Proteoglycans support proper granule formation in pancreatic acinar cells.

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. The molecular mechanisms of their biogenesis and the sorting of zymogens are still incompletely understood. Here, we investigated the role of proteoglycans in granule formation and secretion of zymogens in pancreatic AR42J cells, an acinar model system. Cupromeronic Blue cytochemistry and biochemical studies revealed an association of proteoglycans primarily with the granule membrane. Removal of proteoglycans by carbonate treatment led to a loss of membrane curvature indicating a supportive role in the maintenance of membrane shape and stability. Chemical inhibition of proteoglycan synthesis impaired the formation of normal electron-dense granules in AR42J cells and resulted in the formation of unusually small granule structures. These structures still contained the zymogen carboxypeptidase, a cargo molecule of secretory granules, but migrated to lighter fractions after density gradient centrifugation. Furthermore, the basal secretion of amylase was increased in AR42J cells after inhibitor treatment. In addition, irregular-shaped granules appeared in pancreatic lobules. We conclude that the assembly of a proteoglycan scaffold at the ZG membrane is supporting efficient packaging of zymogens and the proper formation of stimulus-competent storage granules in acinar cells of the pancreas.

Pex11pbeta-mediated growth and division of mammalian peroxisomes follows a maturation pathway.

Peroxisomes are ubiquitous subcellular organelles, which multiply by growth and division but can also form de novo via the endoplasmic reticulum. Growth and division of peroxisomes in mammalian cells involves elongation, membrane constriction and final fission. Dynamin-like protein (DLP1/Drp1) and its membrane adaptor Fis1 function in the later stages of peroxisome division, whereas the membrane peroxin Pex11pbeta appears to act early in the process. We have discovered that a Pex11pbeta-YFP(m) fusion protein can be used as a specific tool to further dissect peroxisomal growth and division. Pex11pbeta-YFP(m) inhibited peroxisomal segmentation and division, but resulted in the formation of pre-peroxisomal membrane structures composed of globular domains and tubular extensions. Peroxisomal matrix and membrane proteins were targeted to distinct regions of the peroxisomal structures. Pex11pbeta-mediated membrane formation was initiated at pre-existing peroxisomes, indicating that growth and division follows a multistep maturation pathway and that formation of mammalian peroxisomes is more complex than simple division of a pre-existing organelle. The implications of these findings on the mechanisms of peroxisome formation and membrane deformation are discussed.

Rab8 is involved in zymogen granule formation in pancreatic acinar AR42J cells.

Zymogen granules (ZGs) are specialized storage organelles in the exocrine pancreas, which allow digestive enzyme storage and regulated apical secretion. To understand the function of these important organelles, we are conducting studies to identify and characterize ZG membrane proteins. Small guanosine triphosphatases (GTPases) of the Rab family are key protein components involved in vesicular/granular trafficking and membrane fusion in eukaryotic cells. In this study, we show by morphological studies that Rab8 (Rab8A) localizes to ZGs in acinar cells of the pancreas. We find that Rab8 is present on isolated ZGs from rat pancreas and in the ZG membrane fraction obtained after granule subfractionation. To address a putative role of Rab8 in granule biogenesis, we conducted RNA interference experiments to 'knock down' the expression of Rab8 in pancreatic AR42J cells. Silencing of Rab8 (but not of Rab3) resulted in a decrease in the number of ZGs and in an accumulation of granule marker proteins within the Golgi complex. By contrast, the trafficking of lysosomal and plasma membrane proteins was not affected. These data provide first evidence for a role of Rab8 early on in ZG formation at the Golgi complex and thus, apical trafficking of digestive enzymes in acinar cells of the pancreas.