Regulated Apical Secretion of Zymogens in Rat Pancreas

INVOLVEMENT OF THE GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED GLYCOPROTEIN GP-2, THE LECTIN ZG16p, AND CHOLESTEROL-GLYCOSPHINGOLIPID-ENRICHED MICRODOMAINS*

Katja SchmidtDagger, Michael SchraderDagger§, Horst-Franz Kern, and Ralf Kleene

From the Department of Cell Biology and Cell Pathology, Philipps University of Marbury, Robert-Koch Strasse 5, 35033 Marburg, Germany

Received for publication, July 13, 2000, and in revised form, December 22, 2000




    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.




    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In endocrine and exocrine cells secretory proteins are stored at high concentrations in secretory granules and are released in response to external stimuli. During the course of granule formation proteins destined for storage and regulated secretion are segregated from those constitutively secreted (1-3). Granule formation and apical sorting in pancreatic acinar cells involves the selective aggregation of a mixture of regulated secretory proteins and the association of these aggregates with specific membrane domains of the trans-Golgi network (TGN),1 which then pinch off as condensing vacuoles. While the selective aggregation of the pancreatic secretory proteins has been well documented (4-6), how these aggregates become bound to the luminal side of the TGN membrane and are ultimately sorted into condensing vacuoles is poorly understood. The zymogen granule (ZG) glycoprotein GP-2, which is linked to the luminal surface via a glycosylphosphatidylinositol (GPI) anchor and represents up to 30% of the total membrane proteins within the ZG membrane, was the first candidate postulated to mediate this sorting event (7). However, two studies have demonstrated that during embryonic development and in partially differentiated acinar carcinoma cell lines, granule formation can occur in the complete absence of GP-2 (8-10). This argues against a role as specific sortase of GP-2, but does not exclude its involvement in granule formation and in apical sorting (7), at least in the adult exocrine pancreas. Scheele et al. (7) have put forward the hypothesis that GP-2, together with attached proteoglycans, forms a submembranous matrix at the luminal side of ZGM. We have recently identified and characterized proteoglycan and glycoprotein components of this matrix (11) and have evidence that it functions in the binding of aggregated enzyme proteins to membranes, a process that is considered as condensation-sorting (12).

The acinar cells of the pancreas are highly polarized cells with regulated secretion directed to the apical domain surrounding the acinus lumen. From studies of polarized protein trafficking performed mainly with MDCK cells, it is known that protein sorting to the apical domain of the plasma membrane is controlled by N- and/or O-glycosylation of protein ectodomains (13, 14), by apical sorting determinants present in the cytoplasmic tail of seven transmembrane proteins (15, 16), or by the incorporation of apically sorted proteins into lipid microdomains, called rafts, in the Golgi complex (17, 18). Inclusion into rafts and subsequent apical sorting has been shown for GPI-anchored proteins at the plasma membrane (17) and for some transmembrane proteins (19, 20). Rafts are membrane microdomains enriched in glycosphingolipids, sphingomyelin, and cholesterol that are thought to be assembled within the exoplasmic leaflet of the Golgi membrane and that have been proposed to act as a sorting platform for the apical delivery of plasma membrane proteins (18, 21). Rafts are defined biochemically by their insolubility in non-ionic detergents such as Triton X-100 at 4 °C (22). The detergent-insoluble glycosphingolipid complexes (DIGs) float to lighter fractions on sucrose density gradients and have been identified in TGN-derived apical transport vesicles (22). Several apical cargo proteins, e.g. the influenza virus proteins hemagglutinin and neuraminidase (19, 20), or GPI-anchored proteins (17) are found in DIGs during their transport to the apical domain, while basolaterally delivered cargo proteins are excluded from DIGs (23). Raft association of proteins is mediated either directly by the GPI moiety or by the transmembrane domains of proteins, e.g. the lectin VIP36, or indirectly by binding to other raft-associated proteins.

It is not clear which of the above mechanisms exist in polarized acinar cells of the pancreas to sort the 15-20 different zymogens to the apical surface and what role the GPI-anchored GP-2 and lipid rafts play in this phenomenon. As lipid microdomains have been described in secretory granules of neuroendocrine cells (24, 25), we investigated whether they were also present in the membranes of zymogen granules of the exocrine pancreas. We obtained evidence that GP-2, the secretory lectin ZG16p, and sulfated proteoglycans were associated with cholesterol-glycosphingolipid-enriched microdomains isolated from membranes of zymogen granules. Furthermore, GP-2 and lipid microdomains were required for proper granule formation and regulated secretion of zymogens.


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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Antibodies-- Polyclonal antibodies to rat GP-2 and pig amylase were used as reported previously (9). Polyclonal antibody ZG16 to recombinant rat ZG16p was described in Cronshagen et al. (26). Polyclonal antibodies to carboxyl ester lipase were raised in chicken. A polyclonal antibody to carboxypeptidase alpha  was obtained from Rockland Immunochemicals (Gilbertsville, PA). Species-specific anti-IgG antibodies conjugated to horseradish peroxidase were obtained from Bio-Rad.

Subcellular Fractionation and Isolation of Zymogen Granules-- ZG were isolated as described previously (6). Briefly, the pancreas was removed from fasted male Wistar rats (200-230 g; Charles River, Sulzfeld, Germany) and homogenized on ice in the following buffer: 0.25 M sucrose, 5 mM MES at pH 6.25, 0.1 mM MgSO4, 1 mM dithiothreitol, 10 µM FOY-305 (Sanol Schwarz, Mannheim, Germany), 2.5 mM Trasylol (Bayer, Leverkusen, Germany), 0.1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 500 × g for 10 min at 4 °C, and the resulting postnuclear supernatant was further centrifuged at 2000 × g for 10 min at 4 °C to pellet the ZG. After removal of the brownish layer of mitochondria on top of the pellet, the ZG were lysed in 50 mM Hepes, pH 8.0, and stored at -20 °C. After thawing, the soluble zymogen granule content proteins were separated from the ZGM by ultracentrifugation (100,000 × g for 30 min, 4 °C). ZGM were resuspended in 50 mM Hepes, pH 8.0, and stored at -20 °C. For bicarbonate treatment of ZGM, 500 µl of 300 mM NaHCO3, pH 11.5, were added to 500 µl of ZGM (corresponding to 200 µg of protein), and the mixture was incubated on ice for 2 h. For PI-PLC treatment of ZGM, 500 milliunits of PI-PLC (Roche Molecular Biochemicals, Mannheim, Germany) were added to ZGM (corresponding to 800 µg of protein), and the samples were incubated at 37 °C for 1 h in a final volume of 500 µl. Treated membranes were reisolated by high-speed centrifugation at 100,000 × g for 30 min. For the preparation of Golgi- and rER-enriched fractions, the postnuclear supernatant was adjusted to 0.5 M sucrose, layered onto a step gradient of 1.4 and 0.9 M sucrose, and was overlaid with 0.25 M sucrose solution containing 10 mM Hepes, pH 6.8, 5 mM MgCl2, and 3 mM CaCl2. After centrifugation for 1 h at 110,000 × g, a Golgi-enriched faction was obtained at the 0.5/0.9 M interphase together with a rER-enriched fraction at the 0.9 M/1.4 M interphase. The collected fractions were adjusted to 0.2 M sucrose with 50 mM Hepes, pH 8.0, and stored at -20 °C. After thawing and centrifugation at 100,000 × g for 30 min at 4 °C, the supernatants containing the soluble Golgi or rER content proteins and the pellets containing Golgi (GM) or rER membranes (rERM) were stored at -20 °C in aliquots for further use.

Analysis of Lipid Composition-- Isolated membranes corresponding to 400 µg of protein were resuspended in 100 µl of 50 mM Hepes, pH 8.0, and mixed with 200 µl of methanol/chloroform (1:1). After Bligh-Dyer two-phase extraction (27) lipids were separated by thin layer chromatography (TLC) using a mixture of chloroform:methanol:H2O (65:25:4). To extract polar lipids (28) membranes corresponding to 2 mg of protein were resuspended in 20 µl of 50 mM Hepes, pH 8.0, and incubated with 500 µl of methanol and 1 ml of n-hexane. After centrifugation at 3000 × g for 5 min, 1 ml of chloroform was added to the methanol phase, and centrifugation was repeated. The resulting supernatant was submitted to TLC using a mixture of chloroform:methanol:0.25% KCl (50:54:10). Lipids were visualized by 20% (v/v) H2SO4 at 150 °C and quantitated. Cholesterol, glucosyl ceramide, lactosyl ceramide, sulfatides, phosphatidylcholine, and sphingomyelin were used as standards.

Triton X-100 Extraction and Sucrose Gradients-- 500 µg of membranes were incubated on ice for 30 min in 50 mM Hepes, pH 8.0, containing 1% (v/v) Triton X-100 and were centrifuged at 120,000 × g for 30 min. The Triton X-100-insoluble pellet fraction was resuspended in 50 mM Hepes, pH 8.0, and adjusted to 1.2 M sucrose. 500 µl of the pellet fraction were overlayed with 1 ml of 1.1 M and 0.5 ml of 0.15 M sucrose. After centrifugation for 1 h at 120,000 × g in a swing out rotor (TLA 120.1, Beckman Instruments, Munich, Germany) fractions of 200 µl were collected from the top of the gradient. For flotation experiments with ZGM and GM, membranes were bicarbonate-treated prior to Triton X-100 extraction to partially remove membrane-associated matrix components which were found to inhibit flotation. Proteins were precipitated by methanol (29) and analyzed by gel electrophoresis. Lipids were extracted by Bligh-Dyer two-phase extraction (27) and analyzed by TLC.

Preparation of Pancreatic Lobules and Drug Treatment-- Using an orogastric tube rats were fed with a single dose of 50 mg/kg of FOY-305 (Sanol-Schwarz, Mohnheim, Germany) dissolved in 1 ml of tap water and were sacrificed 6-9 h after feeding. FOY treatment for up to 6 h results in partial degranulation (40-60%) of the pancreas (30). Pancreatic lobules were prepared as described previously (31). The lobules were incubated at 37 °C under agitation and were supplied with 100% O2 every 15 min. For cholesterol depletion the lobules were incubated for 2.5 h in 5 ml MEM (Sigma, Munich, Germany) containing 25 mM mevalonate acid-lactone (Sigma) and 0.4 mM lovastatin (Merck Sharp & Dohme, Haar, Germany) and thereafter treated with 10 mM methyl-beta -cyclodextrin (CD) for 30 min (Sigma). For inhibition of GPI-anchor biosynthesis lobules were incubated for 3 h either in 5 ml of glucose-deficient MEM (Life Technologies, Inc.) containing 50 mM D-mannosamine (Sigma) or were first incubated in 5 ml of MEM for 2.5 h and afterwards in MEM containing 5 µg/ml YW 3548 (Novartis, Basel, Switzerland) for an additional 30 min. Ceramide biosynthesis was inhibited by treatment with 28 µM fumonisin B1 for 3 h. Treated lobules and controls were either used for electron microscopy or for pulse-chase experiments.

Pulse-Chase Experiments and Stimulation of Exocytosis-- Drug-treated lobules and controls were washed twice with Met/Cys-deficient medium (Life Technologies, Inc.) and were pulse-labeled for 10 min at 37 °C in 5 ml of pulse medium containing 200 µCi/ml Tran35S-label (ICN, Eschwege, Germany) and the appropriate drugs (mevalonate/lovastatin, fumonisin B1, mannosamine, or YW 3548). After washing in MEM the lobules were incubated for different chase times in 5 ml of chase medium (MEM containing 10 × Met/Cys and the appropriate drugs). Exocytosis of zymogen granules was hormonally induced by the addition of 1 ng/ml caerulein, 25 µM carbamoylcholine chloride, 130 nM phorbol 12-myristate 13-acetate (Sigma), and 1 mM cyclic 8-bromo-adenosine-3':5'-monophosphate (Roche Molecular Biochemicals) to the chase medium. For determination of secreted amylase the proteins of 200-500 µl of chase medium were precipitated by trichloroacetic acid, resuspended in the same volume of Laemmli buffer, and separated by SDS-PAGE. Quantitation of radiolabeled amylase was performed on the gels using a Bio-imager FUJIX BAS 1500 and TINATM software, version 2.0 (Raytest, Straubenhardt, Germany). Total amylase was quantitated after Coomassie staining or immunoblotting of the corresponding gels. Alternatively, amylase activity in the medium was assayed according to Ref. 32. 35S-Amylase secretion after different experimental conditions was determined in relation to total normalized amylase secretion. Similar results were obtained when total amylase was either determined by quantitation of gels or by enzyme activity. In Fig. 5 data obtained after quantitation of gels are shown. Lactate dehydrogenase activity was assayed using commercially available test kits (Roche Molecular Biochemicals) and expressed as percent of total lactate dehydrogenase activity in the medium and cell homogenate. Trypsin activity was measured according to Ref. 33. For determination of protein-bound radioactivity, samples were applied to 3MM filters (Whatman, Maidstone, United Kingdom). The filters were swirled in 10% trichloroacetic acid on ice for 30 min and washed with ice-cold 5% trichloroacetic acid and ethanol. After drying of the filters the radioactivity was determined by liquid scintillation counting (Raytest, Straubenhardt, Germany). Protein synthesis and incorporation of radioactivity was reduced after cholesterol depletion (70% of control levels) and after treatment with mannosamine (60% of control levels). However, the ratios of the total protein-bound radioactivity present in the ZG, rER, and Golgi subfractions to total radioactivity incorporated in the postnuclear supernatant were similar after all treatments, indicating that protein delivery to the secretory pathway was not disturbed. Therefore, the protein-bound radioactivity of the individual subfractions was expressed as percent of total protein-bound radioactivity of all subfractions (Sigma  cpm ZG, rER, Golgi) (Table I).

Gel Electrophoresis and Immunoblotting-- Protein samples were separated by SDS-PAGE (12.5-15% acrylamide gels) according to Laemmli (34), transferred to nitrocellulose (Schleicher and Schüll, Dassel, Germany) using a semidry apparatus (Bio-Rad), and analyzed by immunoblotting. Immunoblots were processed using either enhanced chemiluminescence reagents (Amersham Pharmacia Biotech) or diaminobenzidine reagent (0.05% diaminobenzidine, 0.04% CoCl3, 0.05% H2O2 in 10 mM Tris-HCl, pH 7.5). Silver staining of gels was performed according to Hempelmann (35). For quantitation immunoblots were scanned and processed using PC bas software.

Detection of Sulfated Glycosaminoglycans-- Sulfated glycosaminoglycans (GAGs) were detected using the Blyscan assay according to the manufacturer's protocol (Biocolor, Belfast, Ireland). Briefly, a precipitable complex was formed by binding of the Blyscan dye to free GAGs or those bound to proteoglycans. After dissociation of the precipitated complex the samples were photometrically quantified, or in the case of radiolabeled material, samples were analyzed by liquid scintillation counting.

Electron Microscopy-- Pancreatic lobules were fixed in 0.1% cacodylate buffer, pH 7.3, containing 1% glutaraldehyde (Serva, Heidelberg, Germany), postfixed in 1% osmium tetroxide, and embedded in Epon according to standard procedures. Thin sections (70 nm) were stained with uranyl acetate/lead citrate and examined using a Zeiss EM 9S electron microscope. For immunolabeling thin sections of Epon-embedded samples were etched with 10% H2O2 for 5 min, blocked with 1% bovine serum albumin in phosphate-buffered saline, and afterward incubated with an antibody to carboxypeptidase alpha  and visualized using 10 nm protein A-gold. This antibody showed specific labeling of ZG and Golgi cisternae, but was not found to label other intracellular organelles. Quantitative analysis of the intactness of junctional complexes at the apical pole, the integrity of the apical and intracellular membranes (ER, mitochondria, granules), and of morphological alterations of the Golgi complex after different experimental treatments was performed on photographs at ×3000-12,000 (Table II). Sections were systematically scanned at the electron microscope, and each acinus encountered was photographed. A cell (acinus) was defined as intact (polarized) if the apico-lateral junctions and the plasma membrane showed no sign of disruption and if the ZG were still concentrated apically. Furthermore, the integrity of other internal organelles (see above) was comprised.

Statistical Analysis of Data-- Significant differences between experimental groups were detected by analysis of variance for unpaired variables using SigmaStat for Windows (Statistical Products and Service Solutions Inc., Chicago, IL). Data are presented as mean ± S.D., with an unpaired t test used to determine statistical differences. p values <0.05 are considered as significant, and p values <0.01 are considered as highly significant.


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GP-2, the Lectin ZG16p, and Sulfated Proteoglycans from Membranes of Zymogen Granules Are Associated with Triton X-100-insoluble Glycolipid- and Cholesterol-enriched Complexes

Glycosphingolipid-cholesterol rafts form Triton X-100-insoluble glycolipid-rich complexes (DIGs) at 4 °C, which float to lighter fractions on sucrose density gradients (17). To identify DIGs and possible DIG-associated proteins in membranes of isolated ZG, we extracted bicarbonate-treated ZGM with Triton X-100 at 4 °C and analyzed the detergent-insoluble fraction by density gradient centrifugation (Fig. 1, A-D). Lipid analysis of the gradient fractions revealed cholesterol and sphingomyelin to be present mainly in the low density fractions 2 and 3 corresponding to the 0.15/1.1 M interface (Fig. 1A). After SDS-PAGE and silver staining three major protein bands with molecular masses of about 16, 45-50, and 75 kDa were found to float to these lighter fractions (not shown). Immunoblot analysis using a variety of antibodies to different granule proteins (amylase, carboxyl ester lipase, carboxypeptidase, ZG16p, GP-2) revealed the 75-kDa band to be the GPI-anchored glycoprotein GP-2, the major membrane protein of zymogen granules (Fig. 1B). The 16-kDa band was recognized by an antibody to the secretory lectin ZG16p (12, 26) (Fig. 1B), whereas the 45-50-kDa band could not yet be identified. Both ZG16p and GP-2 were mainly present in the lighter fractions 2 and 3 and in the pellet fraction 9 (Fig. 1, B and C). GP-2 showed some variation in distribution and was sometimes found in the heavier fractions, which might be due to changes in the lipid environment (see below). Furthermore, 25-30% of sulfated proteoglycans, which form a submembranous granule matrix (11), were recovered in the low density fractions (Fig. 1D). In addition, proteoglycans were found in fraction 7 representing the 1.1/1.2 M interface (Fig. 1D). When Triton X-100 extraction was performed at 37 °C, which leads to raft disruption, the amount of GP-2, ZG16p, and proteoglycans was increased in the detergent-soluble fraction (not shown). These results indicate that DIGs enriched in cholesterol and sphingomyelin are present in ZGM and that GP-2, the lectin ZG16p, and sulfated proteoglycans are presumably associated with lipid rafts.



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Fig. 1.   Characterization of cholesterol-sphingolipid microdomains from zymogen granules. Equal amounts (corresponding to 1 mg of protein) of bicarbonate-treated ZGM (A-D) or GM (E-H) were incubated with 1% Triton X-100 at 4 °C for 30 min. The detergent-insoluble pellet fraction obtained after high speed centrifugation was adjusted to 1.2 M sucrose, overlayed with 1.1/0.15 M sucrose, and the density gradient was centrifuged at 120,000 × g for 1 h. Fractions were collected from the top of the gradient (fraction 1) and analyzed by TLC (A, E) or by SDS-PAGE and immunoblotting using anti-GP-2 and anti-ZG16p antibodies (B, F). A quantitation of the immunoblots is shown in C and G (, ZG16p; black-square, GP-2). The amount of matrix proteoglycans in the fractions was determined with the Blyscan assay, which specifically precipitates sulfated GAGs on proteoglycans. After dissociation of the precipitated complex the samples were photometrically quantified (D, H). Due to variations in the gradient and fraction volumes DIGs were either concentrated in fraction 2 or 3. The data in D and H are from four independent experiments. Chol, cholesterol; SM, sphingomyelin; X, unknown lipid; PG, proteoglycans; *, unknown protein band recognized mainly in the heavier fractions by the antibody to GP-2; , dimer of ZG16p.

To analyze the formation of lipid rafts in the Golgi complex, a Triton X-100-insoluble fraction was prepared from bicarbonate-treated GM and subjected to sucrose gradient centrifugation (Fig. 1, E-H). Cholesterol and sphingomyelin were recovered in the low density fractions 2 and 3 (Fig. 1E). Most of GP-2, and particularly of ZG16p, remained in the pellet fraction (Fig. 1, F and G), and only low amounts of ZG16p and sulfated proteoglycans floated to lighter fractions (Fig. 1, F-H). Sulfated proteoglycans were mainly present at the 1.1/1.2 M interface (fraction 7) (Fig. 1H), which in addition contained significant amounts of GP-2 and ZG16p (Fig. 1, F and G). These findings imply that the incorporation of GP-2, ZG16p, and proteoglycans into rafts is not complete in GM or that the lipid environments are different.

It has been reported (17) that the GPI moiety of proteins associates with lipid rafts and may function as apical sorting determinant. Therefore, we characterized the association of GP-2 with ZGM and GM in more detail. To determine the amount of raft-associated GP-2, bicarbonate-treated ZGM and GM were Triton X-100-extracted either at 4 °C or at 37 °C. Raft association was defined as detergent insolubility at 4 °C, but detergent solubility at 37 °C (22). To determine the amount of PI-PLC-sensitive GP-2, ZGM and GM were treated with PI-PLC, which has been shown to cleave its GPI-anchor (36). Alternatively, bicarbonate-treated ZGM and GM were first incubated with PI-PLC to remove the PI-PLC-sensitive GP-2 and afterwards extracted with Triton X-100 at 4 or 37 °C to determine the amount of raft-associated, PI-PLC-insensitive GP-2. Quantitation of immunoblots performed with the corresponding insoluble pellet and soluble supernatant fractions after incubation with GP-2 antibodies revealed the existence of four distinct pools of membrane-associated GP-2. In GM 50 ± 3.2% of GP-2 were found to be raft-associated with 15 ± 2.8% insensitive (pool 1) and 35 ± 3.3% sensitive to PI-PLC (pool 2). 34 ± 4.4% of GP-2 were PI-PLC-sensitive, but not raft-associated (pool 3). The remaining 16 ± 2% of GP-2 (pool 4) could not be removed from the membrane neither by PI-PLC nor Triton X-100 treatment (4 and 37 °C). Interestingly, in ZGM only 10 ± 3.6% of GP-2 were raft-associated according to our definition (with 5 ± 0.8% PI-PLC-insensitive (pool 1) and 5 ± 1.1% PI-PLC-sensitive (pool 2)). 21 ± 3.4% were PI-PLC-sensitive, but not raft-associated (pool 3), but 69 ± 3% of GP-2 belonged to the completely detergent-insoluble pool 4, which was not accessible to PI-PLC. These data further indicate that in ZGM and GM the lipid environments of GP-2 are different. We assume that the different pools may represent consecutive steps in lipid raft assembly.

Cholesterol Depletion and Glycosphingolipid Synthesis Inhibition Interferes with Granule Formation and Regulated Secretion of Zymogens

Effects on Granule Formation-- Cholesterol has been postulated to be an essential structural component of lipid rafts (37). Glycosphingolipids are also thought to contribute to raft structure (18). To demonstrate directly the involvement of sphingolipid-cholesterol microdomains in granule formation and secretion of zymogens, we isolated pancreatic lobules and examined these processes upon lipid raft disruption. Disruption was achieved either by reducing the intracellular level of cholesterol or by inhibition of glycosphingolipid biosynthesis. To reduce cholesterol, pancreatic lobules were first treated with mevalonate/lovastatin to inhibit de novo synthesis and were afterward treated with CD, which specifically extracts cholesterol (38-40). Lipid analysis by quantitative TLC revealed a 50% reduction of cholesterol in ZGM (46 ± 3% of controls) and a 30-40% reduction in GM (67 ± 2% of controls) and rER membrane fractions (63 ± 3% of controls) compared with the appropriate controls. Glycosphingolipid biosynthesis was inhibited by fumonisin B1 (41). In membrane fractions isolated from fumonisin B1-treated lobules and analyzed by quantitative TLC, the amount of glycosphingolipids dropped under a detectable level. To study the de novo formation of zymogen granules, pancreatic lobules were radiolabeled upon partial degranulation of the pancreas, which was achieved by feeding rats with a single dose of FOY-305, leading to a hormonally stimulated release of zymogen granules (30). After degranulation, cholesterol depletion, or fumonisin B1 treatment and pulse labeling, fractions enriched in Golgi, rER, or ZG were isolated, and the amount of protein-bound radioactivity was determined. A lower percentage of newly synthesized proteins was directed to ZG after both treatments in comparison with untreated control lobules (Table I). An accumulation of radiolabeled proteins could be detected in the Golgi fraction, but not in the rER fraction (Table I). Electron microscopy of control and treated lobules (Fig. 2) revealed morphological alterations of the Golgi complex, but not of the rER or other intracellular organelles (Fig. 2, B and C), which is consistent with the biochemical data (Table I). After both treatments, ~70% of the Golgi complexes had dilated cisternae (Table II) containing electron dense aggregations of zymogens (Fig. 2, C and inset). Quantitative analysis of the ultrastructural data indicated that based on morphological criteria the majority of the cells was undamaged and still polarized after both treatments (Table II). In 80-90% of the treated cells the plasma membrane and the junctional complexes appeared intact, and the ZG were still concentrated at the apical domain (Table II, Fig. 2). Furthermore, only about 3% of the total lactate dehydrogenase activity were found in the media of both controls (3.7 ± 1.6%) and cholesterol-depleted lobules (2.6 ± 1.2%) after incubation. An increase in lactate dehydrogenase activity up to 6% was measured after pulse labeling and additional 60 min of chase with or without stimulation of exocytosis (control: 4.9 ± 1%; cholesterol depletion: 6.6 ± 2%). These findings further indicate that depletion of cholesterol left most of the cells intact (see also Table II).


                              
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Table I
Effects of inhibition of GPI-anchor biosynthesis and cholesterol depletion on de novo granule formation
Pancreatic lobules were prepared from rats fed with a single dose of FOY-305 leading to hormonally stimulated release of zymogen granules. Degranulated lobules were incubated in the absence (control lobules) or in the presence of mannosamine or the terpenoid lactone YW 3548, two potent inhibitors of GPI-anchor biosynthesis. To disrupt lipid rafts, degranulated lobules were incubated for 2.5 h in the presence of mevalonate/lovastatin followed by incubation with CD for additional 30 min to deplete intracellular cholesterol levels or were treated with fumonisin B1, an inhibitor of glycosphingolipid synthesis. After pulse labeling with Tran35S label, Golgi- and rER-enriched fractions as well as ZG were isolated after 30-min chase. The protein-bound radioactivity of the subfractions was determined and expressed as percent of total (Sigma  cpm ZG, rER, Golgi). The data are from three to four independent experiments and are expressed as mean ± S.D.



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Fig. 2.   Fine structure of pancreatic lobules upon cholesterol depletion or treatment with fumonisin B1. Degranulated pancreatic lobules were incubated for 3 h in the presence of mevalonate/lovastatin/CD (C) to deplete intracellular cholesterol levels, or treated with fumonisin B1 (B), an inhibitor of glycosphingolipid biosynthesis. Lobules were fixed, embedded in Epon, and processed for electron microscopy as described under "Experimental Procedures." A, untreated control lobules. The inset in A shows an acinar lumen formed by the surrounding centro-acinar cells. Note the presence of junctional complexes (arrows) and the apical localization of zymogen granules. After cholesterol depletion or fumonisin B1 treatment individual Golgi cisternae had a dilated appearance, containing electron dense aggregations of zymogens (B, C). Some of the aggregates were labeled with an antibody to carboxypeptidase on Epon-embedded sections (C, inset). ZG, zymogen granules; G, Golgi apparatus. Bars: 1 µm.


                              
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Table II
Quantitative analysis of ultrastructural data
Pancreatic lobules were prepared and incubated with the different inhibitors as described in Table I. Lobules were processed for electron microscopy afterwards, embedded in Epon, and sectioned. The intactness of the junctional complexes at the apical pole, the integrity of the apical and intracellular membranes (ER, mitochondria, granules), and morphological alterations of the Golgi cisternae of photographed cells were quantitated as described under "Experimental Procedures" and expressed as percent of total (mean ± S.D.). Cholesterol depletion or fumonisin B1 treatment caused dilation of individual Golgi cisternae, whereas mannosamine or YW 3548 treatment resulted in the accumulation of vesicles (see Figs. 2 and 6). For the control all unusual alterations of the Golgi complex were counted. The data for each experimental condition are from three to four individual experiments. Chol. dep. = cholesterol depletion; n = number of cells or Golgi complexes.

We recently demonstrated that proteoglycans are components of a submembranous matrix, which forms in the Golgi complex and is involved in sorting of zymogens and granule formation (11). As proteoglycans were found to be associated with DIGs (Fig. 1), we analyzed whether their attachment to the GM was impaired after cholesterol depletion (Fig. 3). Golgi-enriched fractions isolated from treated lobules were separated in a membrane and content fraction, and the amount of proteoglycans in each fraction was determined using the Blyscan assay. Upon treatment with mevalonate/lovastatin and extraction with CD, only 10% of the submembranous proteoglycans were associated with the GM, whereas under control conditions 19% were found to be membrane-associated (Fig. 3).



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Fig. 3.   Effect of cholesterol depletion or inhibition of GPI-anchor biosynthesis on the attachment of proteoglycans to the Golgi membrane. Pancreatic lobules were pulse-labeled for 10 min with 200 µCi/ml Tran35S-label after cholesterol depletion (mevalonate/lovastatin/CD) or inhibition of GPI-anchor biosynthesis by either mannosamine or YW 3548. After 30 min of chase a Golgi-enriched fraction (Golgi) was isolated and separated into a membrane and content fraction by high-speed centrifugation. Sulfated proteoglycans (PG) in the content and membrane fractions were specifically precipitated using the Blyscan assay (see "Experimental Procedures"). The amount of radiolabeled proteoglycans in the precipitate was determined by scintillation counting. The data are from three independent experiments and are expressed as percent of total protein-bound radioactivity in the Golgi fraction (mean ± S.D.). p < 0.01 for all experimental groups.

To study the effect of cholesterol depletion and fumonisin B1 treatment on raft assembly in the Golgi apparatus, detergent-insoluble material of radiolabeled GM isolated from treated lobules was subjected to sucrose gradient centrifugation (Fig. 4). Analysis of the protein-bound radioactivity in the gradient fractions revealed that the amount of newly synthesized proteins in the low density fractions was reduced about 60-70% compared with untreated controls (Fig. 4). These data indicate that after cholesterol depletion of pancreatic lobules or inhibition of glycosphingolipid biosynthesis, raft assembly as well as the association of proteins and proteoglycans to rafts is disturbed. We therefore suggest that the process of granule formation depends on the correct assembly of cholesterol-sphingolipid-enriched rafts.



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Fig. 4.   Effect of cholesterol depletion and inhibition of glycosphingolipid synthesis on the formation of detergent-insoluble, glycosphingolipid-enriched complexes in Golgi membranes. Pancreatic lobules were incubated for 3 h in the absence (control ()) or in the presence of cholesterol depleting substances (mevalonate/lovastatin/CD; ) or fumonisin B1 (×). After pulse labeling (see "Experimental Procedures") radiolabeled GM were isolated and treated with 1% Triton X-100 at 4 °C for 30 min. The detergent-insoluble pellet obtained after high-speed centrifugation was subjected to sucrose density centrifugation. Fractions were collected from the top of the gradients (fraction 1), and the distribution of protein-bound radioactivity in the gradients was determined using scintillation counting. The data are expressed as percent of total protein-bound radioactivity in the insoluble pellet fraction.

Effects on Regulated Secretion-- Next we addressed whether the hormonally induced secretion of zymogens was affected by the reduction of intracellular cholesterol levels. After cholesterol depletion and pulse labeling, pancreatic lobules were stimulated with a mixture of reagents that triggers the hormonally induced release of zymogens, and 35S-amylase secretion was assessed in relation to total amylase secretion (Fig. 5). Stimulated control lobules showed a typical, time-dependent increase in the secretion of newly synthesized 35S-amylase, whereas unstimulated controls exhibited low basic secretion (Fig. 5, A and B). Interestingly, cholesterol depletion resulted in an increase of 35S-amylase secretion in the absence or the presence of a stimulus (Fig. 5, A and B). After 40 min 35S-amylase was increased 20-fold under stimulating and 5-fold under nonstimulating conditions compared with the unstimulated control (Fig. 5A). Similar results were obtained when the total secretion of radiolabeled proteins was assessed (not shown). These kinetic studies reveal that after cholesterol depletion the newly synthesized proteins appear to be constitutively secreted into the medium. When the secretion of radiolabeled proteins was measured after fumonisin B1 treatment and compared with the appropriate controls, an increase was not observed (not shown).



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Fig. 5.   Effect of cholesterol depletion on regulated secretion of zymogens. A and B, cholesterol-depleted pancreatic lobules (Chol. dep.) (triangle , black-triangle) and controls (Co) (, black-square) were pulse-labeled for 10 min with 200 µCi/ml Trans35S-label, and after a handling time of 5 min secretion was either hormonally stimulated (s) (filled symbols) as described under "Experimental Procedures" or measured under nonstimulatory conditions (open symbols). Media were collected after the time points indicated and the proteins precipitated and separated by SDS-PAGE. Total amylase was quantitated after Coomassie Blue staining of the corresponding gels (inset in A) or by determination of amylase activity in the media (see "Experimental Procedures"). Quantitation of 35S-labeled amylase was performed on the gels using a Bio-imager (inset in A). A, 35S-amylase secretion (in relation to total amylase) for the different experimental conditions after 40 min of chase. B, time course of 35S-amylase secretion (in relation to total amylase) for the different experimental conditions (mean ± S.D.). In C the 40-min chase media of the different experimental groups were collected and further incubated at 37 °C for 30 and 60 min. The protein-bound radioactivity in the media was determined by scintillation counting and is expressed as percent of total protein-bound radioactivity in the corresponding 40-min chase medium (mean ± S.D.). The data are from three to four independent experiments.

Surprisingly, amylase secretion of cholesterol-depleted lobules declined after 40 min (Fig. 5B). To examine whether degradation via the activation of zymogens was responsible for the decrease, the chase media collected after 40 min were incubated at 37 °C for different time intervals and assayed for the amount of protein-bound radioactivity and enzymatic activity of the zymogen trypsin. A slight reduction of protein-bound radioactivity was observed in the controls, whereas a decrease of about 20% was found in cholesterol-depleted media after 60 min of incubation (Fig. 5C). The degradation of proteins was reflected by an elevated trypsin activity in the media of cholesterol-depleted lobules (not shown).

Inhibition of GPI-anchor Biosynthesis Disturbs Granule Formation

It has been proposed that rafts serve as sorting platforms for GPI-anchored proteins. In a recent study (11) we obtained first evidence that GP-2 is involved in the tethering of a submembranous matrix composed of proteoglycans and glycoproteins to the ZGM. This protein matrix mediates the binding of aggregated zymogens to the granule membrane, a process that is considered as condensation-sorting. Since condensation and sorting are prerequisites for granule formation, we studied the role of the GPI-anchored GP-2 on de novo granule formation. For this purpose degranulated pancreatic lobules were treated with the amino sugar mannosamine (42, 43) or the terpenoid lactone YW 3548 (44), both of which were shown to inhibit GPI-anchor biosynthesis by blocking the addition of the third mannose to the intermediate structure Man2-GlcN-acyl-PI. After pulse-chase labeling of the lobules, fractions enriched in Golgi, rER, or ZG were isolated, and the protein-bound radioactivity was determined according to Table I. Treatment with mannosamine or YW 3548 resulted in a reduction of protein-bound radioactivity in the granule fraction, which was most pronounced after mannosamine treatment (Table I). Both treatments caused an accumulation of labeled proteins in the Golgi and the rER fractions. Ultrastructural analysis of the lobules revealed an accumulation of vesicles around the Golgi cisternae under both conditions, whereas other intracellular organelles or the integrity of the majority of the cells were not affected (Fig. 6, Table II). These results indicate that inhibition of GPI-anchor biosynthesis in acinar cells interferes with protein trafficking and granule formation.



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Fig. 6.   Fine structure of pancreatic lobules after inhibition of GPI-anchor biosynthesis. Degranulated pancreatic lobules were incubated in the presence of mannosamine (A) or YW 3548 (B), two potent inhibitors of GPI-anchor biosynthesis. After incubation lobules were fixed, embedded in Epon, and processed for electron microscopy as described under "Experimental Procedures." Note the accumulation of vesicles around the Golgi cisternae under both conditions (A and arrows in B) and the irregular shape of some zymogen granules (B, asterisk). Untreated control lobules are shown in Fig. 2A. G, Golgi apparatus. Bar: 1 µm.

Next we addressed whether the membrane association of proteoglycans was also affected upon treatment with mannosamine or YW 3548. Since granule formation starts in the Golgi complex, and ZG formation was reduced upon inhibition of GPI-anchor biosynthesis, Golgi membranes instead of ZGM were isolated and assayed as described in the legend to Fig. 3. Under control conditions ~19% of the proteoglycans were found to be associated with the GM (Fig. 3), whereas in the presence of the inhibitors mannosamine or YW 3548 only 10-13% of the proteoglycans were attached to it. These results suggest that the membrane association of a significant amount of proteoglycans is mediated directly via GPI anchorage or via binding to GPI-anchored membrane proteins, e.g. GP-2. Determination of the amount of DIG-associated radiolabeled proteins after Triton X-100 extraction of YW 3548- or mannosamine-treated GM and density centrifugation revealed only a slight reduction (10-20%, data not shown), indicating that the association of other proteins to DIGs and presumably to rafts is only partially mediated by GPI-anchored proteins.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Lipid Microdomains in Zymogen Granule Membranes-- Early studies addressing the lipid composition of pancreatic ZG showed that ZGM are enriched in cholesterol in comparison with other cellular membranes (45). Since ZG are involved in apical targeting and secretion of zymogens, we hypothesized that the granule membrane may contain cholesterol-rich microdomains similar to those reported to serve as sorting platforms and thus function in apical delivery of proteins in other polarized cell types (46-49).

When we analyzed the lipid composition of ZGM by TLC, a striking enrichment of cholesterol and sphingomyelin relative to phosphatidylcholine and phosphatidylethanolamine was observed when compared with GM or rERM (not shown). Analysis of polar lipids indicated the presence of glucocerebrosides, lactocerebrosides, and sulfatides in ZGM as well as in GM (not shown), which were reported to be minor components of rafts (22). These observations demonstrate that all lipids characteristic for glycosphingolipid-cholesterol rafts are found in ZGM. We could isolate DIGs from ZGM, which floated to lighter fractions on sucrose density gradients and were shown to be enriched in cholesterol and sphingolipids (Fig. 1). Distinct granule proteins such as the major membrane protein GP-2, the lectin ZG16p, and sulfated proteoglycans were found to be associated with these DIGs (Fig. 1). In a recent study (11) we have identified these proteins to be components of a submembranous matrix at the inner surface of ZG, which mediates the binding of aggregated zymogens to the granule membrane. We propose that the submembranous protein matrix has important functions in sorting of zymogens and in granule formation and stability. The GPI-anchored GP-2 is a likely candidate for tethering the submembranous matrix to the lipid bilayer. This notion is supported by our observation that inhibition of GPI-anchor biosynthesis interferes with protein trafficking and granule formation, leading to an accumulation of zymogens in the preceeding compartments, e.g. in the Golgi apparatus and in the rER (Table I, Fig. 6). Furthermore, the attachment of proteoglycans to Golgi membranes was diminished after treatment with mannosamine or YW 3548 (Fig. 3). These results emphasize the role of GPI anchorage and GPI-anchored membrane proteins, e.g. GP-2, in the tethering of a proteoglycan matrix and in granule formation. We propose that anchorage of the matrix to the membrane is a prerequisite for proper sorting of zymogens (11). When the tethering is disturbed, zymogens accumulate and are not packaged into granules. This might disturb the maturation processes in the Golgi cisternae and cause an accumulation of vesicles (Fig. 6). The nature of these vesicles is currently under investigation.

While the association of GP-2 with lipid microdomains is obviously mediated by its GPI-anchor, the association of ZG16p is less clear. ZG16p does not possess a transmembrane domain or a GPI attachment signature. It is likely that its interaction with the membrane is mediated by its lectin domain, possibly by binding to microdomain-associated glycolipids or glycoproteins. Another mode of interaction may be the association of ZG16p with chondroitin sulfate moieties of proteoglycans, which are themselves attached to lipid microdomains (12).

Assembly of Lipid Microdomains and Role in Granule Formation-- The analysis of the association of GP-2 with Golgi membranes or ZGM allowed us to define distinct pools that undergo changes during granule formation and may represent different stages of lipid raft assembly. The data indicate that the lipid environments of the proteins are changing when GM and ZGM are compared. ZGM contained a characteristic pool of GP-2 (pool 4, 69%), which was inaccessible to PI-PLC treatment and insoluble in Triton X-100 even at 37 °C. This pool was also found to be insoluble in detergents like octyl glucoside or deoxycholate (not shown), which are reported to dissolve rafts even at low temperature. It might represent a population of GP-2 present in unusual lipid microdomains with compact packaging and strong interactions between the components. It is further supposed that GP-2 forms tetramers in the membranes of ZG (7), which might account for its inaccessibility. Similar differences between GM and ZGM were obtained when the membrane attachment of ZG16p was analyzed (not shown). These results suggest that the assembly of distinct protein and lipid components into lipid rafts is a progressive process involving different assembly steps. Assembly of lipid rafts is initiated in the Golgi complex when lipids are glycosylated. However, in acinar cells the glycosylation of proteins, and in particular the sulfation of GAGs (and glycoproteins), are a prerequisite for the succeeding assembly steps. According to models proposed for granule biogenesis of neuroendocrine cells (1, 24), we envision that granule formation in the exocrine pancreas is initiated by the interaction of glycolipids, integral and/or GPI-anchored glycoproteins, and proteoglycans via their sugar moities, leading to the formation of lipid/glycoprotein/proteoglycan patches (Fig. 7). The voids of these patches are most likely filled by cholesterol. In the course of granule formation lectins may stabilize these structures and/or mediate the binding of other components, resulting in tightly packaged lipid/protein platforms. The proteoglycans attached to the platforms and other components of the submembranous protein matrix have a function not only in the binding/sorting of aggregated zymogens, but also in the stability of the forming granules (Fig. 7).



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Fig. 7.   A hypothetical model for the formation of zymogen granules in the exocrine pancreas. Zymogen granule formation and apical sorting in pancreatic acinar cells involves the pH-dependent, selective aggregation of the whole mixture of regulated secretory zymogens and the association of these aggregates with specific membrane domains of the trans-Golgi network (TGN), which then pinch off as condensing vacuoles (CV). Assembly of lipid microdomains is initiated in the Golgi complex where lipids are glycosylated and GAGs are attached to matrix proteoglycans (A). Furthermore, the sulfation of GAGs and the association of GPI-anchored proteins with lipid microdomains in the TGN are a prerequisite for granule formation (B). We favor a model in which zymogen aggregates are bound to an intragranular protein matrix. This matrix interacts via linker proteins (e.g. ZG16p) with a submembranous protein matrix composed of proteoglycans and glycoproteins. The proteoglycan/glycoprotein matrix is attached to the membrane via GPI-anchored proteins, e.g. GP-2, which is thought to form tetramers (C). The matrices have a function in the binding/sorting of aggregated zymogens, in granule formation and stability.

A Role for Lipid Microdomains in Regulated Apical Secretion of Zymogens-- We showed that cholesterol depletion of pancreatic lobules as well as inhibition of glycosphingolipid synthesis interfered with the formation of cholesterol-glycosphingolipid microdomains (Fig. 4) and with the association of proteins and proteoglycans (Fig. 3). We propose that the disruption of the tightly packaged lipid/protein platforms interferes with the attachment of proteoglycans, although GP-2 and other GPI-anchored proteins are still present. Interestingly, after cholesterol depletion, and thus lipid microdomain disruption, 35S-amylase secretion was increased in the presence or the absence of a stimulus compared with the appropriate controls (Fig. 5). These findings are indicative for misrouting of secretory proteins to a constitutive pathway and underline the importance of lipid rafts for regulated secretion of zymogens. The fact that amylase secretion was increased nearly 20-fold after cholesterol depletion in combination with a stimulus might indicate that signal transduction processes (mediated by rafts) are as well disturbed. Their disturbance could cause massive and uncontrolled secretion. We have provided biochemical and ultrastructural evidence for the intactness and the polarity of the isolated acinar cells after cholesterol depletion and other treatments (Table II, Figs. 2 and 6). Furthermore, regulated secretion could still be hormonally induced in controls and cholesterol-depleted lobules. Taken together, these findings indicate that the cells are still viable after the different treatments and that the effects we observe on regulated secretion are not due to leaky or otherwise damaged cells.

It is not yet clear if uncoupling of zymogens from the apical sorting platforms directs them to a basolateral pathway. Such missorting would be predicted by the finding that inhibition of sphingolipid synthesis leads to misrouting of GP-2 in MDCK cells (41). However, we did not observe a constitutive secretion upon inhibition of sphingolipid biosynthesis. These findings suggest that cholesterol is the important structural component responsible for the integrity and the function of lipid microdomains in zymogen granules. If specific cholesterol-containing microdomains distinct from the classical cholesterol/glycosphingolipid-containing rafts (50) exist in zymogen granules is currently under investigation. The observed constitutive secretion after cholesterol depletion resulted in the activation of secreted zymogens and in proteolytic degradation after a certain time. The mechanism(s) of this activation is not know, but may be triggered by a proteolytic enzyme present in the basolateral extracellular environment.

In summary, our results demonstrate the existence of lipid rafts in the membranes of zymogen granules and their involvement in granule formation and the trafficking of zymogens in the exocrine pancreas. We propose that the correct assembly of a lipid/glycoprotein/proteoglycan network on the inner surface of the granule membrane plays an essential role in sorting of regulated secretory proteins in acinar cells of the pancreas.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the skillful technical assistance of R. Rösser, W. Sperling, and B. Agricola. V. Kramer helped with the photographic work. YW 3548 was kindly provided by Novartis (Basel, Switzerland). We also thank A. Kenworthy for critical reading of the manuscript.


    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, Germany (SFB 286/B2).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.

Dagger These authors contributed equally to this work.

§ To whom all correspondence should be addressed: Dept. of Cell Biology and Cell Pathology, University of Marburg, Robert-Koch Str. 5, 35033 Marburg, Germany. Tel.: 49-6421-28-63857; Fax: 49-6421-28-66414; E-mail: schrader@mailer.uni-marburg.de.

Current address: Center for Molecular Neurobiology Hamburg (ZMNH), University of Hamburg, Falkenried 94, 20251 Hamburg, Germany.

Published, JBC Papers in Press, January 10, 2001, DOI 10.1074/jbc.M006221200


    ABBREVIATIONS

The abbreviations used are: TGN, trans-Golgi network; CD, methyl-beta -cyclodextrin; DIG, detergent-insoluble, glycolipid-enriched complex; GAG, glycosaminoglycan; GM, membrane of a Golgi-enriched fraction; GPI, glycosylphosphatidylinositol; PI-PLC, phosphatidylinositol-specific phospholipase C; rER, rough endoplasmic reticulum; rERM, membrane of a rough endoplasmic reticulum-enriched fraction; TLC, thin layer chromatography; ZG, zymogen granule(s); ZGM, zymogen granule membrane; MES, 4-morpholineethanesulfonic acid; MEM, minimal essential medium; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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