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S-Acylation of Proteins

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Post-Translational Modification of Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1934))

Abstract

Palmitoylation or S-acylation is the posttranslational attachment of fatty acids to cysteine residues and is common among integral and peripheral membrane proteins. Palmitoylated proteins have been found in every eukaryotic cell type examined (yeast, insect, and vertebrate cells), as well as in viruses grown in these cells. The exact functions of protein palmitoylation are not well understood. Intrinsically hydrophilic proteins, especially signaling molecules, are anchored by long-chain fatty acids to the cytoplasmic face of the plasma membrane. Palmitoylation may also promote targeting to membrane subdomains enriched in glycosphingolipids and cholesterol or affect protein–protein interactions.

This chapter describes (1) a standard protocol for metabolic labeling of palmitoylated proteins and also the procedures to prove a covalent and ester-type linkage of the fatty acids, (2) a simple method to analyze the fatty acid content of S-acylated proteins, (3) two methods to analyze dynamic palmitoylation for a given protein, and (4) protocols to study cell-free palmitoylation of proteins.

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References

  1. Bijlmakers MJ, Marsh M (2003) The on-off story of protein palmitoylation. Trends Cell Biol 13:32–42

    Article  CAS  Google Scholar 

  2. Charollais J, Van der Goot FG (2009) Palmitoylation of membrane proteins. Mol Membr Biol 26:55–66

    Article  CAS  Google Scholar 

  3. Fukata Y, Fukata M (2010) Protein palmitoylation in neuronal development and synaptic plasticity. Nat Rev. Neurosci 11:161–175

    Article  CAS  Google Scholar 

  4. Greaves J, Chamberlain LH (2007) Palmitoylation-dependent protein sorting. J Cell Biol 176:249–254

    Article  CAS  Google Scholar 

  5. Kordyukova LV, Serebryakova MV, Veit M (2013) Palmitoylation of influenza virus proteins. Biochem Soc Trans 41:50–55

    Article  Google Scholar 

  6. Levental I, Grzybek M, Simons K (2010a) Greasing their way: lipid modifications determine protein association with membrane rafts. Biochemistry 49:6305–6316

    Article  CAS  Google Scholar 

  7. Linder ME, Deschenes RJ (2007) Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 8:74–84

    Article  CAS  Google Scholar 

  8. Smotrys JE, Linder ME (2004) Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem 73:559–587

    Article  CAS  Google Scholar 

  9. Resh MD (2004a) Membrane targeting of lipid modified signal transduction proteins. Subcell Biochem 37:217–232

    Article  CAS  Google Scholar 

  10. Veit M (2012) Palmitoylation of virus proteins. Biol Cell 104:493–515

    Article  CAS  Google Scholar 

  11. Veit M, Engel S, Thaa B et al (2013) Lipid domain association of influenza virus proteins detected by dynamic fluorescence microscopy techniques. Cell Microbiol 15:179–189

    Article  CAS  Google Scholar 

  12. Veit M, Thaa B (2011) Association of influenza virus proteins with membrane rafts. Adv Virol 2011:370606

    Article  Google Scholar 

  13. Dekker FJ, Rocks O, Vartak N et al (2010) Small-molecule inhibition of APT1 affects Ras localization and signaling. Nat Chem Biol 6:449–456

    Article  CAS  Google Scholar 

  14. Duncan JA, Gilman AG (1998) A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J Biol Chem 273:15830–15837

    Article  CAS  Google Scholar 

  15. Tomatis VM, Trenchi A, Gomez GA, Daniotti JL (2010) Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43. PLoS One 5:e15045

    Article  CAS  Google Scholar 

  16. Veit M, Schmidt MF (2001) Enzymatic depalmitoylation of viral glycoproteins with acyl-protein thioesterase 1 in vitro. Virology 288:89–95

    Article  CAS  Google Scholar 

  17. Kordyukova LV, Serebryakova MV, Baratova LA, Veit M (2008) S acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine. J Virol 82:9288–9292

    Article  CAS  Google Scholar 

  18. Veit M, Herrler G, Schmidt MF et al (1990) The hemagglutinating glycoproteins of influenza B and C viruses are acylated with different fatty acids. Virology 177:807–811

    Article  CAS  Google Scholar 

  19. Veit M, Reverey H, Schmidt MF (1996) Cytoplasmic tail length influences fatty acid selection for acylation of viral glycoproteins. Biochem J 318. (Pt 1:163–172

    Article  CAS  Google Scholar 

  20. Jennings BC, Lindner ME (2012) DHHC protein S-acyltransferases use a similar ping-pong kinetic mechanism but display different acyl-CoA specificities. J Biol Chem

    Google Scholar 

  21. Hou H, John Peter AT, Meiringer C et al (2009) Analysis of DHHC acyltransferases implies overlapping substrate specificity and a two-step reaction mechanism. Traffic 10:1061–1073

    Article  CAS  Google Scholar 

  22. Lobo S, Greentree WK, Linder ME, Deschenes RJ (2002) Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae. J Biol Chem 277:41268–41273

    Article  CAS  Google Scholar 

  23. Mitchell DA, Vasudevan A, Linder ME, Deschenes RJ (2006) Protein palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res 47:1118–1127

    Article  CAS  Google Scholar 

  24. Ohno Y, Kihara A, Sano T, Igarashi Y (2006) Intracellular localization and tissue-specific distribution of human and yeast DHHC cysteine-rich domain-containing proteins. Biochim Biophys Acta 1761:474–483

    Article  CAS  Google Scholar 

  25. Roth AF, Feng Y, Chen L, Davis NG (2002) The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase. J Cell Biol 159:23–28

    Article  CAS  Google Scholar 

  26. Roth AF, Wan J, Bailey AO et al (2006) Global analysis of protein palmitoylation in yeast. Cell 125:1003–1013

    Article  CAS  Google Scholar 

  27. Duncan JA, Gilman AG (1996) Autoacylation of G protein alpha subunits. J Biol Chem 271:23594–23600

    Article  CAS  Google Scholar 

  28. Kummel D, Heinemann U, Veit M (2006) Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A 103:12701–12706

    Article  Google Scholar 

  29. Kümmel D, Walter J, Heck M et al (2010) Characterisation of the self-palmitoylation activity of the transport protein particle component Bet3. Cell Mol Life Sci 67:2653–2664

    Article  Google Scholar 

  30. Kordyukova LV, Serebryakova MV (2012) Mass spectrometric approaches to study enveloped viruses: new possibilities for structural biology and prophylactic medicine. Biochemistry (Mosc) 77(8):830–842

    Article  CAS  Google Scholar 

  31. Kordyukova LV, Serebryakova MV, Baratova LA, Veit M (2010) Site-specific attachment of palmitate or stearate to cytoplasmic versus transmembrane cysteines is a common feature of viral spike proteins. Virology 398:49–56

    Article  CAS  Google Scholar 

  32. Kordyukova LV, Ksenofontov AL, Serebryakova MV et al (2004) Influenza A hemagglutinin C-terminal anchoring peptide: identification and mass spectrometric study. Protein Pept Lett 11:385–391

    Article  CAS  Google Scholar 

  33. Serebryakova MV, Kordyukova LV, Baratova LA, Markushin SG (2006) Mass spectrometric sequencing and acylation character analysis of C-terminal anchoring segment from Influenza A hemagglutinin. Eur J Mass Spectrom (Chichester, Eng) 12:51–62

    Article  CAS  Google Scholar 

  34. Kordyukova LV, Serebryakova MV, Polyansky AA et al (2011) Linker and/or transmembrane regions of influenza A/Group-1, A/Group-2, and type B virus hemagglutinins are packed differently within trimers. Biochim Biophys Acta 1808:1843–1854

    Article  CAS  Google Scholar 

  35. Brett K, Kordyukova LV, Serebryakova MV, Mintaev RR, Alexeevski AV, Veit M (2014) Site specific S-acylation of influenza virus hemagglutinin: the location of the acylation site relative to the membrane border is the decisive factor for attachment of stearate. J Biol Chem 289:34978–34989

    Article  Google Scholar 

  36. Martinek K, Klyachko NL, Kabanov AV et al (1989) The second E.C. Slater lecture. Micellar enzymology: its relation to membranology. Biochim Biophys Acta 981(2):161–172. Review

    Article  CAS  Google Scholar 

  37. Serebryakova MV, Kordyukova LV, Semashko TA, Ksenofontov AL, Rudneva IA, Kropotkina EA, Filippova IY, Veit M, Baratova LA (2011) Influenza virus hemagglutinin spike neck architectures and interaction with model enzymes evaluated by MALDI-TOF mass spectrometry and bioinformatics tools. Virus Res 160:294–304

    Article  CAS  Google Scholar 

  38. Mitchell DA, Mitchell G, Ling Y et al (2010) Mutational analysis of Saccharomyces cerevisiae Erf2 reveals a two-step reaction mechanism for protein palmitoylation by DHHC enzymes. J Biol Chem 285:38104–38114

    Article  CAS  Google Scholar 

  39. Kim Y, Tanner KG, Denu JM (2000) A continuous, nonradioactive assay for histone acetyltransferases. Anal Biochem 280:308–314

    Article  CAS  Google Scholar 

  40. Molnos J, Gardiner R, Dale GE, Lange R (2003) A continuous coupled enzyme assay for bacterial malonyl–CoA:acyl carrier protein transacylase (FabD). Anal Biochem 319:171–176

    Article  CAS  Google Scholar 

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Acknowledgments

The German Research Foundation (DFG) and the Russian Foundation for Basic Research, respectively, are acknowledged for providing grants SFB 740 (TP C3) and Ve 141-10 to M.V. and No. 15-04-05044 and No. 18-54-00019 to L.K.

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Authors and Affiliations

  1. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russia

    Larisa Kordyukova & Marina Serebryakova

  2. Freie Universität Berlin, Fachbereich Veterinärmedizin, Zentrum für Infektionsmedizin, Institut für Virologie, Berlin, Germany

    Ludwig Krabben & Michael Veit

Authors
  1. Larisa Kordyukova
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  2. Ludwig Krabben
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  3. Marina Serebryakova
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  4. Michael Veit
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Corresponding authors

Correspondence to Larisa Kordyukova or Michael Veit .

Editor information

Editors and Affiliations

  1. Recombinant Research and Development, Octapharma Biopharmaceuticals GmbH, Heidelberg, Germany

    Christoph Kannicht

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Kordyukova, L., Krabben, L., Serebryakova, M., Veit, M. (2019). S-Acylation of Proteins. In: Kannicht, C. (eds) Post-Translational Modification of Proteins. Methods in Molecular Biology, vol 1934. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9055-9_17

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  • DOI: https://doi.org/10.1007/978-1-4939-9055-9_17

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9053-5

  • Online ISBN: 978-1-4939-9055-9

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