Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

By E. Mandon, I. Ehses, J. Rother, G. Echten, K. Sandhoff. 1992

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.

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Citations in the corpus

• Mammalian Ceramide Synthases (2010, background): “The subcellular location of the CerS proteins is the ER (8, 10, 11, 20, 23, 26, 27), although it should be emphasized that Table 1 Comparison of human CerS”
• Mitochondrial Ceramide and the Induction of Apoptosis (2005, background): “The de novo synthesis of ceramide occurs in the endoplasmic recticulum (ER) (Mandon et al., 1992) and in mitochondria (Bionda et al., 2004; Shimeno et al., 1998) and begins with the condensation of serine and palmitoylCoA to form 3-ketosphinganine as catalyzed by serine palmitoyl transferase.”
• LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia Published, JLR Papers in Press, March 16, 2005. DOI 10.1194/jlr.M500001-JLR200 (2005, background): “The enzyme localizes to the endoplasmic reticulum, and its activity is inhibited by fumonisins, fungal inhibitors that produce pulmonary edema (16, 17).”
• Differential expression of (dihydro)ceramide synthases in mouse brain: oligodendrocyte-specific expression of CerS2/Lass2 (2008, unknown): “24), membrane-bound enzymes of the endoplasmic reticulum (Mandon et al. 1992; Hirschberg et al. 1993).”
• Discovery of the molecular machinery CERT for endoplasmic reticulum-to-Golgi trafficking of ceramide (2006, background): “These reactions to produce ceramide occur at the cytosolic surface of the ER [8].”
• An overview of sphingolipid metabolism: from synthesis to breakdown (2010, background): “This reaction occurs through cytosolic serine and palmitoyl CoA condensation to produce 3-ketodihydrosphingosine [1].”