title: Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis.
abstract: Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase.
authors.affiliation: Membrane Enzymology, Bijvoet Center, and Department of Cell Biology, University Medical Center Utrecht, Institute of Biomembranes, Utrecht University, 3584 Utrecht, Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Halter
authors.forename: Sylvia
authors.initials: S
authors.lastname: Neumann
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: van Dijk
authors.forename: Jasja
authors.initials: J
authors.lastname: Wolthoorn
authors.forename: Ann M
authors.initials: AM
authors.lastname: de Mazière
authors.forename: Otilia V
authors.initials: OV
authors.lastname: Vieira
authors.forename: Peter
authors.initials: P
authors.lastname: Mattjus
authors.forename: Judith
authors.initials: J
authors.lastname: Klumperman
authors.forename: Gerrit
authors.initials: G
authors.lastname: van Meer
authors.forename: Hein
authors.initials: H
authors.lastname: Sprong
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D020126:Brefeldin A / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005963:Glucosylceramides / Q000378:metabolism*; D006028:Glycosphingolipids / Q000096:biosynthesis*; D016695:Glycosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007790:Lactosylceramides / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D054328:Proton Pump Inhibitors / Q000494:pharmacology; D051381:Rats
chemical_list: D015703:Antigens, CD; D002352:Carrier Proteins; D005963:Glucosylceramides; D006028:Glycosphingolipids; D007790:Lactosylceramides; D018942:Macrolides; D054328:Proton Pump Inhibitors; D020126:Brefeldin A; C009744:CDw17 antigen; C036978:concanamycin A; D016695:Glycosyltransferases; D017346:Protein Serine-Threonine Kinases