Brefeldin A inhibits the constitutive-like secretion of a sulfated protein in pancreatic acinar cells.
Sulfation is a common posttranslational modification of secretory proteins and serves as a valuable marker of constitutive and regulated secretory pathways. We investigated the cellular localization and the secretory behavior of sulfated macromolecules in the mouse pancreatic acinar cell. The major sulfated proteins of the cell were present in isolated zymogen granules, as determined by metabolic labeling with [35S]sulfate and subcellular fractionation. The sulfated proteins fell into three groups: gp300 is not secreted and is a component of the zymogen granule membrane; pancreatic lipase (56 kDa) and a 40 kDa protein are soluble and exhibit regulated secretion kinetics; and p82 is initially granule membrane associated, but is released from the cell with constitutive-like kinetics as a 75 kDa protein (p75). Secretion of p75 could be stimulated for up to 4 h after pulse labeling, presumably from immature secretory granules, but not after 6 h of chase. Treatment of cells with brefeldin A (BFA) at the start of the [35S]sulfate pulse resulted in almost total inhibition of sulfation. Addition of BFA during the chase (0-2 h) allowed normal basal and stimulated secretion of regulated secretory proteins, but reversibly inhibited the constitutive-like secretion of p75. In this case, the behavior of p75 was maintained as that of a regulated secretory protein for up to 6 h of chase. In untreated cells, immunofluorescence of p82/p75 was along the acinar lumen, and in small punctate structures in the apical cytoplasm. In BFA-treated cells, immunolabeling of p82/p75 was lost from the acinar lumen, and cytoplasmic labeling was finer and appeared to be associated with the secretory granule membranes. These data suggest a role for brefeldin A-sensitive coat formation in maturation of secretory granules after they bud from the TGN.
De Lisle
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Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City 66160, USA.
Bansal
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D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A; D016764:Cell Polarity; D003517:Cyclopentanes / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010179:Pancreas / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology*; D011506:Proteins / Q000378:metabolism*; D017421:Sequence Analysis; D013347:Subcellular Fractions
D003517:Cyclopentanes; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D020126:Brefeldin A