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. 1996 Aug 15;318(Pt 1):163–172. doi: 10.1042/bj3180163

Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins.

M Veit 1, H Reverey 1, M F Schmidt 1
PMCID: PMC1217603  PMID: 8761467

Abstract

We report remarkable differences in the fatty acid content of thioester-type acylated glycoproteins of enveloped viruses from mammalian cells. The E2 glycoprotein of Semliki Forest virus contains mainly palmitic acid like most other palmitoylated proteins analysed so far. However, the other glycoprotein (E1) of the same virus, as well as the HEF (haemagglutinin esterase fusion) glycoprotein of influenza C virus, are unique in this respect because they are acylated primarily with stearic acid. Comparative radiolabelling of uninfected cells with different fatty acids suggests that stearate may also be the prevailing fatty acid in some cellular acylproteins. To look for further differences between palmitoylated and stearoylated glycoproteins we characterized stearoylation in more detail. We identified the acylation site of HEF as a cysteine residue located at the boundary between the transmembrane region and the cytoplasmic tail. The attachment of stearate to HEF and E1 occurs post-translationally in a pre-Golgi compartment. Thus, stearoylated and palmitoylated proteins cannot be discriminated on the basis of the fatty acid linkage site or the intracellular compartment, where acylation occurs. However, stearoylated acylproteins contain a very short, positively charged cytoplasmic tail, whereas in palmitoylated proteins this molecular region is longer. Replacing the short cytoplasmic tail of stearoylated HEF with the long influenza A virus haemagglutinin (HA) tail in an HEF-HA chimera, and subsequent vaccinia T7 expression in CV-1 cells, yielded proteins with largely palmitic acid bound. The reverse chimera, HA-HEF with a short cytoplasmic tail was not fatty acylated at all during expression, indicating that conformational or topological constraints control fatty acid transfer.

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Selected References

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  1. Arumugham R. G., Seid R. C., Jr, Doyle S., Hildreth S. W., Paradiso P. R. Fatty acid acylation of the fusion glycoprotein of human respiratory syncytial virus. J Biol Chem. 1989 Jun 25;264(18):10339–10342. [PubMed] [Google Scholar]
  2. Bach R., Konigsberg W. H., Nemerson Y. Human tissue factor contains thioester-linked palmitate and stearate on the cytoplasmic half-cystine. Biochemistry. 1988 Jun 14;27(12):4227–4231. doi: 10.1021/bi00412a004. [DOI] [PubMed] [Google Scholar]
  3. Barth B. U., Suomalainen M., Liljeström P., Garoff H. Alphavirus assembly and entry: role of the cytoplasmic tail of the E1 spike subunit. J Virol. 1992 Dec;66(12):7560–7564. doi: 10.1128/jvi.66.12.7560-7564.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berger M., Schmidt M. F. Identification of acyl donors and acceptor proteins for fatty acid acylation in BHK cells infected with Semliki Forest virus. EMBO J. 1984 Apr;3(4):713–719. doi: 10.1002/j.1460-2075.1984.tb01874.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berthiaume L., Resh M. D. Biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins. J Biol Chem. 1995 Sep 22;270(38):22399–22405. doi: 10.1074/jbc.270.38.22399. [DOI] [PubMed] [Google Scholar]
  6. Bonatti S., Migliaccio G., Simons K. Palmitylation of viral membrane glycoproteins takes place after exit from the endoplasmic reticulum. J Biol Chem. 1989 Jul 25;264(21):12590–12595. [PubMed] [Google Scholar]
  7. 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]
  8. Camp L. A., Hofmann S. L. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J Biol Chem. 1993 Oct 25;268(30):22566–22574. [PubMed] [Google Scholar]
  9. Casey P. J. Lipid modifications of G proteins. Curr Opin Cell Biol. 1994 Apr;6(2):219–225. doi: 10.1016/0955-0674(94)90139-2. [DOI] [PubMed] [Google Scholar]
  10. Casey W. M., Gibson K. J., Parks L. W. Covalent attachment of palmitoleic acid (C16:1 delta 9) to proteins in Saccharomyces cerevisiae. Evidence for a third class of acylated proteins. J Biol Chem. 1994 Jan 21;269(3):2082–2085. [PubMed] [Google Scholar]
  11. Cierniewski C. S., Krzeslowska J., Pawlowska Z., Witas H., Meyer M. Palmitylation of the glycoprotein IIb-IIIa complex in human blood platelets. J Biol Chem. 1989 Jul 25;264(21):12158–12164. [PubMed] [Google Scholar]
  12. Crise B., Rose J. K. Identification of palmitoylation sites on CD4, the human immunodeficiency virus receptor. J Biol Chem. 1992 Jul 5;267(19):13593–13597. [PubMed] [Google Scholar]
  13. Eason M. G., Jacinto M. T., Theiss C. T., Liggett S. B. The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation. Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):11178–11182. doi: 10.1073/pnas.91.23.11178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. FOLCH J., LEES M. Proteolipides, a new type of tissue lipoproteins; their isolation from brain. J Biol Chem. 1951 Aug;191(2):807–817. [PubMed] [Google Scholar]
  15. Fuerst T. R., Niles E. G., Studier F. W., Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122–8126. doi: 10.1073/pnas.83.21.8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fujimoto T., Stroud E., Whatley R. E., Prescott S. M., Muszbek L., Laposata M., McEver R. P. P-selectin is acylated with palmitic acid and stearic acid at cysteine 766 through a thioester linkage. J Biol Chem. 1993 May 25;268(15):11394–11400. [PubMed] [Google Scholar]
  17. Funke C., Becker S., Dartsch H., Klenk H. D., Mühlberger E. Acylation of the Marburg virus glycoprotein. Virology. 1995 Apr 1;208(1):289–297. doi: 10.1006/viro.1995.1151. [DOI] [PubMed] [Google Scholar]
  18. Gordon J. I., Duronio R. J., Rudnick D. A., Adams S. P., Gokel G. W. Protein N-myristoylation. J Biol Chem. 1991 May 15;266(14):8647–8650. [PubMed] [Google Scholar]
  19. Grand R. J. Acylation of viral and eukaryotic proteins. Biochem J. 1989 Mar 15;258(3):625–638. doi: 10.1042/bj2580625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Hensel J., Hintz M., Karas M., Linder D., Stahl B., Geyer R. Localization of the palmitoylation site in the transmembrane protein p12E of Friend murine leukaemia virus. Eur J Biochem. 1995 Sep 1;232(2):373–380. doi: 10.1111/j.1432-1033.1995.373zz.x. [DOI] [PubMed] [Google Scholar]
  22. Herrler G., Klenk H. D. Structure and function of the HEF glycoprotein of influenza C virus. Adv Virus Res. 1991;40:213–234. doi: 10.1016/S0065-3527(08)60280-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ivanova L., Schlesinger M. J. Site-directed mutations in the Sindbis virus E2 glycoprotein identify palmitoylation sites and affect virus budding. J Virol. 1993 May;67(5):2546–2551. doi: 10.1128/jvi.67.5.2546-2551.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jensen A. A., Pedersen U. B., Kiemer A., Din N., Andersen P. H. Functional importance of the carboxyl tail cysteine residues in the human D1 dopamine receptor. J Neurochem. 1995 Sep;65(3):1325–1331. doi: 10.1046/j.1471-4159.1995.65031325.x. [DOI] [PubMed] [Google Scholar]
  25. Jing S. Q., Trowbridge I. S. Identification of the intermolecular disulfide bonds of the human transferrin receptor and its lipid-attachment site. EMBO J. 1987 Feb;6(2):327–331. doi: 10.1002/j.1460-2075.1987.tb04758.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Lobigs M., Zhao H. X., Garoff H. Function of Semliki Forest virus E3 peptide in virus assembly: replacement of E3 with an artificial signal peptide abolishes spike heterodimerization and surface expression of E1. J Virol. 1990 Sep;64(9):4346–4355. doi: 10.1128/jvi.64.9.4346-4355.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mack D., Kruppa J. Fatty acid acylation at the single cysteine residue in the cytoplasmic domain of the glycoprotein of vesicular-stomatitis virus. Biochem J. 1988 Dec 15;256(3):1021–1027. doi: 10.1042/bj2561021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Magee A. I., Courtneidge S. A. Two classes of fatty acid acylated proteins exist in eukaryotic cells. EMBO J. 1985 May;4(5):1137–1144. doi: 10.1002/j.1460-2075.1985.tb03751.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Magee A. I. Lipid modification of proteins and its relevance to protein targeting. J Cell Sci. 1990 Dec;97(Pt 4):581–584. doi: 10.1242/jcs.97.4.581. [DOI] [PubMed] [Google Scholar]
  32. McIlhinney R. A., Chadwick J. K., Pelly S. J. Studies on the cellular location, physical properties and endogenously attached lipids of acylated proteins in human squamous-carcinoma cell lines. Biochem J. 1987 May 15;244(1):109–115. doi: 10.1042/bj2440109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McIlhinney R. A., Pelly S. J., Chadwick J. K., Cowley G. P. Studies on the attachment of myristic and palmitic acid to cell proteins in human squamous carcinoma cell lines: evidence for two pathways. EMBO J. 1985 May;4(5):1145–1152. doi: 10.1002/j.1460-2075.1985.tb03752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. McIlhinney R. A. The fats of life: the importance and function of protein acylation. Trends Biochem Sci. 1990 Oct;15(10):387–391. doi: 10.1016/0968-0004(90)90237-6. [DOI] [PubMed] [Google Scholar]
  35. Mollner S., Beck K., Pfeuffer T. Acylation of adenylyl cyclase catalyst is important for enzymic activity. FEBS Lett. 1995 Sep 11;371(3):241–244. doi: 10.1016/0014-5793(95)00864-6. [DOI] [PubMed] [Google Scholar]
  36. Moss B., Elroy-Stein O., Mizukami T., Alexander W. A., Fuerst T. R. Product review. New mammalian expression vectors. Nature. 1990 Nov 1;348(6296):91–92. doi: 10.1038/348091a0. [DOI] [PubMed] [Google Scholar]
  37. Muszbek L., Laposata M. Covalent modification of proteins by arachidonate and eicosapentaenoate in platelets. J Biol Chem. 1993 Aug 25;268(24):18243–18248. [PubMed] [Google Scholar]
  38. Naeve C. W., Williams D. Fatty acids on the A/Japan/305/57 influenza virus hemagglutinin have a role in membrane fusion. EMBO J. 1990 Dec;9(12):3857–3866. doi: 10.1002/j.1460-2075.1990.tb07604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. O'Dowd B. F., Hnatowich M., Caron M. G., Lefkowitz R. J., Bouvier M. Palmitoylation of the human beta 2-adrenergic receptor. Mutation of Cys341 in the carboxyl tail leads to an uncoupled nonpalmitoylated form of the receptor. J Biol Chem. 1989 May 5;264(13):7564–7569. [PubMed] [Google Scholar]
  41. Okubo K., Hamasaki N., Hara K., Kageura M. Palmitoylation of cysteine 69 from the COOH-terminal of band 3 protein in the human erythrocyte membrane. Acylation occurs in the middle of the consensus sequence of F--I-IICLAVL found in band 3 protein and G2 protein of Rift Valley fever virus. J Biol Chem. 1991 Sep 5;266(25):16420–16424. [PubMed] [Google Scholar]
  42. Omary M. B., Trowbridge I. S. Biosynthesis of the human transferrin receptor in cultured cells. J Biol Chem. 1981 Dec 25;256(24):12888–12892. [PubMed] [Google Scholar]
  43. Ovchinnikov YuA, Abdulaev N. G., Bogachuk A. S. Two adjacent cysteine residues in the C-terminal cytoplasmic fragment of bovine rhodopsin are palmitylated. FEBS Lett. 1988 Mar 28;230(1-2):1–5. doi: 10.1016/0014-5793(88)80628-8. [DOI] [PubMed] [Google Scholar]
  44. Ponimaskin E., Schmidt M. F. Acylation of viral glycoproteins: structural requirements for palmitoylation of transmembrane proteins. Biochem Soc Trans. 1995 Aug;23(3):565–568. doi: 10.1042/bst0230565. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. Resh M. D. Myristylation and palmitylation of Src family members: the fats of the matter. Cell. 1994 Feb 11;76(3):411–413. doi: 10.1016/0092-8674(94)90104-x. [DOI] [PubMed] [Google Scholar]
  47. Rose J. K., Adams G. A., Gallione C. J. The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2050–2054. doi: 10.1073/pnas.81.7.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schmidt M. F. Acylation of viral spike glycoproteins: a feature of enveloped RNA viruses. Virology. 1982 Jan 15;116(1):327–338. doi: 10.1016/0042-6822(82)90424-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Schmidt M. F., Burns G. R. On the enzymes which make "fatty proteins". Behring Inst Mitt. 1991 Jul;(89):185–197. [PubMed] [Google Scholar]
  50. Schmidt M. F. Fatty acylation of proteins. Biochim Biophys Acta. 1989 Dec 6;988(3):411–426. doi: 10.1016/0304-4157(89)90013-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schmidt M. F., McIlhinney R. A., Burns G. R. Palmitoylation of endogenous and viral acceptor proteins by fatty acyltransferase (PAT) present in erythrocyte ghosts and in placental membranes. Biochim Biophys Acta. 1995 Aug 3;1257(3):205–213. doi: 10.1016/0005-2760(95)00062-h. [DOI] [PubMed] [Google Scholar]
  52. Schmidt M. F., Schlesinger M. J. Fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein. Cell. 1979 Aug;17(4):813–819. doi: 10.1016/0092-8674(79)90321-0. [DOI] [PubMed] [Google Scholar]
  53. Schmidt M. F., Schlesinger M. J. Relation of fatty acid attachment to the translation and maturation of vesicular stomatitis and Sindbis virus membrane glycoproteins. J Biol Chem. 1980 Apr 25;255(8):3334–3339. [PubMed] [Google Scholar]
  54. Schmidt M. F. The transfer of myristic and other fatty acids on lipid and viral protein acceptors in cultured cells infected with Semliki Forest and influenza virus. EMBO J. 1984 Oct;3(10):2295–2300. doi: 10.1002/j.1460-2075.1984.tb02129.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schmidt M., Schmidt M. F., Rott R. Chemical identification of cysteine as palmitoylation site in a transmembrane protein (Semliki Forest virus E1). J Biol Chem. 1988 Dec 15;263(35):18635–18639. [PubMed] [Google Scholar]
  56. Steinhauer D. A., Wharton S. A., Wiley D. C., Skehel J. J. Deacylation of the hemagglutinin of influenza A/Aichi/2/68 has no effect on membrane fusion properties. Virology. 1991 Sep;184(1):445–448. doi: 10.1016/0042-6822(91)90867-b. [DOI] [PubMed] [Google Scholar]
  57. Sudo Y., Valenzuela D., Beck-Sickinger A. G., Fishman M. C., Strittmatter S. M. Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. EMBO J. 1992 Jun;11(6):2095–2102. doi: 10.1002/j.1460-2075.1992.tb05268.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Szepanski S., Veit M., Pleschka S., Klenk H. D., Schmidt M. F., Herrler G. Post-translational folding of the influenza C virus glycoprotein HEF: defective processing in cells expressing the cloned gene. J Gen Virol. 1994 May;75(Pt 5):1023–1030. doi: 10.1099/0022-1317-75-5-1023. [DOI] [PubMed] [Google Scholar]
  59. Towler D., Glaser L. Acylation of cellular proteins with endogenously synthesized fatty acids. Biochemistry. 1986 Feb 25;25(4):878–884. doi: 10.1021/bi00352a021. [DOI] [PubMed] [Google Scholar]
  60. Veit M., Herrler G., Schmidt M. F., Rott R., Klenk H. D. The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids. Virology. 1990 Aug;177(2):807–811. doi: 10.1016/0042-6822(90)90554-5. [DOI] [PubMed] [Google Scholar]
  61. Veit M., Kretzschmar E., Kuroda K., Garten W., Schmidt M. F., Klenk H. D., Rott R. Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin. J Virol. 1991 May;65(5):2491–2500. doi: 10.1128/jvi.65.5.2491-2500.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. 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]
  63. Veit M., Schmidt M. F., Rott R. Different palmitoylation of paramyxovirus glycoproteins. Virology. 1989 Jan;168(1):173–176. doi: 10.1016/0042-6822(89)90417-0. [DOI] [PubMed] [Google Scholar]
  64. Veit M., Schmidt M. F. Timing of palmitoylation of influenza virus hemagglutinin. FEBS Lett. 1993 Dec 27;336(2):243–247. doi: 10.1016/0014-5793(93)80812-9. [DOI] [PubMed] [Google Scholar]
  65. 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]

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