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. 2007 Oct 8;179(1):101-15.
doi: 10.1083/jcb.200704091.

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

David Halter  1 , Sylvia Neumann, Suzanne M van Dijk, Jasja Wolthoorn, Ann M de Mazière, Otilia V Vieira, Peter Mattjus, Judith Klumperman, Gerrit van Meer, Hein Sprong
Affiliations

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

David Halter et al. J Cell Biol. .

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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Figures

Figure 1.
Figure 1.
Cellular localization of enzymes in Golgi sphingolipid synthesis by confocal fluorescence microscopy. HeLa cells were transiently transfected with the epitope-tagged enzymes (Table I). After 18 h, cells were fixed, permeabilized, and labeled with rabbit antibodies against the HA or V5 epitopes and with mouse antibody against GM130, a cis-Golgi matrix protein. Cells were counterstained with FITC-labeled anti–rabbit (left) and Texas red–labeled anti–mouse (GM130) antisera. Overlapping distributions appear as yellow in the merged images. After brefeldin A (BFA) treatment for 0.5 h, the glycosyltransferases were labeled with the specific antibodies followed by Texas red–labeled secondary antibodies. BFA fuses the Golgi stack to the ER. GCS, GlcCer synthase; LCS, LacCer synthase; GM3S, GM3 synthase; SMS1, SM synthase 1. For GCS, the transfected cells displayed 6.4× the activity of wild-type cells. Bars, 10 μm.
Figure 2.
Figure 2.
Cellular localization of enzymes in Golgi sphingolipid synthesis by IEM. (A) HeLa cells stably transfected with the epitope-tagged enzymes (Table I) were double labeled for GM130 (10 nm protein A–gold; indicated by arrows) as a cis-Golgi marker and the specific enzyme (15 nm protein A–gold). Only a little labeling was present outside the Golgi in all cases, and this was similar to the signal found on untransfected cells. Clathrin coats, which are indicated by arrowheads, were often present on vesicles or tubules close to the TGN. CST, CMP–sialic acid transporter (SLC35A1); NGT, UDP-N-acetylglucosamine transporter (SLC35A3); UGT, UDP-Gal transporter (SLC35A2). (B) For each enzyme, the gold labeling was quantified in Golgi stacks that contained five cisternae. The cisterna labeled with the cis-Golgi reference GM130 was denoted as 1. The number of gold particles per cisterna was expressed as a percentage of the total gold particles within that Golgi stack: mean percentage ± SEM (error bars; n = 20). Bars, 200 nm.
Figure 3.
Figure 3.
Natural GlcCer translocates less efficiently across the Golgi than across the ER membrane. PNSs were prepared from HeLa cells that had been (mock) treated with 1 μg/ml BFA. Equal volumes of PNS were incubated with or without 5 μM PSC833, 20 μM indomethacin (indometh), 1 mM vanadate, or 5 nM concanamycin A (ConcA) for 10 min at 37°C followed by a 1-h incubation with 2 mM UDP-Gal and 1 μM C6-NBD–GlcCer complexed to BSA or 66 kBq/ml [3H]GlcCer solubilized with recombinant GLTP. Each panel represents an independent set of experiments (n = 3). Lipids were extracted, analyzed, and expressed as the percentage of LacCer synthesized in the control. Error bars represent SEM.
Figure 4.
Figure 4.
BFA completely blocks transport to the surface of complex glycolipids but not of GlcCer and GalCer. (A) 2D TLC radiogram of a typical experiment in which MF cells pretreated ± 1 μg/ml BFA and labeled with [14C]palmitate for 1.5 h were incubated for 45 min at 37°C with 1.5 mg/ml GLTP. The lipids were extracted from cells and medium, separated by 2D TLC, and analyzed by phosphorimaging (see Materials and methods). The marked lipids are (1) GlcCer, (2) GM3, (3) phosphatidylcholine, (4) phosphatidylethanolamine, (5) SM, and (6) precursor [14C]palmitate. The origin is marked (o). First dimension, NH4OH solvent. (B) CHO-GalCS cells were labeled with [14C]galactose for 1.5 h ±1 μg/ml BFA, and the transport of GlcCer, GalCer, and GM3 was assessed in a subsequent 45-min incubation with 1.5 mg/ml GLTP (medium), again ±BFA. Lipid numbering as in A, plus (7) GalCer and (8) galactosyldiacylglycerol. First dimension, NH4OH solvent; borate plates. (C) MF, CHO-GalCS, and D6P2T cells were treated as in A and B. Lipid transport as a percentage of that lipid found in the medium was calculated from at least three independent experiments for each lipid, each in triplicate. HeLa cells synthesized Gb3 instead of GM3, which could not be tested because its synthesis is interrupted by BFA (Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200704091/DC1). Error bars represent SEM. (D) MF cells were incubated with or without 1.5 mg/ml GLTP for 45 min at 37°C, fixed, stained with FITC-conjugated annexin V, and analyzed by fluorescence microscopy. As a positive control, MF cells were forced into apoptosis by overnight growth in serum-free medium, exposure to 200 J/m2 UV light, and incubation in regular medium for 2 h at 37°C. The pictures were taken using identical settings. Bars, 10 μm.
Figure 5.
Figure 5.
Effect of the expression levels of FAPP2 and GLTP on GSL transport to the cell surface. (A) MEB4 cells stably expressing RNAi plasmids against FAPP2 or lamin were preincubated with or without 1 μg/ml BFA for 0.5 h and labeled with [14C]palmitic acid for 1.5 h. [14C]GSLs were extracted from the cell surface during an additional 45-min incubation with 1.5 mg/ml GLTP in the medium. The lipids in the cells and media combined with the washes were analyzed and quantified. Transport is expressed as the percentage of GlcCer recovered in the medium. (B and C) D6P2T cells stably expressing RNAi plasmids against GLTP or empty vector (B) or stably transfected with GLTP (C) were treated with BFA and labeled with [14C]galactose, and transport of [14C]GlcCer to the cell surface was measured as in A. Error bars represent SEM. (D) Western blot analysis from D6P2T clones expressing either RNAi plasmids (B) against GLTP and empty vector as control or pCDNA3.1 plasmids with GLTP-HA (C) using a rabbit antiserum against mouse GLTP. A background band at ∼40 kD (BG) and a blot against glyceraldehyde-3 phosphate dehydrogenase (GAPDH) were used as loading controls.
Figure 6.
Figure 6.
In contrast to C6-NBD–GlcCer, endogenous GlcCer is translocated by a mechanism that does not depend on multidrug transporters but is blocked by concanamycin A. (A) MF cells, TKO cells, an MF line with a triple knockout for the multidrug transporters Abcb1a, -b1b, and -c1, and TKO cells transfected with human ABCB1 were incubated with C6-NBD–ceramide for 1 h or [14C]palmitic acid for 1.5 h at 37°C. Labeled GlcCer was extracted from the cell surface by BSA and GLTP and quantitatively analyzed as described in Materials and methods. The same experiment was performed in the presence of BFA (not depicted). (B) MF cells were preincubated with 1 μg/ml BFA ± 1 mM vanadate or 5 nM concanamycin A (ConcA) for 0.5 h at 37°C and incubated with C6-NBD–ceramide with BSA or [14C]palmitate followed by GLTP, all in the presence of the inhibitors. 5 μM PSC833, 20 μM indomethacin, and 50 μM glibenclamide gave similar results to vanadate, both +BFA and –BFA. (C) MF cells (GlcCer and GM3) or D6P2T cells (GalCer) were preincubated with or without 1 μg/ml BFA and 5 nM concanamycin A for 0.5 h at 37°C followed by incubations with [14C]palmitate and GLTP, all in the presence of the inhibitors. Labeled GlcCer was extracted from the cell surface and quantified as in A. Transport was calculated as the percentage of the specific lipid that was recovered in the medium. Error bars represent SEM.
Figure 7.
Figure 7.
Transport of newly synthesized GlcCer to the lumen of the ER and its conversion to GM3 are greatly reduced by knocking down FAPP2. (A) MEB4 cells were transiently transfected with a construct containing the sulfo-GalCS and the PAPS transporter (SGCS). After 18 h, the cells were fixed and labeled with rabbit antibodies against the HA tag of SGCS and with mouse antibodies against GM130 followed by FITC (left) and Texas red secondary antibodies (right). (B) Cells (mock) transfected with SGCS were washed and lysed in 50 mM sodium citrate, pH 5.5, 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.1% SDS, and protease inhibitors. Half of the transfected sample received endoglycosidase H (EndoH), and all samples were incubated for 6 h at 30°C. Proteins were resolved on 7.5% SDS-PAGE followed by Western blot detection with rabbit anti-HA antibodies. The housekeeping enzyme glyceraldehyde-3 phosphatase dehydrogenase (GAPDH) served as a loading control. (C) MEB4 cells (mock) transfected with SGCS were incubated with 600 kBq [35S]H2SO4/105 cells. After 24 h, the lipids were extracted, separated by 1D TLC, and stained with iodine to visualize the bulk lipid phosphatidylcholine (PC) as a loading control. [35S]lipids were visualized by phosphorimager screens. Western blot analysis with anti-HA antibodies was performed on parallel dishes. (D) D6P2T cells were labeled overnight with [35S]H2SO4 in the presence or absence of 40 μM of the GCS inhibitor N-butyldeoxygalactonojirimycin (NB-DGJ), after which the lipids were analyzed by TLC and phosphorimaging. The more hydrophilic spot was most likely SLacCer. (E) Semiquantitative RT-PCR on RNAs from MEB4 clones stably expressing RNAi plasmids against PCR fragments of FAPP2 or lamin as a control to detect expression levels of FAPP2 and GAPDH as a control. (F) MEB4 cells stably expressing RNAi plasmids against FAPP2 or lamin were transiently transfected with SGCS. After 18 h, cells were labeled with 3,600 kBq/ml [35S]H2SO4 for 8 h. The lipids were analyzed as in C. Parallel dishes had an equal expression of SGCS as checked by Western blotting against HA (not depicted). (G and H) MEB4 cells stably expressing RNAi plasmids against FAPP2 or lamin and MDCKII cells infected with empty virus (control) or with retrovirus-mediated RNAi against FAPP2 were labeled overnight with 72 kBq/ml [14C]galactose. Lipids were extracted and separated by 2D TLC, and radiolabeled lipids were quantified using a phosphorimager as described in Materials and methods. GSL, LacCer plus GM3 (MEB4) and a mix of complex GSLs (MDCKII). Values are expressed as a percentage of total [14C]lipids and are the mean of three experiments in triplicate. Error bars represent SEM. Bar, 10 μm.
Figure 8.
Figure 8.
Synthesis and transport of GSLs. (A) In the traditional model, ceramide moves to the cis-Golgi via vesicular transport and is converted to SM and GlcCer. GlcCer is further processed by transferases in the trans-Golgi and TGN. (B) In the updated model, most ceramide is transported to the trans-Golgi by CERT, SM is produced in the lumen, and GlcCer is produced on the cytosolic surface. Most GlcCer is transported back to the ER via FAPP2, whereas another fraction reaches the cytosolic surface of the plasma membrane (or endosome), where it is translocated. The ER GlcCer (our assays do not strictly exclude the possibility that this is actually the cis-Golgi) enters the secretory pathway and is converted to LacCer and GM3 in the trans-Golgi followed by further modification in the TGN, depending on cell type. It remains unclear whether any GlcCer translocates directly across the Golgi membrane.
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