Abstract
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.
Highlights
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
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
These results indicate that detergent-insoluble glycosphingolipid complexes (DIGs) enriched in cholesterol and sphingomyelin are present in Pancreatic lobules were prepared from rats fed with a single dose of FOY-305 leading to hormonally stimulated release of zymogen granules
Summary
Antibodies—Polyclonal antibodies to rat GP-2 and pig amylase were used as reported previously [9]. To extract polar lipids [28] membranes corresponding to 2 mg of protein were resuspended in 20 l of 50 mM Hepes, pH 8.0, and incubated with 500 l of methanol and 1 ml of n-hexane. 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 ␣ 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. Data are presented as mean Ϯ S.D., with an unpaired t test used to determine statistical differences. p values Ͻ0.05 are considered as significant, and p values Ͻ0.01 are considered as highly significant
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