abolished palmitoylation. Co-translational myristoylation is thus an absolute requirement for alpha i1 to be post-translationally palmitoylated. The non-palmitoylated alpha i1-C3S showed reduced membrane binding to the same extent as the non-myristoylated/non-palmitoylated alpha i1-G2A and alpha i1-G2A/C3S mutants, indicating that the attachment of palmitic acid is necessary for proper interaction with the membrane.

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Selected References

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

  1. Alland L., Peseckis S. M., Atherton R. E., Berthiaume L., Resh M. D. Dual myristylation and palmitylation of Src family member p59fyn affects subcellular localization. J Biol Chem. 1994 Jun 17;269(24):16701–16705. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Eide B., Gierschik P., Milligan G., Mullaney I., Unson C., Goldsmith P., Spiegel A. GTP-binding proteins in brain and neutrophil are tethered to the plasma membrane via their amino termini. Biochem Biophys Res Commun. 1987 Nov 13;148(3):1398–1405. doi: 10.1016/s0006-291x(87)80287-5. [DOI] [PubMed] [Google Scholar]
  5. Gallego C., Gupta S. K., Winitz S., Eisfelder B. J., Johnson G. L. Myristoylation of the G alpha i2 polypeptide, a G protein alpha subunit, is required for its signaling and transformation functions. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9695–9699. doi: 10.1073/pnas.89.20.9695. [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. Gutierrez L., Magee A. I. Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system. Biochim Biophys Acta. 1991 Jun 24;1078(2):147–154. doi: 10.1016/0167-4838(91)99003-b. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Hallak H., Muszbek L., Laposata M., Belmonte E., Brass L. F., Manning D. R. Covalent binding of arachidonate to G protein alpha subunits of human platelets. J Biol Chem. 1994 Feb 18;269(7):4713–4716. [PubMed] [Google Scholar]
  10. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  11. Hancock J. F., Paterson H., Marshall C. J. A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell. 1990 Oct 5;63(1):133–139. doi: 10.1016/0092-8674(90)90294-o. [DOI] [PubMed] [Google Scholar]
  12. Jones D. T., Reed R. R. Molecular cloning of five GTP-binding protein cDNA species from rat olfactory neuroepithelium. J Biol Chem. 1987 Oct 15;262(29):14241–14249. [PubMed] [Google Scholar]
  13. 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]
  14. Kochel T. J., Sinden R. R. Analysis of trimethylpsoralen photoreactivity to Z-DNA provides a general in vivo assay for Z-DNA: analysis of the hypersensitivity of (GT)n B-Z junctions. Biotechniques. 1988 Jun;6(6):532–543. [PubMed] [Google Scholar]
  15. Kokame K., Fukada Y., Yoshizawa T., Takao T., Shimonishi Y. Lipid modification at the N terminus of photoreceptor G-protein alpha-subunit. Nature. 1992 Oct 22;359(6397):749–752. doi: 10.1038/359749a0. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Linder M. E., Middleton P., Hepler J. R., Taussig R., Gilman A. G., Mumby S. M. Lipid modifications of G proteins: alpha subunits are palmitoylated. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3675–3679. doi: 10.1073/pnas.90.8.3675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liu Y., Fisher D. A., Storm D. R. Analysis of the palmitoylation and membrane targeting domain of neuromodulin (GAP-43) by site-specific mutagenesis. Biochemistry. 1993 Oct 12;32(40):10714–10719. doi: 10.1021/bi00091a023. [DOI] [PubMed] [Google Scholar]
  19. Mariani M., Maretzki D., Lutz H. U. A tightly membrane-associated subpopulation of spectrin is 3H-palmitoylated. J Biol Chem. 1993 Jun 15;268(17):12996–13001. [PubMed] [Google Scholar]
  20. Milligan G. Multiple heterotrimeric guanine nucleotide binding proteins: roles in the determination of cellular signalling specificity. Biochem Soc Trans. 1992 Feb;20(1):135–140. doi: 10.1042/bst0200135. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Neubert T. A., Johnson R. S., Hurley J. B., Walsh K. A. The rod transducin alpha subunit amino terminus is heterogeneously fatty acylated. J Biol Chem. 1992 Sep 15;267(26):18274–18277. [PubMed] [Google Scholar]
  23. Parenti M., Viganó M. A., Newman C. M., Milligan G., Magee A. I. A novel N-terminal motif for palmitoylation of G-protein alpha subunits. Biochem J. 1993 Apr 15;291(Pt 2):349–353. doi: 10.1042/bj2910349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schmidt M. F., Burns G. R. On the enzymes which make "fatty proteins". Behring Inst Mitt. 1991 Jul;(89):185–197. [PubMed] [Google Scholar]
  25. Skene J. H., Virág I. Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43. J Cell Biol. 1989 Feb;108(2):613–624. doi: 10.1083/jcb.108.2.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Taussig R., Iñiguez-Lluhi J. A., Gilman A. G. Inhibition of adenylyl cyclase by Gi alpha. Science. 1993 Jul 9;261(5118):218–221. doi: 10.1126/science.8327893. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Veit M., Nürnberg B., Spicher K., Harteneck C., Ponimaskin E., Schultz G., Schmidt M. F. The alpha-subunits of G-proteins G12 and G13 are palmitoylated, but not amidically myristoylated. FEBS Lett. 1994 Feb 14;339(1-2):160–164. doi: 10.1016/0014-5793(94)80406-0. [DOI] [PubMed] [Google Scholar]
  30. Wedegaertner P. B., Chu D. H., Wilson P. T., Levis M. J., Bourne H. R. Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha. J Biol Chem. 1993 Nov 25;268(33):25001–25008. [PubMed] [Google Scholar]

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