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Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis

¹ Membrane Enzymology, Bijvoet Center, and ² Department of Cell Biology, University Medical Center Utrecht, Institute of Biomembranes, Utrecht University, 3584 Utrecht, Netherlands
³ Max Planck Institute for Molecular Cell Biology and Genetics, 01307 Dresden, Germany
⁴ Department of Biochemistry and Pharmacy, Åbo Akademi University, FI-20520 Turku, Finland

Correspondence to Gerrit van Meer: g.vanmeer{at}uu.nl

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.

D. Halter's present address is Dept. of Bio-Molecular Engineering, Philips Research Laboratories Eindhoven, 5656 AA Eindhoven, Netherlands.

J. Wolthoorn's present address is TNO Quality of Life, Analytical Research Department, 3700 AJ Zeist, Netherlands.

O.V. Vieira's present address is Centro de Neurociencias e Biologia Celular, Universidade de Coimbra, 3000 Coimbra, Portugal.

H. Sprong's present address is Laboratory for Zoonoses and Environmental Microbiology, National Institute of Public Health and Environment (RIVM), 3720 BA Bilthoven, Netherlands.

Abbreviations used in this paper: BFA, brefeldin A; C₆-NBD, N-6-NBD-aminohexanoyl; CERT, ceramide transport protein; CST, CMP–sialic acid transporter; GalCer, galactosylceramide; GalCS, GalCer synthase; GCS, GlcCer synthase; GlcCer, glucosylceramide; GLTP, glycolipid transfer protein; GM3S, GM3 synthase; GSL, glycosphingolipid; IEM, immuno-EM; LacCer, lactosylceramide; LCS, LacCer synthase; MF, mouse fibroblast; PAPS, 3'-phosphoadenosine 5'-phosphosulfate; PNS, postnuclear supernatant; SGalCer, GalCer sulfate; SM, sphingomyelin; SMS, SM synthase; TKO, triple knockout; UGT, UDP-Gal transporter.

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