Abstract
Metabolic labeling with tritiated palmitate is a direct method for monitoring posttranslational modification of Ras proteins with this fatty acid. Advances in intensifying screens have allowed for the easy visualization of tritium without the need for extended exposure times. While more energetic radioisotopes are easier to visualize, the lack of commercial source and need for shielding make them more difficult to work with. Since radiolabeled palmitate is directly incorporated into Ras, its loss can be monitored by traditional pulse-chase experiments that cannot be accomplished with the method of acyl-exchange chemistry. As such, tritiated palmitate remains a readily accessible and direct method for monitoring the palmitoylation status of Ras proteins under a multitude of conditions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Willumsen BM, Norris K, Papageorge AG et al (1984) Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus. EMBO J 3:2581–2585
Wright LP, Philips MR (2006) Thematic review series: lipid posttranslational modifications. CAAX modification and membrane targeting of Ras. J Lipid Res 47:883–891
Choy E, Chiu VK, Silletti J et al (1999) Endomembrane trafficking of ras: the CAAX motif targets proteins to the ER and Golgi. Cell 98:69–80
Bivona TG, Quatela SE, Bodemann BO et al (2006) PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol Cell 21:481–493
Goodwin JS, Drake KR, Rogers C et al (2005) Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway. J Cell Biol 170:261–272
Smotrys JE, Linder ME (2004) Palmitoylation of intracellular signaling proteins: regulation and function. Annu Rev Biochem 73:559–587
Sefton BM, Trowbridge IS, Cooper JA et al (1982) The transforming proteins of Rous sarcoma virus, Harvey sarcoma virus and Abelson virus contain tightly bound lipid. Cell 31: 465–474
Hancock JF (1995) Prenylation and palmitoylation analysis. Methods Enzymol 55:237–245
Peseckis SM, Deichaite I, Resh MD (1993) Iodinated fatty acids as probes for myristate processing and function. Incorporation into pp 60v-src. J Biol Chem 268:5107–5114
Liang X, Nazarian A, Erdjument-Bromage H et al (2001) Heterogeneous fatty acylation of Src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction. J Biol Chem 276:30987–30994
Drisdel RC, Green WN (2004) Labeling and quantifying sites of protein palmitoylation. Biotechniques 36:276–285
Drisdel RC, Alexander JK, Sayeed A et al (2006) Assays of protein palmitoylation. Methods 40:127–134
Kang R, Wan J, Arstikaitis P et al (2008) Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation. Nature 456:904–909
Roth AF, Wan J, Bailey AO et al (2006) Global analysis of protein palmitoylation in yeast. Cell 125:1003–1013
Charron G, Zhang MM, Yount JS et al (2009) Robust fluorescent detection of protein fatty-acylation with chemical reporters. J Am Chem Soc 131:4967–4975
Yang YY, Ascano JM, Hang HC (2010) Bioorthogonal chemical reporters for monitoring protein acetylation. J Am Chem Soc 132:3640–3641
Ahearn IM, Tsai FD, Court H et al (2011) FKBP12 binds to acylated H-ras and promotes depalmitoylation. Mol Cell 41:173–185
Veit M, Ponimaskin E, Schmidt MF (2008) Analysis of S-acylation of proteins. Methods Mol Biol 446:163–182
Qanbar R, Bouvier M (2004) Determination of protein-bound palmitate turnover rates using a three-compartment model that formally incorporates [3H]palmitate recycling. Biochemistry 43:12275–12288
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Tsai, F.D., Wynne, J.P., Ahearn, I.M., Philips, M.R. (2014). Metabolic Labeling of Ras with Tritiated Palmitate to Monitor Palmitoylation and Depalmitoylation. In: Trabalzini, L., Retta, S. (eds) Ras Signaling. Methods in Molecular Biology, vol 1120. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-791-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-62703-791-4_3
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-790-7
Online ISBN: 978-1-62703-791-4
eBook Packages: Springer Protocols