Abstract
Analysis of lipid biosynthesis by radioactive precursor incorporation provides information on metabolic rates and the identity of rate-limiting enzymes and transporters. The biosynthesis of sphingolipids in cultured cells is initiated in the endoplasmic reticulum (ER) by the formation of a sphingoid base from serine and palmitoyl-CoA. N-acylation of the sphingoid base produces ceramide, which is transported to the Golgi apparatus where phosphocholine or carbohydrate headgroups are added to form sphingomyelin (SM) and complex glycosphingolipids (GSLs), respectively. Herein is described a protocol to measure ceramide and SM biosynthesis in cultured cells based on [3H]serine incorporation at the first step in the pathway. The method can be used to assay the effect of pharmacological and genetic manipulations on ceramide synthesis and transport to the Golgi apparatus.
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
Tidhar R, Futerman AH (2013) The complexity of sphingolipid biosynthesis in the endoplasmic reticulum. Biochim Biophys Acta 1833:2511–2518
Hanada K, Kumagai K, Tomishige N, Yamaji T (2009) CERT-mediated trafficking of ceramide. Biochim Biophys Acta 1791:684–691
Hanada K, Kumagai K, Yasuda S, Miura Y, Kawano M, Fukasawa M, Nishijima M (2003) Molecular machinery for non-vesicular trafficking of ceramide. Nature 426:803–809
Smith ER, Merrill AH Jr (1995) Differential roles of de novo sphingolipid biosynthesis and turnover in the “burst” of free sphingosine and sphinganine, and their 1-phosphates and N-acyl-derivatives, that occurs upon changing the medium of cells in culture. J Biol Chem 270:18749–18758
Lowther J, Naismith JH, Dunn TM, Campopiano DJ (2012) Structural, mechanistic and regulatory studies of serine palmitoyltransferase. Biochem Soc Trans 40:547–554
Siow DL, Wattenberg BW (2012) Mammalian ORMDL proteins mediate the feedback response in ceramide biosynthesis. J Biol Chem 287:40198–40204
Perry RJ, Ridgway ND (2006) Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein. Mol Biol Cell 17:2604–2616
Wang E, Norred WP, Bacon CW, Riley RT, Merrill AH (1991) Inhibition of sphingolipid biosynthesis by fumonisins-implications for diseases associated with Fusarium-Moniliforme. J Biol Chem 266:14486–14490
Ridgway ND (1995) 25-Hydroxycholesterol stimulates sphingomyelin synthesis in Chinese hamster ovary cells. J Lipid Res 36:1345–1358
Perry RJ, Ridgway ND (2004) The role of de novo ceramide synthesis in the mechanism of action of the tricyclic xanthate D609. J Lipid Res 45:164–173
Badiani K, Byers DM, Cook HW, Ridgway ND (1996) Effect of fumonisin B1 on phosphatidylethanolamine biosynthesis in Chinese hamster ovary cells. Biochim Biophys Acta 1304:190–196
Burgett AW, Poulsen TB, Wangkanont K, Anderson DR, Kikuchi C, Shimada K, Okubo S, Fortner KC, Mimaki Y, Kuroda M, Murphy JP, Schwalb DJ, Petrella EC, Cornella-Taracido I, Schirle M, Tallarico JA, Shair MD (2011) Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nat Chem Biol 7:639–647
Goto A, Liu X, Robinson CA, Ridgway ND (2012) Multisite phosphorylation of oxysterol-binding protein regulates sterol binding and activation of sphingomyelin synthesis. Mol Biol Cell 23:3624–3635
Acknowledgements
Our studies were supported by the Canadian Institutes of Health Research. Mark Charman assisted in editing of this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Ridgway, N.D. (2016). Analysis of Sphingolipid Synthesis and Transport by Metabolic Labeling of Cultured Cells with [3H]Serine. In: Waugh, M. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 1376. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3170-5_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3170-5_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3169-9
Online ISBN: 978-1-4939-3170-5
eBook Packages: Springer Protocols