Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1992 Jun 15;284(Pt 3):835–840. doi: 10.1042/bj2840835

Prenylated protein methyltransferases do not distinguish between farnesylated and geranylgeranylated substrates.

D Pérez-Sala 1, B A Gilbert 1, E W Tan 1, R R Rando 1
PMCID: PMC1132615  PMID: 1622400

Abstract

Proteins that are post-translationally modified by prenylation can be either farnesylated (C-15) or geranylgeranylated (C-20) by separate prenyltransferase enzymes. Prenylated proteins are also methylated at their C-terminal residue by S-adenosylmethionine-linked methylation. In this paper we show that the methylation of farnesylated and geranyl-geranylated substrates can be accounted for by the presence of a single enzyme. It is demonstrated that the Km and Vmax. values for the retinal rod outer segment methyltransferase, measured with small molecule farnesylated and geranylgeranylated substrates, are identical. These substrates mutually inhibit each other's methylation, with KI values being equal to their Km values. The Km for S-adenosylmethionine was measured to be the same with either farnesylated or geranylgeranylated substrates. Competitive inhibitors of the methyltransferase containing either a geranylgeranyl or a farnesyl group equally block the methylation of synthetic geranylgeranylated and farnesylated substrates of the enzyme. Importantly, these inhibitors are also equipotent at inhibiting the methylation of the physiological substrates of the rod outer segment methyltransferase. These substrates are both farnesylated and geranylgeranylated. One of these substrates had previously been identified as the farnesylated gamma subunit of transducin. Therefore it appears that the same enzymic activity can methylate both farnesylated and geranylgeranylated substrates.

Full text

PDF
836

Images in this article

838Fig. 5.
on p.838

Selected References

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

  1. Aktories K., Braun U., Rösener S., Just I., Hall A. The rho gene product expressed in E. coli is a substrate of botulinum ADP-ribosyltransferase C3. Biochem Biophys Res Commun. 1989 Jan 16;158(1):209–213. doi: 10.1016/s0006-291x(89)80199-8. [DOI] [PubMed] [Google Scholar]
  2. Aktories K., Hall A. Botulinum ADP-ribosyltransferase C3: a new tool to study low molecular weight GTP-binding proteins. Trends Pharmacol Sci. 1989 Oct;10(10):415–418. doi: 10.1016/0165-6147(89)90191-0. [DOI] [PubMed] [Google Scholar]
  3. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chardin P., Boquet P., Madaule P., Popoff M. R., Rubin E. J., Gill D. M. The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells. EMBO J. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Christy K. G., Jr, LaTart D. B., Osterhoudt H. W. Modifications for SDS-PAGE of proteins. Biotechniques. 1989 Jul-Aug;7(7):692–693. [PubMed] [Google Scholar]
  6. Clarke S., Vogel J. P., Deschenes R. J., Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643–4647. doi: 10.1073/pnas.85.13.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Didsbury J., Weber R. F., Bokoch G. M., Evans T., Snyderman R. rac, a novel ras-related family of proteins that are botulinum toxin substrates. J Biol Chem. 1989 Oct 5;264(28):16378–16382. [PubMed] [Google Scholar]
  8. Fukada Y., Takao T., Ohguro H., Yoshizawa T., Akino T., Shimonishi Y. Farnesylated gamma-subunit of photoreceptor G protein indispensable for GTP-binding. Nature. 1990 Aug 16;346(6285):658–660. doi: 10.1038/346658a0. [DOI] [PubMed] [Google Scholar]
  9. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  11. Hancock J. F., Cadwallader K., Marshall C. J. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. doi: 10.1002/j.1460-2075.1991.tb07992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Hrycyna C. A., Sapperstein S. K., Clarke S., Michaelis S. The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins. EMBO J. 1991 Jul;10(7):1699–1709. doi: 10.1002/j.1460-2075.1991.tb07694.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. Lai R. K., Perez-Sala D., Cañada F. J., Rando R. R. The gamma subunit of transducin is farnesylated. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7673–7677. doi: 10.1073/pnas.87.19.7673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Madaule P., Axel R., Myers A. M. Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Feb;84(3):779–783. doi: 10.1073/pnas.84.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maltese W. A. Posttranslational modification of proteins by isoprenoids in mammalian cells. FASEB J. 1990 Dec;4(15):3319–3328. doi: 10.1096/fasebj.4.15.2123808. [DOI] [PubMed] [Google Scholar]
  18. Maltese W. A., Sheridan K. M., Repko E. M., Erdman R. A. Post-translational modification of low molecular mass GTP-binding proteins by isoprenoid. J Biol Chem. 1990 Feb 5;265(4):2148–2155. [PubMed] [Google Scholar]
  19. Manne V., Roberts D., Tobin A., O'Rourke E., De Virgilio M., Meyers C., Ahmed N., Kurz B., Resh M., Kung H. F. Identification and preliminary characterization of protein-cysteine farnesyltransferase. Proc Natl Acad Sci U S A. 1990 Oct;87(19):7541–7545. doi: 10.1073/pnas.87.19.7541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mumby S. M., Casey P. J., Gilman A. G., Gutowski S., Sternweis P. C. G protein gamma subunits contain a 20-carbon isoprenoid. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5873–5877. doi: 10.1073/pnas.87.15.5873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pérez-Sala D., Tan E. W., Cañada F. J., Rando R. R. Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3043–3046. doi: 10.1073/pnas.88.8.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reiss Y., Goldstein J. L., Seabra M. C., Casey P. J., Brown M. S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell. 1990 Jul 13;62(1):81–88. doi: 10.1016/0092-8674(90)90242-7. [DOI] [PubMed] [Google Scholar]
  23. Reiss Y., Stradley S. J., Gierasch L. M., Brown M. S., Goldstein J. L. Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):732–736. doi: 10.1073/pnas.88.3.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Schaber M. D., O'Hara M. B., Garsky V. M., Mosser S. C., Bergstrom J. D., Moores S. L., Marshall M. S., Friedman P. A., Dixon R. A., Gibbs J. B. Polyisoprenylation of Ras in vitro by a farnesyl-protein transferase. J Biol Chem. 1990 Sep 5;265(25):14701–14704. [PubMed] [Google Scholar]
  25. Schafer W. R., Kim R., Sterne R., Thorner J., Kim S. H., Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989 Jul 28;245(4916):379–385. doi: 10.1126/science.2569235. [DOI] [PubMed] [Google Scholar]
  26. Seabra M. C., Reiss Y., Casey P. J., Brown M. S., Goldstein J. L. Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell. 1991 May 3;65(3):429–434. doi: 10.1016/0092-8674(91)90460-g. [DOI] [PubMed] [Google Scholar]
  27. Simonds W. F., Butrynski J. E., Gautam N., Unson C. G., Spiegel A. M. G-protein beta gamma dimers. Membrane targeting requires subunit coexpression and intact gamma C-A-A-X domain. J Biol Chem. 1991 Mar 25;266(9):5363–5366. [PubMed] [Google Scholar]
  28. Stephenson R. C., Clarke S. Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate. J Biol Chem. 1990 Sep 25;265(27):16248–16254. [PubMed] [Google Scholar]
  29. Swanson R. J., Applebury M. L. Methylation of proteins in photoreceptor rod outer segments. J Biol Chem. 1983 Sep 10;258(17):10599–10605. [PubMed] [Google Scholar]
  30. Tan E. W., Pérez-Sala D., Cañada F. J., Rando R. R. Identifying the recognition unit for G protein methylation. J Biol Chem. 1991 Jun 15;266(17):10719–10722. [PubMed] [Google Scholar]
  31. 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]
  32. Wessling-Resnick M., Johnson G. L. Allosteric behavior in transducin activation mediated by rhodopsin. Initial rate analysis of guanine nucleotide exchange. J Biol Chem. 1987 Mar 15;262(8):3697–3705. [PubMed] [Google Scholar]
  33. Wieland T., Ulibarri I., Aktories K., Gierschik P., Jakobs K. H. Interaction of small G proteins with photoexcited rhodopsin. FEBS Lett. 1990 Apr 24;263(2):195–198. doi: 10.1016/0014-5793(90)81372-u. [DOI] [PubMed] [Google Scholar]
  34. Yamane H. K., Farnsworth C. C., Xie H. Y., Evans T., Howald W. N., Gelb M. H., Glomset J. A., Clarke S., Fung B. K. Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):286–290. doi: 10.1073/pnas.88.1.286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Yamane H. K., Farnsworth C. C., Xie H. Y., Howald W., Fung B. K., Clarke S., Gelb M. H., Glomset J. A. Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5868–5872. doi: 10.1073/pnas.87.15.5868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yamane H. K., Fung B. K. The membrane-binding domain of a 23-kDa G-protein is carboxyl methylated. J Biol Chem. 1989 Nov 25;264(33):20100–20105. [PubMed] [Google Scholar]

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

RESOURCES