Metabolic Labeling with Fatty Acids

Caroline S. Jackson1, Anthony I. Magee1

1 National Institute For Medical Research, London, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.4
DOI:  10.1002/0471143030.cb0704s05
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Covalent attachment of radiolabeled fatty acids (e.g., [3H]myristate or palmitate) is an alternative method for labeling proteins. This unit contains methods for biosynthetic labeling with fatty acids, analysis of the fatty acid linkage with protein, analysis of total proteinā€bound fatty acid level in cell extracts, and analysis of the identity of the bound fatty acid.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Biosynthetic Labeling with Fatty Acids
  • Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein
  • Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract
  • Basic Protocol 4: Analysis of Fatty Acid Label Identity
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Biosynthetic Labeling with Fatty Acids

  Materials
  • Cells for culture
  • Complete tissue culture medium appropriate for cells
  • Labeling medium: complete tissue culture medium containing the relevant dialyzed serum and 5 mM sodium pyruvate, 37°C
  • 5 to 10 µCi/µl [9,10(n)‐3H]fatty acid, e.g., [9,10(n) ‐3H]palmitic acid or [9,10(n)‐3H]myristic acid (30 to 60 Ci/mmol; Amersham International, American Radiolabeled Chemicals, or NEN Research Products) in ethanol
  • PBS, pH 7.2 ( appendix 2A), ice‐cold
  • 1% (w/v) SDS or SDS sample buffer (for SDS‐PAGE, when using adherent or nonadherent cells respectively; unit 6.1) or RIPA lysis buffer (for immunoprecipitation; see recipe)
  • 5× SDS sample buffer (see recipe)
  • Cell scrapers
  • Nitrogen gas
  • Additional reagents and equipment for culturing cells (unit 1.1), immunoprecipitation (unit 7.2), SDS‐PAGE (unit 6.1), treating a gel with sodium salicylate (unit 6.3), and fluorography (unit 6.3)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media (e.g., DMEM) may require altered levels of CO 2 to maintain pH 7.4.

Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein

  Materials
  • Lysate or immunoprecipitate from [3H]fatty acid–labeled cells (see protocol 1, step )
  • 0.2 M potassium hydroxide (KOH) in methanol
  • Methanol
  • 1 M hydroxylamine⋅HCl, titrated to pH 7.5 with NaOH
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 2A)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), treating a gel with sodium salicylate (unit 6.3), and fluorography (unit 7.2)

Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract

  Materials
  • 0.1 M HCl/acetone, −20°C
  • Lysate from [3H]fatty acid–labeled cells in 1% SDS (see protocol 1, step or )
  • 1% (w/v) SDS
  • 2:1 (v/v) chloroform/methanol
  • Diethyl ether
  • 6 M HCl (concentrated HCl diluted 1:1 with H 2O)
  • Hexane
  • 5 to 10 µCi/µl [9,10(n)‐3H]fatty acid standards (30 to 60 Ci/mmol; Amersham International, American Radiolabeled Chemicals, or NEN Research Products) in ethanol
  • 90:10 (v/v) acetonitrile/acetic acid
  • EN3HANCE spray (NEN Research Products)
  • 15‐ml polypropylene centrifuge tubes
  • Mistral 3000i benchtop centrifuge with swing‐out four‐bucket rotor or equivalent
  • Nitrogen gas
  • 30‐ml thick‐walled Teflon container with an air‐tight screw top
  • 110°C oven
  • Thin‐layer chromatography tank
  • RP18 thin‐layer chromatography plate (e.g., Merck)
  • Kodak XAR‐5 film, preflashed

Basic Protocol 4: Analysis of Fatty Acid Label Identity

  Materials
  • SDS‐PAGE gel of lysate from [3H]fatty acid–labeled cells
  • Additional reagents and equipment for analysis of protein‐bound label (see protocol 3)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Aitken, A., 1992. Structure determination of acylated proteins. In Lipid Modification of Proteins, A Practical Approach (N.M. Hooper and A.J. Turner, eds.) pp. 63‐88. Oxford University Press, Oxford.
   Camp, L.A. and Hoffmann, S.L. 1993. Purification and properties of a palmitoyl‐protein thioesterase that cleaves palmitate from H‐Ras. J. Biol. Chem. 268: 22566‐22574.
   DeGrella, R.F. and Light, R.J. 1980. Uptake and metabolism of fatty acids by dispersed adult rat heart myocytes. J. Biol. Chem. 255: 9739‐9745.
   Dunphy, J.T. and Linder, H.E. 1998. Signaling functions of protein palmitoylation. Biochim. Biophys. Acta 1436: 245‐261.
   French, S.A., Christakis, H., O'Neill, R.R., and Miller, S.P.F. 1994. An assay for myristoyl‐CoA:protein N‐myristoyltransferase activity based on ion‐exchange exclusion of [3H]myristoyl peptide . Anal. Biochem. 220: 115‐121.
   Glover, C.J., Goddard, C., and Felsted, R.L. 1988. N‐Myristoylation of p60src. Biochem. J. 250: 485‐491.
   Gordon, J.I., Duronio, R.J., Rudnick, D.A., Adams, S.P., and Gokel, G.W. 1991. Protein N‐myristoylation. J. Biol. Chem. 266: 8647‐8650.
   James, G. and Olson, E.N. 1989. Identification of a novel fatty acylated protein that partitions between the plasma membrane and cytosol and is deacylated in response to serum and growth factor stimulation. J. Biol. Chem. 264: 20988‐21006.
   Jochen, A., Hays, J., Lianos, E., and Hager, S. 1991. Insulin stimulates fatty acid acylation of adipocyte proteins. Biochem. Biophys. Res. Commun. 177: 797‐801.
   Kamps, M.P., Buss, J.E., and Sefton, B.M. 1986. Rous sarcoma virus transforming protein lacking myristic acid phosphorylates known polypeptide substrates without inducing transformation. Cell 45: 105‐112.
   King, M.J. and Sharma, R.K. 1991. N‐Myristoyl transferase assay using phosphocellulose paper binding. Anal. Biochem. 199: 149‐153.
   Magee, A.I., Wootton, J., and de Bony, J. (1995). Optimized methods for detecting radiolabeled lipid‐modified proteins in polyacrylamide gels. Methods in Enzymology 250: 330‐336.
   Moench, S.J., Terry, C.E., and Dewey, T.G. 1994a. Fluorescence labeling of the palmitoylation sites of rhodopsin. Biochemistry 33: 5783‐5790.
   Moench, S.J., Moreland, J., Stewart, D.H. and Dewey, T.G. 1994b. Fluorescence studies of the location and membrane accessibility of the palmitoylation sites of rhodopsin. Biochemistry 33: 5791‐5796.
   Muszbek, L. and Laposata, M. 1993. Myristoylation of proteins in platelets occurs predominantly through thioester linkages. J. Biol. Chem. 268: 8251‐8255.
   Newman, C.M.H. and Magee, A.I. 1993. Post‐translational processing of the ras superfamily of small GTP‐binding proteins. Biochim. Biophys. Acta 1155: 79‐96.
   Peseckis, S.M., Deichaite, I. and Resh, M.D., 1993. Iodinated fatty acids as probes for myristate processing and function. J. Biol. Chem. 268: 5107‐5114.
   Resh, M.D. 1994. Myristoylation and palmitoylation of Src family members: The fats of the matter. Cell 76: 411‐413.
   Rodgers, W., Crise, B., and Rose, J.K. 1994. Signals determining protein tyrosine kinase and glycosyl‐phosphatidylinositol‐anchored protein targeting to a glycolipid‐enriched membrane fraction. Mol. Cell. Biol. 14: 5384‐5391.
   Rudnick, D.A., McWherter, C.A., Rocque, W.J., Lennon, P.J., Getman, D.P., and Gordon, J.I. 1991. Kinetic and structural evidence for a sequential ordered bi bi mechanism of catalysis by Saccharomycescerevisiae myristoyl‐CoA:protein N‐myristoyltransferase. J. Biol. Chem. 266: 9732‐9739.
   Rudnick, D.A., Duronio, R.J., and Gordon, J.I. 1992. Methods for studying myristoyl‐CoA:protein N‐myristoyltransferase. In Lipid Modification of Proteins, A Practical Approach (N.M. Hooper and A.J. Turner, eds.) pp. 37‐61. Oxford University Press, Oxford.
   Tomoda, H., Igarashi, K., and Ømura, S. 1987. Inhibition of acyl‐CoA synthetase by triacsins. Biochim. Biophys. Acta 921: 595‐598.
   Wedegaertner, P.B., Wilson, P.T., and Bourne, H.B. 1995. Lipid modifications of trimeric G proteins. J. Biol. Chem. 270: 503‐506.
Key Reference
  Casey P.J. and Buss J.E. 1995. Lipid modification of proteins. Methods Enzymol. Vol. 250.
  A compilation of methods used in studying lipid modification of proteins.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library