Metabolic Labeling of Prenyl and Carboxyl‐Methyl Groups

Anthony I. Magee1

1 National Institute of Medical Research, London, United Kingdom
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.5
DOI:  10.1002/0471143030.cb0705s05
Online Posting Date:  May, 2001
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Abstract

This unit provides protocols for prenylation and carboxy‐methylation of proteins in cultured cells. These modifications often accompany fatty acid acylation. Cultured cells can be labeled biosynthetically using radiolabeled mevalonate, a precursor, to label intermediates that are incorporated as prenoids‐‐e.g., farnesyl and geranylgeranyl. Carboxy‐methylation often accompanies prenylation. The methyl group can be labeled using [3H‐methyl]methionine.

     
 
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Table of Contents

  • Basic Protocol 1: Prenylation of Proteins in Cultured Cells
  • Basic Protocol 2: Carboxyl‐Methylation of Proteins in Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Prenylation of Proteins in Cultured Cells

  Materials
  • Cells
  • Complete tissue culture medium appropriate for the cells, 37°C
  • Complete tissue culture medium containing dialyzed serum, 37°C
  • 10 mM mevinolin (see recipe)
  • 1 µCi/µl R‐[5‐3H]mevalonic acid (10 to 40 Ci/mmol; DuPont NEN or American Radiolabeled Chemicals)
  • PBS, pH 7.2 ( appendix 2A) ice‐cold
  • 1× SDS‐PAGE sample buffer (unit 6.1) or RIPA lysis buffer for immunprecipitation (unit 7.4)
  • Nitrogen gas
  • Additional reagents and equipment for immunoprecipitation (unit 7.2), SDS‐PAGE (unit 6.1), treating a gel with sodium salicylate (unit 6.3), and fluorography (unit 6.3)

Basic Protocol 2: Carboxyl‐Methylation of Proteins in Cultured Cells

  Materials
  • Cells
  • Complete tissue culture medium (CM) appropriate for the cells
  • Methionine‐free tissue culture medium containing dialyzed serum (MFM) 95:5 (v/v) MFM/complete culture medium with dialyzed serum (95:5 MFM/CM)
  • 1 µCi/µl L‐[3H‐methyl]methionine (∼80 Ci/mmol, Amersham)
  • PBS, pH 7.2 ( appendix 2A), ice‐cold
  • 1× SDS‐PAGE sample buffer (unit 6.1) or RIPA lysis buffer (unit 7.4)
  • 1 M NaOH
  • 1 M HCl
  • Warm room or incubator at 37°C
  • Preflashed film (unit 6.3)
  • Additional reagents and equipment for immunoprecipitation (unit 7.2), treating a gel with sodium solicylate (unit 6.3), SDS‐PAGE (unit 6.1), and fluorography (unit 6.3)
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Figures

Videos

Literature Cited

Literature Cited
   Aitken, A. 1992. Structure determination of acylated proteins. In Lipid Modifications of Proteins: A Practical Approach (N.M. Hooper and A.J. Turner. eds.) pp. 63‐ 88. IRL Press, Oxford.
   Alberts, A.W. 1988. Discovery, biochemistry and biology of lovastatin. Am. J. Cardiol. 62:10J‐15J.
  Casey, P. (ed.) 1990. Covalent Modification of Proteins by Lipids. Methods, Volume 1.
  Casey, P. and Buss, J. (eds.) 1995. Lipid Modifications of Proteins. Methods Enzymol., Volume 250 .
   Chamberlain, J.P. 1979. Fluorographic detection of radioactivity in polyacrylamide gels with the water‐soluble fluor, sodium salicylate. Anal. Biochem. 98:132‐135.
   Chelsky, D., Ruskin, B., and Koshland, D.E. Jr. 1985. Methyl‐esterified proteins in a mammalian cell line. Biochemistry 24:6651‐6658.
   Clarke, S. 1992. Protein isoprenylation and methylation at carboxyl‐terminal cysteine residues. Annu. Rev. Biochem. 61:355‐386.
   Farnsworth, C.C., Casey, P.J., Howald, W.N., Glomset, J.A., and Gelb, M.H. 1990. Structural characterization of prenyl groups attached to proteins. Methods 1:231‐240.
   Giannakouros, T., Armstrong, J., and Magee, A.I. 1992. Protein prenylation in Schizosaccharomyces pombe. FEBS Letts. 297:103‐106.
   Giannakouros, T., Newman, C.M.H., Craighead, M.W., Armstrong, J., and Magee, A.I. 1993. Post‐translational processing of S. pombe YPT5 protein: In vitro and in vivo analysis of processing mutants. J. Biol. Chem. 268:24467‐24474.
   Goldstein, J.L. and Brown, M.S. 1990. Regulation of the mevalonate pathway. Nature 43:425‐430.
   Gutierrez, L., Magee, A.I., Marshall, C.J., and Hancock, J.F. 1989. Post‐translational processing of p21ras is two‐step and involves carboxyl‐methylation and carboxy‐terminal proteolysis. EMBO J. 8:1093‐1098.
   Hancock, J.F., Cadwallader, K., and Marshall, C.J. 1991. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K‐ras(B). EMBO J. 10:641‐646.
   Hooper, N.M. and Turner, A.J. eds. 1992. Lipid Modification of Proteins: A Practical Approach. IRL Press, Oxford.
   Kita, T., Brown, M.S., and Goldstein, J.L. 1980. Feedback regulation of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase in livers of mice treated with mevinolin, a competitive inhibitor of the reductase. J. Clin. Invest. 66:1094‐1107.
   Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680‐685.
   Laskey, R.A. 1980. The use of intensifying screens or organic scintillators for visualizing radioactive molecules resolved by gel electrophoresis Methods Enzymol. 65:363‐369.
   Magee, A.I., Wootton, J., and de Bony, J. 1995. Optimized methods for detecting radiolabeled lipid‐modified proteins in polyacrylamide gels. Methods Enzymol. 250:330‐336.
   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.
   Newman, C.M.H., Giannakouros, T., Hancock, J.F., Fawell, E.H., Armstrong, J., and Magee, A.I. 1992. Post‐translational processing of Schizosaccharomyces pombe YPT proteins. J. Biol. Chem. 267:11329‐11336.
   Parenti, M. and Magee, A.I. 1995. Fatty acid‐ and isoprenoid‐linked membrane proteins. In Biomembranes, Vol.1 (A.G. Lee, ed.) pp. 79‐105. JAI Press, Greenwich, Connecticut.
Key References
   Casey, P. (ed.) 1990. See above.
  These references contain compilations of methods for studying lipid modifications of proteins.
   Casey, P. and Buss, J. (eds.) 1995. See above.
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