Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis.
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.
Halter
David
D
Membrane Enzymology, Bijvoet Center, and Department of Cell Biology, University Medical Center Utrecht, Institute of Biomembranes, Utrecht University, 3584 Utrecht, Netherlands.
Neumann
Sylvia
S
van Dijk
Suzanne M
SM
Wolthoorn
Jasja
J
de Mazière
Ann M
AM
Vieira
Otilia V
OV
Mattjus
Peter
P
Klumperman
Judith
J
van Meer
Gerrit
G
Sprong
Hein
H
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
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