Nonradioactive Analysis of Dynamic Protein Palmitoylation

Brent R. Martin1

1 University of Michigan, Ann Arbor, Michigan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.15
DOI:  10.1002/0471140864.ps1415s73
Online Posting Date:  September, 2013
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Abstract

Methods to study protein S‐palmitoylation dynamics have previously relied on metabolic labeling with [14C]palmitate, which requires additional safety precautions and long exposures. Nonradioactive alkynyl palmitate analogs have been developed for in‐gel fluorescence detection and affinity purification. Cells metabolically labeled with the commercially available analog 17‐octadynoic acid are lysed and then combined with azide‐linked reporter tags for efficient conjugation by copper‐catalyzed click chemistry in phosphate buffer. This approach has been demonstrated to label hundreds of endogenous palmitoylated proteins and is compatible with traditional pulse‐chase methods. This protocol describes the reagents and procedures for labeling and detection of dynamic palmitoylation in mammalian cells. Curr. Protoc. Protein Sci. 73:14.15.1‐14.15.9. © 2013 by John Wiley & Sons, Inc.

Keywords: palmitoylation; click chemistry; post‐translational modifications

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cultured mammalian cells
  • Culture medium (e.g., DMEM, Invitrogen, cat. no. 10564‐011), 37°C
  • DPBS, calcium and magnesium free (Invitrogen, cat. no. 14190‐136), 37° and 4°C (do not use EDTA)
  • 17‐ODYA labeling medium (see recipe), 37°C
  • Palmitic acid chase medium (see recipe), 37°C
  • Protein thioesterase inhibitor (select one):
    • Phenylmethylsulfonylfluoride (PMSF, Sigma, cat. no. P7626)
    • Hexadecylsulfonyl fluoride (HDSF, Calbiochem, cat. no. 373250)
    • Hexadecylfluorophosphonate (HDFP, available upon request, synthetic methods described in Martin et al., )
  • 50 mM copper(II) sulfate (CuSO 4, Sigma‐Aldrich, cat. no. 451657) in water
  • 50 mM tris 2‐carboxyethyl phosphine (TCEP, Sigma‐Aldrich, cat. no. 93284), freshly prepared in DPBS
  • 1× TBTA ligand solution (see recipe)
  • 1 mM rhodamine‐azide: tetramethylrhodamine (TAMRA) azide [tetramethylrhodamine 5‐carboxamido‐(6‐azidohexanyl), 5‐isomer, Invitrogen, cat. no. T10182] in dimethyl sulfoxide (DMSO)
  • 50% (v/v) hydroxylamine (Alfa Aesar, cat no. B22202 or L16990), adjusted to pH 7 in PBS
  • Cell scraper
  • Branson S‐250A sonifier cell disruptor with 1/8‐in. tapered microtip
  • Beckman Optima Max ultracentrifuge (or equivalent) with TLA‐100.3 rotor
  • 1.5‐ml thick‐walled ultracentrifuge tubes (Beckman, cat. no. 357448)
  • 1.5‐ml microcentrifuge tubes
  • Flatbed fluorescence gel scanner, Hitachi FMBIO II or equivalent
  • Additional reagents and equipment for protein quantification (unit 3.4), immunoprecipitation (unit 9.8, optional), SDS‐PAGE (unit 10.1), scanning gels (unit 10.12), electroblotting (unit 10.7, optional), and detection of proteins on blots (unit 10.10, optional)
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Figures

Videos

Literature Cited

Literature Cited
  Ahearn, I.M., Tsai, F.D., Court, H., Zhou, M., Jennings, B.C., Ahmed, M., Fehrenbacher, N., Linder, M.E., and Philips, M.R. 2011. FKBP12 binds to acylated H‐Ras and promotes depalmitoylation. Mol. Cell 41:173-185.
  Charron, G., Zhang, M.M., Yount, J.S., Wilson, J., Raghavan, A.S., Shamir, E., and Hang, H.C. 2009. Robust fluorescent detection of protein fatty‐acylation with chemical reporters. J. Am. Chem. Soc. 131:4967-4975.
  Das, A.K., Bellizzi, J.J., III, Tandel, S., Biehl, E., Clardy, J., and Hofmann, S.L. 2000. Structural basis for the insensitivity of a serine enzyme (palmitoyl‐protein thioesterase) to phenylmethylsulfonyl fluoride. J. Biol. Chem. 275:23847-23851.
  Dekker, F.J., Rocks, O., Vartak, N., Menninger, S., Hedberg, C., Balamurugan, R., Wetzel, S., Renner, S., Gerauer, M., Schölermann, B., Rusch, M., Kramer, J.W., Rauh, D., Coates, G.W., Brunsveld, L., Bastiaens, P.I.H., and Waldmann, H. 2010. Small‐molecule inhibition of APT1 affects Ras localization and signaling. Nat. Chem. Biol. 6:449-456.
  Drisdel, R.C. and Green, W.N. 2004. Labeling and quantifying sites of protein palmitoylation. Biotechniques 36:276-285.
  Forrester, M.T., Hess, D.T., Thompson, J.W., Hultman, R., Moseley, M.A., Stamler, J.S., and Casey, P.J. 2011. Site‐specific analysis of protein S‐acylation by resin‐assisted capture. J. Lipid Res. 52:393-398.
  Hang, H.C., Geutjes, E.J., Grotenbreg, G., Pollington, A.M., Bijlmakers, M.J., and Ploegh, H.L. 2007. Chemical probes for the rapid detection of fatty‐acylated proteins in mammalian cells. J. Am. Chem. Soc. 129:2744-2745.
  Kang, R., Wan, J., Arstikaitis, P., Takahashi, H., Huang, K., Bailey, A.O., Thompson, J.X., Roth, A.F., Drisdel, R.C., Mastro, R., Green, W.N., Yates Iii, J.R., Davis, N.G., and El‐Husseini, A. 2008. Neural palmitoyl‐proteomics reveals dynamic synaptic palmitoylation. Nature 456:904-909.
  Kostiuk, M.A., Corvi, M.M., Keller, B.O., Plummer, G., Prescher, J.A., Hangauer, M.J., Bertozzi, C.R., Rajaiah, G., Falck, J.R., and Berthiaume, L.G. 2008. Identification of palmitoylated mitochondrial proteins using a bio‐orthogonal azido‐palmitate analog. FASEB J. 22:721-732.
  Martin, B.R. and Cravatt, B.F. 2009. Large‐scale profiling of protein palmitoylation in mammalian cells. Nat. Methods 6:135-138.
  Martin, B.R., Wang, C., Adibekian, A., Tully, S.E., and Cravatt, B.F. 2012. Global profiling of dynamic protein palmitoylation. Nat. Methods 9:84-89.
  Roth, A.F., Wan, J., Bailey, A.O., Sun, B., Kuchar, J.A., Green, W.N., Phinney, B.S., Yates, J.R., III, and Davis, N.G. 2006. Global analysis of protein palmitoylation in yeast. Cell 125:1003-1013.
  Rusch, M., Zimmermann, T.J., Burger, M., Dekker, F.J., Gormer, K., Triola, G., Brockmeyer, A., Janning, P., Bottcher, T., Sieber, S.A., Vetter, I.R., Hedberg, C., and Waldmann, H. 2011. Identification of acyl protein thioesterases 1 and 2 as the cellular targets of the Ras‐signaling modulators palmostatin B and M. Angew Chem. Int. Ed. Engl. 50:9838-9842.
  Schlesinger, M., Magee, A., and Schmidt, M. 1980. Fatty acid acylation of proteins in cultured cells. J. Biol. Chem. 255:10021-10024.
  Schmidt, M.F., Bracha, M., and Schlesinger, M.J. 1979. Evidence for covalent attachment of fatty acids to Sindbis virus glycoproteins. Proc. Natl. Acad. Sci. U.S.A. 76:1687-1691.
  Wan, J., Roth, A.F., Bailey, A.O., and Davis, N.G. 2007. Palmitoylated proteins: Purification and identification. Nat. Protoc. 2:1573-1584.
  Yount, J.S., Moltedo, B., Yang, Y.‐Y., Charron, G., Moran, T.M., López, C.B., and Hang, H.C. 2010. Palmitoylome profiling reveals S‐palmitoylation‐dependent antiviral activity of IFITM3. Nat. Chem. Biol. 6:610-614.
  Zhang, M.M., Tsou, L.K., Charron, G., Raghavan, A.S., and Hang, H.C. 2010. Tandem fluorescence imaging of dynamic S‐acylation and protein turnover. Proc. Natl. Acad. Sci. U.S.A. 107:8627-8632.
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