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

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          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.

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          Most cited references57

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          Molecular machinery for non-vesicular trafficking of ceramide.

          Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.
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            Characterization of a cis-Golgi matrix protein, GM130

            Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure.
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              Identification of a family of animal sphingomyelin synthases.

              Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.
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                Author and article information

                Journal
                J Cell Biol
                jcb
                The Journal of Cell Biology
                The Rockefeller University Press
                0021-9525
                1540-8140
                8 October 2007
                : 179
                : 1
                : 101-115
                Affiliations
                [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
                Author notes

                Correspondence to Gerrit van Meer: g.vanmeer@ 123456uu.nl

                Article
                200704091
                10.1083/jcb.200704091
                2064740
                17923531
                411671dd-2f0f-489d-83e8-c4bec2d28533
                Copyright © 2007, The Rockefeller University Press
                History
                : 17 April 2007
                : 6 September 2007
                Categories
                Research Articles
                Article

                Cell biology
                Cell biology

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