From the Biochemie Zentrum Heidelberg, Biosynthesis of plasma membrane sphingolipids
involves the coordinate action of enzymes localized to individual
compartments of the biosynthetic secretory pathway of proteins. These
stations include the endoplasmic reticulum and the Golgi apparatus.
Although a precise localization of all the enzymes that synthesize
glycosphingolipids has not been achieved to date, it is assumed that
the sequence of events in glycosphingolipid biosynthesis resembles that
in glycoprotein biosynthesis, i.e. that early reactions
occur in early stations (endoplasmic reticulum and cis/medial Golgi) of the pathway, and late reactions occur in late stations (trans Golgi/trans Golgi network).
Using truncated analogues of ceramide and glucosylceramide that allow
measurement of enzyme activities in intact membrane fractions, we have
reinvestigated the localization of individual enzymes involved in
glycosphingolipid biosynthesis and for the first time studied the
localization of lactosylceramide synthase after partial separation of
Golgi membranes as previously described (Trinchera, M., and Ghidoni, R. (1989) J. Biol. Chem. 264, 15766-15769). Here, we
show that the reactions involved in higher glycosphingolipid biosynthesis, including lactosylceramide synthesis, all reside in the
lumen of the late Golgi compartments from rat liver.
Sphingomyelin (SM)1 and
glycosphingolipids (GSLs) are major constituents of the outer leaflet
of the plasma membrane in eukaryotic cells. Their involvement is
discussed in neuronal growth (1), in signal transduction (2-4), cell
growth (5), cell-cell recognition (2, 6), and the receptor-mediated
endocytosis of bacterial toxins and viruses (6, 7). Although a variety
of reports exist on the subcellular localization of individual enzymes
involved in their biosynthesis and on their biosynthetic transport to
the plasma membrane (reviewed in Ref. 8), our picture of the functional organization of the endomembrane systems involved in GSL biosynthesis is not complete.
The lipid moiety of sphingolipids, ceramide, is produced on the
endoplasmic reticulum (9, 10) and, within the lumen of the ER, part of
it in some cell types is converted to form galactosylceramide (11, 12).
For further sphingolipid synthesis, ceramide is then carried to the
Golgi apparatus in either a vesicular (13, 14) or a nonvesicular
transport step (15, 16). The Golgi apparatus comprises stacks of
various cisternae, the cis Golgi network, cis, medial, and trans
cisternae, and the trans Golgi network (TGN) (17). Ceramide is then
converted to sphingomyelin in the lumen of the early (cis/medial) Golgi
(18, 19) by transfer of the phosphorylcholine head group of
phosphatidylcholine to ceramide (20). In some cell types, a minor part
of SM seems to be generated in the outer leaflet of the plasma
membrane (21).
In glycoprotein biosynthesis, functions of the various Golgi
subcompartments are well established: early reactions in glycoprotein trimming and processing take place in the lumen of the early
(cis/medial) Golgi, and late reactions take place in the trans
Golgi/trans Golgi network (summarized in Refs. 17, 22, and 23).
Biosynthesis of complex glycosphingolipids, like that of glycoproteins,
involves sequential glycosyltransferase reactions, starting with the
formation of glucosylceramide, and it was assumed that the various
transferases are functionally organized within the Golgi in a way
similar to protein glycosyltransferases (24). This view results from
experiments including partial separation of Golgi subcompartments by
sucrose density gradient centrifugation and subsequent determination of the various enzymatic activities in the presence of detergent (25-27)
or the use of inhibitors that block various steps in vesicular transport, like the fungal macrolide brefeldin A (BFA) (28-31) and the
antibiotic monensin (32, 33). However, differences exist in the
topology of these reactions. Rather than lumenal, GlcCer is synthesized
on the cytoplasmic face of the Golgi (34-36), not only in the early
but also in the late subcompartment of this organelle (36, 37). GlcCer
is then converted to LacCer by LacCer synthase (38-40), a lumenal
Golgi enzyme (12, 39), the localization of which to Golgi
subcompartments has not been described to date. This has prompted us to
investigate the localization within the Golgi of this enzyme and to
reinvestigate the topography of various additional glycosyltransferases
involved in GSL biosynthesis using sucrose density gradient
centrifugation. To this end, the conditions for assaying enzyme
activities in partially separated Golgi subfractions (25, 26, 41) were
optimized. Using a truncated analogue of ceramide with only eight
carbon atoms in both its sphingosine and fatty acid moieties
(C8C8-ceramide), transferase activities can be
measured in the absence of detergent, because the truncated substrates
readily permeate membranes (39, 42). Assaying intact membranes has the
advantage that overall higher transferase activities are obtained and
the enzymes are characterized functionally, because in intact membranes
their activities depend on functionally intact translocators for their respective substrates, such as, e.g. CMP-NeuAc or UDP-Gal
(43, 44). Moreover, care was taken to inhibit Materials--
Conduritol B epoxide, Dextran (average molecular
weight, 250,000), Synthesis of 3H-Truncated Ceramide
Derivatives--
[3H]t-Cer and
[3H]t-GlcCer were synthesized as described (42).
[3H]t-LacCer was purified by high performance liquid
chromatography as a byproduct of the semipreparative purification of
[3H]t-Cer derivatives (42).
Isolation of Golgi Membranes--
For enzyme activity
determinations without further partial separation into subcompartments,
intact Golgi membranes from livers of fasted rats (Wistar male rats,
180-200 g) were isolated according to the method described in Ref. 45
with some modifications. All sucrose solutions were in 10 mM Tris-maleate buffer, pH 7.4. The homogenization buffer
contained 0.5 M sucrose, 5 mM EDTA, and 5 mM dithiothreitol.
Institut für Anatomie und Zellbiologie,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-glucosidase, which is
present at different levels of activity in the various subfractions examined, because degradation of the substrate added or the product formed in an enzyme assay may profoundly change the result. Here we
show that, in contrast to the functional organization of the Golgi
apparatus in glycoprotein biosynthesis, all lumenal activities investigated for the formation of more complex GSLs reside in the late
Golgi, including LacCer synthesis.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-amylase type X-A from Aspergillus
orizae and type VIII-A from barley malt, ovalbumin (chicken egg
albumin, grade V), and neuraminidase from Vibrio cholerae
were from Sigma. Triton CF-54, CMP-N-acetylneuraminic acid,
UDP-N-acetylglucosamine,
UDP-N-acetylgalactosamine, UDP-galactose, and UDP-glucose
were purchased from Boehringer Mannheim. Glucosylceramide,
lactosylceramide, and GM3 were obtained from Matreya, Inc.
[3H]CMP-N-acetylneuraminic acid,
[3H]UDP-galactose,
[3H]UDP-N-acetylglucosamine, and
[3H]UDP-N-acetylgalactosamine were from NEN
Life Science Products. Polyvinylpyrrolidone (molecular weight, 30,000)
and agar (extra pure) were purchased from Merck (Darmstadt,
Germany).
Treatment of Golgi with Pronase E--
Pronase E treatment of
membranes was as described (36, 39), with the following modifications:
100 µg membrane protein in 10 mM Tris-maleate, pH 7.4, containing 50 mM NaCl and 250 mM sucrose was
incubated with Pronase E (protein/protease = 5:1) in a total
volume of 100 µl at 37 °C for various time intervals. After
incubation, the samples were diluted 3-fold with ice-cold buffer,
loaded on 1 ml of 10 mM Tris-maleate, pH 7.4, containing 500 mM sucrose and 2 mg of bovine serum albumin/ml, and the
membranes were pelleted by centrifugation (30 min at 170,000 × g and 4 °C). The pellets were resuspended in the above
buffer, and aliquots were used for the determination of the following
enzyme-activities: for GlcCer and SM synthase,
[3H]t-Cer was used as a substrate, and for
-glucosidase, [3H]t-GlcCer was used.
Isolation and Subfractionation of Golgi Membranes for Topographical Studies-- To obtain partially purified Golgi subcompartments, a protocol was followed as described in Ref. 25, and the following markers were used to characterize the subfractions obtained: esterase and galactosyltransferase (Gal-T) as described (46) and N-acetylglucosaminyl-phosphotransferase (GlcNAc-P-T) according to the method described in Ref. 47. Of each 1-ml fraction, the following aliquots were used: 10 µl for esterase, 20 µl for Gal-T, and 20 µl for GlcNAc-P-T. In addition, various types of membranes were analyzed immunologically with antibodies directed against dipeptidylpeptidase IV (DPP IV) (rat liver plasma membranes (48, 49)), rab5 (early endosomes (50, 51)), and TGN38 (trans Golgi network (52)). Several independent gradients were run with nearly the same results. A partial separation of early and late Golgi membranes was always found, with the maxima of marker activities separated by three fractions. Fractionation of the gradients allowed a reproducibility of the maxima range of ±1 fraction without affecting their distance. Three gradients that showed highly reproducible patterns are shown under "Results"; they were used for the immunological and activity assays presented. Immunological analyses were performed twice in independent gradients and identical results were obtained (see Fig. 2).
Assays of Sphingolipid Biosynthesis with Truncated Substrates-- Activities of SM and GlcCer synthase were determined as described (36). A standard assay to follow t-GSL synthesis in Golgi membranes contained in a total volume of 10 µl, 1-5 µg of protein, 50 µM [3H]t-GlcCer (specific activity, 0.2 µCi/nmol), 1 mM conduritol B epoxide (CBE), 100 mM KOAc, 1 mM Mg(OAc)2, 1 mM MnCl2, 5 mM NaCl, and 2 mM UDP-Gal in 50 mM Hepes/KOH, pH 6.5, to measure LacCer synthase. Further addition of 2 mM CMP-NeuAc leads to the synthesis of t-GM3 and t-GD3. These conditions were found to be optimal after variations of buffer, pH, ion concentrations, and concentration of substrates. Enzyme activities were found to be linear for up to 2 h. For standard assays, samples were incubated for 40 min at 37 °C. To measure these enzyme activities across the gradients, 4 µl of each fraction was used. To determine the activities in the presence of detergent, a final concentration of 0.05% Triton CF54 was used. The reaction was stopped by the addition of an equal volume of 2-propanol, and then the samples were centrifuged for 5 min at 10,000 × g, and an aliquot (10 µl) of the supernatants was analyzed by TLC.
Identification of t-GM3 and t-GD3-- t-GM3 was isolated by scraping the band from a preparative TLC and hydrolysis with sialidase from V. cholerae (Sigma). Only t-LacCer was found as the product. When the isolated t-GM3 was used as a substrate, Golgi membranes in the presence of detergent converted this ganglioside into t-GD3 as evidenced by an additional spot on the TLC that appeared only after the addition of CMP-NeuAc.
Assay of -Glucosidase Activity--
Activity of
-glucosidase was determined using [3H]t-GlcCer (50 µM) as substrate, under conditions described above, in
the absence of CBE and detergent. A total volume of 10 µl contained 4-µl gradient fractions. The hydrolysing activity was calculated from
the sum of [3H]t-SM and [3H]t-Cer
formed.
Assays of Ganglioside Biosynthesis with Long Chain Substrates and Detergent-treated Membranes-- GM3 synthase (SAT-I) was determined with minor modifications according to the method described in Ref. 41. In a total volume of 20 µl, 200 µM long chain LacCer, 0.05% Triton CF 54, 50 mM Hepes/KOH, pH 6.5, 100 mM KOAc, 5 mM Mg(OAc)2, 10 mM MnCl2, 1 mM [3H]CMP-NeuAc (4000-6000 cpm/nmol), and 10 µl of gradient-fractions were incubated for 30 min at 37 °C. The reaction was stopped by the addition of 20 µl of chloroform/methanol (2:1), and an aliquot (10 µl) of the organic phase was analyzed by TLC. Identification of GM3 was performed by comparison with an authentic standard substance.
GalNAc-T (GA2, GM2, and GD2 synthase) was measured with minor modifications according to the method described in Ref. 53. In a total volume of 20 µl, 200 µM GM3, 0.15% Triton X-100, 40 mM Hepes/KOH, pH 6.5, 10 mM MnCl2, 5 mM Mg(OAc)2, 100 mM KOAc, 200 µM [3H]UDP-GalNAc (25,000-45,000 cpm/nmol), and 14 µl of each membrane fraction was incubated at 37 °C for 30 min. The reaction was stopped by the addition of 20 µl of chloroform/methanol (2:1), and an aliquot (10 µl) of the organic phase was analyzed by TLC. GM2 was characterized by TLC and autoradiography after using either GM3 and [3H]UDP-GalNAc as substrates or [3H]GM3 and UDP-GalNAc. In both cases, superimposed spots were obtained.TLC and Evaluation of Radioactivity-- Whatman silica gel plates (LK 6) were developed with a mixture of chloroform, methanol, and 0.22% CaCl2 in water (65:35:8) as a solvent. Identification of the various glycosphingolipids was performed either by incorporation of 3H-labeled sugars from their nucleotide-activated precursors (long chain GSLs) or by hydrolysis with the appropriate specific glycosidases (t-GSLs). For fluorography, the chromatograms were prepared according to the method described in Ref. 54. For determination of radioactivity, the chromatograms were evaluated on an automatic TLC two-dimensional analyzer (digital autoradiograph) (Berthold, Wildbad, Germany). The scanner counts the radioactivity of 3H-labeled spots on the TLC plate with a yield of 0.88% compared with liquid counting. When the spots counted for 1 h (cph) in the scanner (e.g. 2700 cph) are measured by liquid scintillation counting, they yield 113-fold of the radioactivity registered (in our example, 5085 cpm). In this system a significant signal is obtained down to 100 cph, representing 188 cpm in the scintillation system.
SDS-Polyacrylamide Gel Electrophoresis and Immunoblot Analysis-- Proteins were separated on SDS-10% polyacrylamide gels under reducing conditions (55). Western blot analysis was performed according to the method described in Ref. 56 with antibodies directed against DPP IV, rab5, and TGN38. Immunoreactions were visualized by chemiluminescence (ECL, Amersham Corp.).
Electron Microscopy-- For morphological evaluations, aliquots of fractions 15 and 18 were diluted 10-fold with 10 mM Tris/maleate, pH 7.4, and the membranes were sedimented by centrifugation (60 min at 4 °C and 100,000 × g). The pellets were fixed with 2.5% glutaraldehyde in 0.1 M sodium-cacodylate buffer, pH 7.6, containing 4% polyvinylpyrrolidone (Mr 30,000) and 0.05% calcium chloride. After washing with 0.1 M cacodylate buffer, pH 7.6, three times, the pellets were embedded in agar (extra pure) and cut into thin slices. Postfixation with osmium tetroxide and tannic acid treatment were according to the method described in Ref. 57. After washing in cacodylate buffer, the slices were dehydrated in a gradient of ethanol and embedded in Epon 812. Ultra-thin sections were stained with alkaline lead citrate and analyzed using a Zeiss EM 10 electron microscope.
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RESULTS |
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-Glucosidase Activity in Partially Separated Golgi
Subfractions--
To obtain valid activity quantifications,
GSL-degrading activities need to be excluded in the corresponding
assays. The observation of
-glucosidase activity in intact Golgi
membranes (35, 39) prompted us to characterize the topography and
topology of this activity in partially separated Golgi subcompartments
isolated by sucrose density gradient centrifugation according to the
method described in Ref. 25. Addition of [3H]t-GlcCer and
UDP-Gal to nonfractionated Golgi membranes led to the formation of
t-LacCer (Fig. 1). In addition, free
[3H]t-Cer was formed (indicative for the presence of
-glucosidase), part of which was converted into
[3H]t-SM (Fig. 1, lane 2). In the presence of
CBE hydrolysis of [3H]t-GlcCer was efficiently inhibited
(Fig. 1, lane 3) (58, 59). These conditions allowed the
determination of the
-glucosidase (without added CBE) or LacCer
synthase (in presence of CBE).
-Glucosidase activity was found by
protease latency experiments to be lumenal and did not follow typical
Golgi peak activities after subfractionation (data not shown). Part of
this activity may be present in the lumen of Golgi subcompartments en
route to the lysosomes.
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Lactosylceramide Is Synthesized in the Lumen of the Late Golgi-- The activity profile of LacCer synthase after partial separation of Golgi subcompartments is shown in Fig. 2A with a peak in fraction 18. The following marker proteins were quantitated immunologically to determine membrane contaminants within the fractions across the gradient: for liver plasmamembranes DPP IV (48, 49) (Fig. 2B), for early endosomal membranes rab5 (50, 51) (Fig. 2C), and for the trans Golgi network TGN38 (52) (Fig. 2D). As an ER marker, the activity of esterase was determined (46) (Fig. 2E). Golgi subcompartments were characterized by GlcNAc-P-T (cis Golgi (60)) and by Gal-T (trans Golgi (61)), as shown in Fig. 2E, and confirm their distribution across the gradient as described earlier (18, 25, 26, 36). In Fig. 2F, the sucrose density and protein profiles of the gradients investigated are given. The activity for t-LacCer synthesis is restricted to late Golgi subfractions, and the peak activity coincides with the marker activity for trans Golgi, protein-Gal-T (compare Fig. 2, A and E). Clearly, contaminating plasma membranes, as well as early endosomes, peak differently in the gradient (see Fig. 2, B and C) excluding these membranes as residences for LacCer synthase.
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GM3, GD3, and GM2 Synthase Activities Reside in the Lumen of the Late Golgi-- Thus, surprisingly, LacCer synthase, the first lumenal activity in the synthesis of gangliosides, is a late Golgi enzyme. In light of reports that SAT-I (catalyzing the biosynthetic step after LacCer) resides in the early Golgi (25-27), an exclusive late Golgi activity for LacCer synthesis would imply a retrograde transport step for LacCer. This has prompted us to reinvestigate the localization of the enzyme activities for steps in ganglioside biosynthesis subsequent to LacCer.
To this end, we analyzed the activity profiles for GM3 and GD3 synthesis in the partially separated Golgi subfractions. This was performed with [3H]t-GlcCer as a substrate in the presence of both UDP-Gal and CMP-NeuAc. As a result, t-GM3 formation was observed in the late Golgi with a peak activity coincident with the peak of t-LacCer synthesis (not shown; see Fig. 2A). Under these conditions, no intermediate t-LacCer was observed, indicating that t-LacCer formation represents the rate-limiting step for t-GM3 synthesis. This result shows GM3 synthase activity in the late Golgi but does not exclude the presence of additional GM3 biosynthesis steps in earlier subcompartments of the organelle, because t-GM3 formation under the conditions used for the assay strictly depends on LacCer synthase activity, which in turn is restricted to the late Golgi. Therefore, GM3 synthase activity was followed using an assay system with long chain LacCer and [3H]CMP-NeuAc as substrates, in the presence of detergent (41). Under these conditions, part of the resulting GM3 was further sialylated to yield GD3 (not shown), and therefore the formation of both GM3 and GD3 reflects the overall activity of GM3 synthase. Those activities show a profile (Fig. 4A) nearly identical with that of LacCer synthase (depicted in Fig. 2A), irrespective of the experimental conditions. Thus, both LacCer and GM3 synthases peak in the late Golgi. Likewise, the activity profile for GM2 synthase in the presence of detergent shows localization of this enzyme to the late subcompartments of the organelle (Fig. 4B). In addition, all of these activity profiles peak with the established marker activity of the trans Golgi, protein-Gal-T (61) (Fig. 2E).
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DISCUSSION |
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Characterization of Early and Late Golgi-- We have characterized the partial separation of Golgi subcompartments obtained by sucrose density gradient centrifugation according to the method described in Ref. 25 by use of the following markers: the activity profile of GlcNAc-P-T (60, 62, 63) peaks in fraction 15 representing the early Golgi, and the late Golgi is characterized by a peak in fraction 18 of protein Gal-T, a marker for the trans Golgi (61, 64-66). Additional markers for membranes known to contaminate the Golgi fractions show that neither ER nor early endosomal or plasma membranes peak with early or late Golgi markers. TGN38, a marker for the TGN (52, 67) shows its strongest signal coincident with the peak of Gal-T.
Localization of Lumenal Enzymes Involved in GSL Biosynthesis-- The activity profiles for synthesis of t-LacCer and GM3 colocalize both with each other and with the late Golgi markers Gal-T and TGN38. The activity profile for GM2 synthesis peaks with the same marker enzymes. GM2 synthase was found also to catalyze the formation of GA2 and GD2 (53). These results were confirmed by Lutz et al. (68), who characterized the cDNA for GalNAc-T and showed that these activities are catalyzed by the same enzyme. Thus, the activities for formation of GA2, GD2, and GM2 are likely to colocalize with the late Golgi markers.
LacCer synthase has been shown to represent the first activity in the lumen of the Golgi during step by step formation of more complex GSLs (12, 39), but it had not previously been localized to an individual Golgi subsite. The localization of LacCer synthase and SAT-I to the late Golgi came as a surprise, because SAT-I, the enzyme needed to convert LacCer into GM3, had been described as an early Golgi resident in rat liver (25, 26). In addition, in primary cultured cerebellar neurons of 6-day-old mice, SAT-I has also been attributed to an early Golgi site (27). In the same system, however, SM synthase, an early Golgi enzyme (18, 19), had been localized to the late Golgi (29). These alterations may be due to a maturation process within the organelles of the secretory pathway in the developing brain, similar to that described in chick embryo retina cells (69). Two factors may account for the discrepancy between earlier results (25, 26) and those described here: (i) in the earlier reports, detergent concentrations of 0.2% Triton CF54 were used for determination of SAT-I, too high to detect significant enzyme activity, as shown in a subsequent publication (41). These authors describe 0.05% of the detergent (Triton CF54) as optimal for SAT-I activity and show complete inactivation already at 0.15%. In addition, another sialyltransferase, SAT-IV, localized to the late Golgi (25, 26), was reported to be less sensitive to detergent and to have a broad substrate specificity, including conversion of LacCer to GM3 (41). This raises the question why SAT-I activity (catalyzed by SAT-IV) was not found in both the early and the late Golgi in earlier studies (25, 26). (ii) The use of detergent may allow access of substrates to SAT-I present in the early Golgi in transit to the late Golgi. However, the latter possibility is unlikely, because in this study, early Golgi SAT-I activity was not detectable in assays both with and without detergent. This issue cannot be resolved at present. A variety of reports deal with the intra-Golgi localization of individual enzymes involved in GSL biosynthesis, analyzed by use of inhibitors of intracellular vesicular transport, like the antibiotics monensin (32, 33) and BFA (28-31), and by investigation of mitotic cells (16). These reports describe blocks of GSL biosynthesis in steps after GM3 and/or GD3 formation and localize to the early Golgi the enzymes acting before this block. In chick embryo retina cells, increased amounts of GM3, GD3, and GT3 were found in the presence of BFA (31). In the presence of monensin, an inhibition of GT3 formation was observed, whereas the amounts of GM3 and GD3 remained unchanged (33). The authors concluded that synthesis of GM3 and GD3 takes place in the cis/medial Golgi and that synthesis of GT3 takes place in the trans Golgi, and they postulated the existence of individual enzymes for these sialylation reactions. However, Nakayama et al. (70) found that an expression-cloned cDNA coding for GT3 synthase was identical with the cDNA for GD3 synthase and concluded that both products are formed by the same enzyme. Thus, the different results obtained with monensin and BFA may need to be explained by some direct or indirect effect on GD3/GT3 synthase of monensin, such as, for example, a shift of pH, as discussed in Ref. 33.A Revised Model for the Functional Organization of the Golgi Apparatus in Sphingolipid Biosynthesis-- In the above mentioned studies, localization of the biosynthetic activities for LacCer, GM3, GD3, and GT3 was assigned dependent on SAT-I as a reference enzyme, localized to the cis Golgi earlier (25, 26). With our finding that all lumenal activities in GSL biosynthesis reside in the late Golgi, the above results would easily and consistently fit into a model as depicted in Scheme I: GlcCer is synthesized at the cytosolic sides of both the early and the late Golgi. If it is translocated into the lumen of the early Golgi, GlcCer may be carried to the late Golgi by vesicular transport, where it can serve as a substrate for LacCer synthesis. GlcCer expressed at the cell surface possibly stems from the cytosolic side of the Golgi and represents molecules that have not been translocated into the lumen but transported monomolecularly to the inner leaflet of the plasma membrane. A very recent report (71) indicates that ATP binding cassette proteins in the plasma membrane are able to translocate various GlcCer-species to the outer leaflet, thus expressing these monohexosyl lipids at the cell surface.
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ACKNOWLEDGEMENTS |
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We are indebted to Drs. Gerhilde van Echten-Deckert and Konrad Sandhoff (Bonn, Germany), Gerrit van Meer (Utrecht, Netherlands), and Bernd Helms (Heidelberg, Germany) for critical reading of the manuscript and helpful discussions. We thank the following colleagues for their generous gifts of antibodies: Drs. Werner Reutter (Berlin, Germany) for DPP IV, George Banting (Bristol, United Kingdom) for TGN38, and Marino Zerial (Heidelberg, Germany) for rab5. We thank also Dr. Jürgen Kopitz (Heidelberg, Germany) for his generous gift of tritiated endogenous GM3.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 352).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 49-6221-54-41 37; Fax: 49-6221-54-4366; E-mail: CI1{at}ix.urz.uni-heidelberg.de.
1 The abbreviations used are: SM, sphingomyelin; BFA, brefeldin A; CBE, conduritol B epoxide; CMP-NeuAc, CMP-N-acetylneuraminic acid; DPP IV, dipeptidylpeptidase IV; Gal-T, protein galactosyltransferase; GalNAc-T, N-actetyl-galactosaminyltransferase (GM2 synthase); GlcCer, glucosylceramide; GlcNAc-P-T, N-acetylglucosaminyl-phospho-transferase; GSL, glycosphingolipid; LacCer, lactosylceramide; SAT-I, sialyltransferase I (GM3 synthase); t, truncated; t-Cer, C8,C8-ceramide; TGN, trans Golgi network; cph, counts per hour.
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REFERENCES |
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