title: Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.
abstract: 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%.
authors.affiliation: Institut für Organische Chemie und Biochemie, Universität Bonn, Germany.
authors.forename: E C
authors.initials: EC
authors.lastname: Mandon
authors.forename: I
authors.initials: I
authors.lastname: Ehses
authors.forename: J
authors.initials: J
authors.lastname: Rother
authors.forename: G
authors.initials: G
authors.lastname: van Echten
authors.forename: K
authors.initials: K
authors.lastname: Sandhoff
mesh_terms: D000217:Acyltransferases / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D015536:Down-Regulation; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005952:Glucose-6-Phosphatase / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D051379:Mice; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis; D051049:Sphingosine N-Acyltransferase
chemical_list: D013107:Sphingolipids; D010171:Palmitoyl Coenzyme A; D000429:Alcohol Oxidoreductases; C046333:3-dehydrosphinganine reductase; D000217:Acyltransferases; D051049:Sphingosine N-Acyltransferase; D051102:Serine C-Palmitoyltransferase; C495088:Sptlc1 protein, mouse; D005952:Glucose-6-Phosphatase