Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

The EMBO Journal logoLink to The EMBO Journal
. 1999 Apr 15;18(8):2057–2065. doi: 10.1093/emboj/18.8.2057

Flagellar protein localization mediated by a calcium-myristoyl/palmitoyl switch mechanism.

L M Godsel 1, D M Engman 1
PMCID: PMC1171290  PMID: 10205160

Abstract

The mechanisms by which proteins are targeted to flagella and cilia are poorly understood. We set out to determine the basis for the specific localization of a 24 kDa flagellar calcium-binding protein (FCaBP) expressed in all life cycle stages of Trypanosoma cruzi. Through the study of trypanosome transfectants expressing various FCaBP deletion mutants, we found that the N-terminal 24 amino acids of the protein are necessary and sufficient for flagellar localization. Subsequent experiments revealed that FCaBP is myristoylated and palmitoylated and, in fact, is one of very few proteins in the cell possessing these acyl modifications. Both fatty acids are required for flagellar localization, suggesting that FCaBP localization may be mediated through association with the flagellar plasma membrane. Indeed, FCaBP associates with the flagellar membrane in a calcium-dependent manner, reminiscent of the recoverin family of calcium-myristoyl switch proteins. Thus, FCaBP is a novel member of the calcium-acyl switch protein family and is the only member described to date that requires two fatty acid modifications for specific membrane association. Its unique localization mechanism is the first described for any flagellar protein. The existence of such a protein in this protozoan suggests that acylation and calcium switch mechanisms for regulated membrane association are conserved among eukaryotes.

Full Text

The Full Text of this article is available as a PDF (234.3 KB).

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. Arreaza G., Melkonian K. A., LaFevre-Bernt M., Brown D. A. Triton X-100-resistant membrane complexes from cultured kidney epithelial cells contain the Src family protein tyrosine kinase p62yes. J Biol Chem. 1994 Jul 22;269(29):19123–19127. [PubMed] [Google Scholar]
  3. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  4. Calvert P. D., Klenchin V. A., Bownds M. D. Rhodopsin kinase inhibition by recoverin. Function of recoverin myristoylation. J Biol Chem. 1995 Oct 13;270(41):24127–24129. doi: 10.1074/jbc.270.41.24127. [DOI] [PubMed] [Google Scholar]
  5. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  6. De Souza W. Structural organization of the cell surface of pathogenic protozoa. Micron. 1995;26(5):405–430. doi: 10.1016/0968-4328(95)00010-0. [DOI] [PubMed] [Google Scholar]
  7. Degtyarev M. Y., Spiegel A. M., Jones T. L. Increased palmitoylation of the Gs protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin. J Biol Chem. 1993 Nov 15;268(32):23769–23772. [PubMed] [Google Scholar]
  8. Dizhoor A. M., Nekrasova E. R., Philippov P. P. The binding of G proteins to immobilized delipidated rhodopsin. Biochem Biophys Res Commun. 1989 Jul 14;162(1):544–549. doi: 10.1016/0006-291x(89)92031-7. [DOI] [PubMed] [Google Scholar]
  9. Dizhoor A. M., Ray S., Kumar S., Niemi G., Spencer M., Brolley D., Walsh K. A., Philipov P. P., Hurley J. B., Stryer L. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science. 1991 Feb 22;251(4996):915–918. doi: 10.1126/science.1672047. [DOI] [PubMed] [Google Scholar]
  10. Engman D. M., Krause K. H., Blumin J. H., Kim K. S., Kirchhoff L. V., Donelson J. E. A novel flagellar Ca2+-binding protein in trypanosomes. J Biol Chem. 1989 Nov 5;264(31):18627–18631. [PubMed] [Google Scholar]
  11. García-Cardeña G., Oh P., Liu J., Schnitzer J. E., Sessa W. C. Targeting of nitric oxide synthase to endothelial cell caveolae via palmitoylation: implications for nitric oxide signaling. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6448–6453. doi: 10.1073/pnas.93.13.6448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Godsel L. M., Olson C. L., Lacava Z. G., Engman D. M. Comparison of the 24 kDa flagellar calcium-binding protein cDNA of two strains of Trypanosoma cruzi. J Eukaryot Microbiol. 1995 May-Jun;42(3):320–322. doi: 10.1111/j.1550-7408.1995.tb01587.x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Han K. K., Martinage A. Possible relationship between coding recognition amino acid sequence motif or residue(s) and post-translational chemical modification of proteins. Int J Biochem. 1992 Sep;24(9):1349–1363. doi: 10.1016/0020-711x(92)90060-e. [DOI] [PubMed] [Google Scholar]
  15. Hanakam F., Albrecht R., Eckerskorn C., Matzner M., Gerisch G. Myristoylated and non-myristoylated forms of the pH sensor protein hisactophilin II: intracellular shuttling to plasma membrane and nucleus monitored in real time by a fusion with green fluorescent protein. EMBO J. 1996 Jun 17;15(12):2935–2943. [PMC free article] [PubMed] [Google Scholar]
  16. Hanakam F., Gerisch G., Lotz S., Alt T., Seelig A. Binding of hisactophilin I and II to lipid membranes is controlled by a pH-dependent myristoyl-histidine switch. Biochemistry. 1996 Aug 27;35(34):11036–11044. doi: 10.1021/bi960789j. [DOI] [PubMed] [Google Scholar]
  17. Kirchhoff L. V., Neva F. A. Chagas' disease in Latin American immigrants. JAMA. 1985 Dec 6;254(21):3058–3060. [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. 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]
  21. 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]
  22. Olson C. L., Nadeau K. C., Sullivan M. A., Winquist A. G., Donelson J. E., Walsh C. T., Engman D. M. Molecular and biochemical comparison of the 70-kDa heat shock proteins of Trypanosoma cruzi. J Biol Chem. 1994 Feb 4;269(5):3868–3874. [PubMed] [Google Scholar]
  23. Parkin E. T., Turner A. J., Hooper N. M. Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes. Biochem J. 1996 Nov 1;319(Pt 3):887–896. doi: 10.1042/bj3190887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Randazzo P. A., Terui T., Sturch S., Fales H. M., Ferrige A. G., Kahn R. A. The myristoylated amino terminus of ADP-ribosylation factor 1 is a phospholipid- and GTP-sensitive switch. J Biol Chem. 1995 Jun 16;270(24):14809–14815. doi: 10.1074/jbc.270.24.14809. [DOI] [PubMed] [Google Scholar]
  25. Resh M. D. Interaction of tyrosine kinase oncoproteins with cellular membranes. Biochim Biophys Acta. 1993 Dec 23;1155(3):307–322. doi: 10.1016/0304-419x(93)90012-2. [DOI] [PubMed] [Google Scholar]
  26. Robbins S. M., Quintrell N. A., Bishop J. M. Myristoylation and differential palmitoylation of the HCK protein-tyrosine kinases govern their attachment to membranes and association with caveolae. Mol Cell Biol. 1995 Jul;15(7):3507–3515. doi: 10.1128/mcb.15.7.3507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Robinson D. R., Sherwin T., Ploubidou A., Byard E. H., Gull K. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J Cell Biol. 1995 Mar;128(6):1163–1172. doi: 10.1083/jcb.128.6.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Saborio J. L., Manuel Hernandez J., Narayanswami S., Wrightsman R., Palmer E., Manning J. Isolation and characterization of paraflagellar proteins from Trypanosoma cruzi. J Biol Chem. 1989 Mar 5;264(7):4071–4075. [PubMed] [Google Scholar]
  30. 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]
  31. Shenoy-Scaria A. M., Gauen L. K., Kwong J., Shaw A. S., Lublin D. M. Palmitylation of an amino-terminal cysteine motif of protein tyrosine kinases p56lck and p59fyn mediates interaction with glycosyl-phosphatidylinositol-anchored proteins. Mol Cell Biol. 1993 Oct;13(10):6385–6392. doi: 10.1128/mcb.13.10.6385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Simons K., Ikonen E. Functional rafts in cell membranes. Nature. 1997 Jun 5;387(6633):569–572. doi: 10.1038/42408. [DOI] [PubMed] [Google Scholar]
  33. Tanaka T., Ames J. B., Harvey T. S., Stryer L., Ikura M. Sequestration of the membrane-targeting myristoyl group of recoverin in the calcium-free state. Nature. 1995 Aug 3;376(6539):444–447. doi: 10.1038/376444a0. [DOI] [PubMed] [Google Scholar]
  34. Thelen M., Rosen A., Nairn A. C., Aderem A. Regulation by phosphorylation of reversible association of a myristoylated protein kinase C substrate with the plasma membrane. Nature. 1991 May 23;351(6324):320–322. doi: 10.1038/351320a0. [DOI] [PubMed] [Google Scholar]
  35. Tibbetts R. S., Klein K. G., Engman D. M. A rapid method for protein localization in trypanosomes. Exp Parasitol. 1995 May;80(3):572–574. doi: 10.1006/expr.1995.1071. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Towler D. A., Gordon J. I., Adams S. P., Glaser L. The biology and enzymology of eukaryotic protein acylation. Annu Rev Biochem. 1988;57:69–99. doi: 10.1146/annurev.bi.57.070188.000441. [DOI] [PubMed] [Google Scholar]
  38. Wedegaertner P. B., Bourne H. R. Activation and depalmitoylation of Gs alpha. Cell. 1994 Jul 1;77(7):1063–1070. doi: 10.1016/0092-8674(94)90445-6. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. Weimbs T., Low S. H., Chapin S. J., Mostov K. E. Apical targeting in polarized epithelial cells: There's more afloat than rafts. Trends Cell Biol. 1997 Oct;7(10):393–399. doi: 10.1016/S0962-8924(97)01130-6. [DOI] [PubMed] [Google Scholar]
  42. Wong S., Kretsinger R. H., Campbell D. A. Identification of a new EF-hand superfamily member from Trypanosoma brucei. Mol Gen Genet. 1992 May;233(1-2):225–230. doi: 10.1007/BF00587583. [DOI] [PubMed] [Google Scholar]
  43. Yoshida N., Mortara R. A., Araguth M. F., Gonzalez J. C., Russo M. Metacyclic neutralizing effect of monoclonal antibody 10D8 directed to the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi. Infect Immun. 1989 Jun;57(6):1663–1667. doi: 10.1128/iai.57.6.1663-1667.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Zozulya S., Stryer L. Calcium-myristoyl protein switch. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11569–11573. doi: 10.1073/pnas.89.23.11569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. da Cunha e Silva N. L., Hassón-Voloch A., de Souza W. Isolation and characterization of a highly purified flagellar membrane fraction from trypanosomatids. Mol Biochem Parasitol. 1989 Nov;37(1):129–136. doi: 10.1016/0166-6851(89)90109-6. [DOI] [PubMed] [Google Scholar]
  46. da Silva A. C., Reinach F. C. Calcium binding induces conformational changes in muscle regulatory proteins. Trends Biochem Sci. 1991 Feb;16(2):53–57. doi: 10.1016/0968-0004(91)90024-p. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES