Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Sep 15;342(Pt 3):513–518.

Characterization of prenylated protein methyltransferase in Leishmania.

M P Hasne 1, F Lawrence 1
PMCID: PMC1220491  PMID: 10477261

Abstract

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-l-[methyl-(3)H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans, trans-farnesyl-l-cysteine and N-acetyl-S-all-trans-geranylgeranyl-l-cysteine, but N-acetyl-S-trans, trans-geranyl-l-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5'-[gamma-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-l-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5'-[gamma-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

Full Text

The Full Text of this article is available as a PDF (146.4 KB).

Selected References

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

  1. Ben Baruch G., Paz A., Marciano D., Egozi Y., Haklai R., Kloog Y. The uniquely distributed isoprenylated protein methyltransferase activity in the rat brain is highly expressed in the cerebellum. Biochem Biophys Res Commun. 1993 Aug 31;195(1):282–288. doi: 10.1006/bbrc.1993.2042. [DOI] [PubMed] [Google Scholar]
  2. Boivin D., Potier M., Béliveau R. Functional size of C-terminal protein carboxyl methyltransferase from kidney basolateral plasma membranes. Biochim Biophys Acta. 1994 Jul 20;1207(1):114–119. doi: 10.1016/0167-4838(94)90059-0. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Cappai R., Osborn A. H., Gleeson P. A., Handman E. Cloning and characterization of a Golgi-associated GTP-binding protein homologue from Leishmania major. Mol Biochem Parasitol. 1993 Nov;62(1):73–82. doi: 10.1016/0166-6851(93)90179-2. [DOI] [PubMed] [Google Scholar]
  5. Chelsky D., Olson J. F., Koshland D. E., Jr Cell cycle-dependent methyl esterification of lamin B. J Biol Chem. 1987 Mar 25;262(9):4303–4309. [PubMed] [Google Scholar]
  6. Chelsky D., Sobotka C., O'Neill C. L. Lamin B methylation and assembly into the nuclear envelope. J Biol Chem. 1989 May 5;264(13):7637–7643. [PubMed] [Google Scholar]
  7. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  8. Coombs G. H., Craft J. A., Hart D. T. A comparative study of Leishmania mexicana amastigotes and promastigotes. Enzyme activities and subcellular locations. Mol Biochem Parasitol. 1982 Mar;5(3):199–211. doi: 10.1016/0166-6851(82)90021-4. [DOI] [PubMed] [Google Scholar]
  9. Coso O. A., Díaz Añel A., Martinetto H., Muschietti J. P., Kazanietz M., Fraidenraich D., Torres H. N., Flawia M. M. Characterization of a Gi-protein from Trypanosoma cruzi epimastigote membranes. Biochem J. 1992 Oct 15;287(Pt 2):443–446. doi: 10.1042/bj2870443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coulter L. J., Hide G. Trypanosoma brucei: characterisation of a life cycle stage-specific G-protein. Exp Parasitol. 1995 Mar;80(2):308–318. doi: 10.1006/expr.1995.1037. [DOI] [PubMed] [Google Scholar]
  11. Dai Q., Choy E., Chiu V., Romano J., Slivka S. R., Steitz S. A., Michaelis S., Philips M. R. Mammalian prenylcysteine carboxyl methyltransferase is in the endoplasmic reticulum. J Biol Chem. 1998 Jun 12;273(24):15030–15034. doi: 10.1074/jbc.273.24.15030. [DOI] [PubMed] [Google Scholar]
  12. Field H., Blench I., Croft S., Field M. C. Characterisation of protein isoprenylation in procyclic form Trypanosoma brucei. Mol Biochem Parasitol. 1996 Nov 12;82(1):67–80. doi: 10.1016/0166-6851(96)02723-5. [DOI] [PubMed] [Google Scholar]
  13. Field M. C., Boothroyd J. C. Trypanosoma brucei: molecular cloning of homologues of small GTP-binding proteins involved in vesicle trafficking. Exp Parasitol. 1995 Nov;81(3):313–320. doi: 10.1006/expr.1995.1122. [DOI] [PubMed] [Google Scholar]
  14. Field M. C., Field H., Boothroyd J. C. A homologue of the nuclear GTPase ran/TC4 from Trypanosoma brucei. Mol Biochem Parasitol. 1995 Jan;69(1):131–134. doi: 10.1016/0166-6851(94)00209-6. [DOI] [PubMed] [Google Scholar]
  15. Gingras D., Boivin D., Béliveau R. Subcellular distribution and guanine nucleotide dependency of COOH-terminal methylation in kidney cortex. Am J Physiol. 1993 Aug;265(2 Pt 2):F316–F322. doi: 10.1152/ajprenal.1993.265.2.F316. [DOI] [PubMed] [Google Scholar]
  16. Gottlieb M., Dwyer D. M. Leishmania donovani: surface membrane acid phosphatase activity of promastigotes. Exp Parasitol. 1981 Aug;52(1):117–128. doi: 10.1016/0014-4894(81)90067-9. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Hrycyna C. A., Clarke S. Modification of eukaryotic signaling proteins by C-terminal methylation reactions. Pharmacol Ther. 1993 Sep;59(3):281–300. doi: 10.1016/0163-7258(93)90071-k. [DOI] [PubMed] [Google Scholar]
  19. Hrycyna C. A., Yang M. C., Clarke S. Protein carboxyl methylation in Saccharomyces cerevisiae: evidence for STE14-dependent and STE14-independent pathways. Biochemistry. 1994 Aug 16;33(32):9806–9812. doi: 10.1021/bi00198a053. [DOI] [PubMed] [Google Scholar]
  20. Inglese J., Glickman J. F., Lorenz W., Caron M. G., Lefkowitz R. J. Isoprenylation of a protein kinase. Requirement of farnesylation/alpha-carboxyl methylation for full enzymatic activity of rhodopsin kinase. J Biol Chem. 1992 Jan 25;267(3):1422–1425. [PubMed] [Google Scholar]
  21. Klein Z., Ben-Baruch G., Marciano D., Solomon R., Altaras M., Kloog Y. Characterization of the prenylated protein methyltransferase in human endometrial carcinoma. Biochim Biophys Acta. 1994 Jul 18;1226(3):330–336. doi: 10.1016/0925-4439(94)90045-0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Law R. E., Stimmel J. B., Damore M. A., Carter C., Clarke S., Wall R. Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5'-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions. Mol Cell Biol. 1992 Jan;12(1):103–111. doi: 10.1128/mcb.12.1.103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lawrence F., Robert-Gero M. Distribution of macromolecular methylations in promastigotes of Leishmania donovani and impact of sinefungin. J Eukaryot Microbiol. 1993 Sep-Oct;40(5):581–589. doi: 10.1111/j.1550-7408.1993.tb06111.x. [DOI] [PubMed] [Google Scholar]
  25. Leiser M., Efrat S., Fleischer N. Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion. Endocrinology. 1995 Jun;136(6):2521–2530. doi: 10.1210/endo.136.6.7750474. [DOI] [PubMed] [Google Scholar]
  26. Li G., Kowluru A., Metz S. A. Characterization of prenylcysteine methyltransferase in insulin-secreting cells. Biochem J. 1996 May 15;316(Pt 1):345–351. doi: 10.1042/bj3160345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Luján H. D., Mowatt M. R., Chen G. Z., Nash T. E. Isoprenylation of proteins in the protozoan Giardia lamblia. Mol Biochem Parasitol. 1995 Jun;72(1-2):121–127. doi: 10.1016/0166-6851(94)00070-4. [DOI] [PubMed] [Google Scholar]
  28. Metz S. A., Rabaglia M. E., Stock J. B., Kowluru A. Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins. Biochem J. 1993 Oct 1;295(Pt 1):31–40. doi: 10.1042/bj2950031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ong O. C., Ota I. M., Clarke S., Fung B. K. The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terminal cysteine. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9238–9242. doi: 10.1073/pnas.86.23.9238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Otsuka K., Roullet C. M., McDougal P., McCarron D. A., Roullet J. B. Protein carboxyl methylation controls intracellular pH in human platelets. J Hypertens. 1998 Sep;16(9):1261–1266. doi: 10.1097/00004872-199816090-00006. [DOI] [PubMed] [Google Scholar]
  31. Paolantonacci P., Lawrence F., Lederer F., Robert-Gero M. Protein methylation and protein methylases in Leishmania donovani and Leishmania tropica promastigotes. Mol Biochem Parasitol. 1986 Oct;21(1):47–54. doi: 10.1016/0166-6851(86)90078-2. [DOI] [PubMed] [Google Scholar]
  32. Parish C. A., Smrcka A. V., Rando R. R. Functional significance of beta gamma-subunit carboxymethylation for the activation of phospholipase C and phosphoinositide 3-kinase. Biochemistry. 1995 Jun 13;34(23):7722–7727. doi: 10.1021/bi00023a019. [DOI] [PubMed] [Google Scholar]
  33. Phelouzat M. A., Lawrence F., Robert-Gero M. Characterization of sinefungin-resistant Leishmania donovani promastigotes. Parasitol Res. 1993;79(8):683–689. doi: 10.1007/BF00932511. [DOI] [PubMed] [Google Scholar]
  34. Philips M. R., Pillinger M. H., Staud R., Volker C., Rosenfeld M. G., Weissmann G., Stock J. B. Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils. Science. 1993 Feb 12;259(5097):977–980. doi: 10.1126/science.8438158. [DOI] [PubMed] [Google Scholar]
  35. Pillinger M. H., Volker C., Stock J. B., Weissmann G., Philips M. R. Characterization of a plasma membrane-associated prenylcysteine-directed alpha carboxyl methyltransferase in human neutrophils. J Biol Chem. 1994 Jan 14;269(2):1486–1492. [PubMed] [Google Scholar]
  36. Remaley A. T., Das S., Campbell P. I., LaRocca G. M., Pope M. T., Glew R. H. Characterization of Leishmania donovani acid phosphatases. J Biol Chem. 1985 Jan 25;260(2):880–886. [PubMed] [Google Scholar]
  37. Romano J. D., Schmidt W. K., Michaelis S. The Saccharomyces cerevisiae prenylcysteine carboxyl methyltransferase Ste14p is in the endoplasmic reticulum membrane. Mol Biol Cell. 1998 Aug;9(8):2231–2247. doi: 10.1091/mbc.9.8.2231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schlenk F. Methylthioadenosine. Adv Enzymol Relat Areas Mol Biol. 1983;54:195–265. doi: 10.1002/9780470122990.ch4. [DOI] [PubMed] [Google Scholar]
  39. Stephenson R. C., Clarke S. Characterization of a rat liver protein carboxyl methyltransferase involved in the maturation of proteins with the -CXXX C-terminal sequence motif. J Biol Chem. 1992 Jul 5;267(19):13314–13319. [PubMed] [Google Scholar]
  40. 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]
  41. Volker C., Pillinger M. H., Philips M. R., Stock J. B. Prenylcysteine analogs to study function of carboxylmethylation in signal transduction. Methods Enzymol. 1995;250:216–225. doi: 10.1016/0076-6879(95)50074-x. [DOI] [PubMed] [Google Scholar]
  42. Yokoyama K., Lin Y., Stuart K. D., Gelb M. H. Prenylation of proteins in Trypanosoma brucei. Mol Biochem Parasitol. 1997 Jul;87(1):61–69. doi: 10.1016/s0166-6851(97)00043-1. [DOI] [PubMed] [Google Scholar]

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

RESOURCES