Differential effects of temperature blockade on the proteolytic processing of three secretory granule-associated proteins.
Vesicular transport within the secretory pathway can be arrested by incubating cells at 15 degrees C or 20 degrees C to block exit from the endoplasmic reticulum or trans-Golgi network, respectively. Using this powerful tool we have compared the intracellular sites of endoproteolytic processing of proopiomelanocortin and two prohormone processing enzymes in AtT-20 mouse pituitary corticotrope tumor cells. For comparison, proopiomelanocortin processing was also evaluated in primary neurointermediate pituitary cultures. AtT-20 cells synthesize and store endogenous proopiomelanocortin and prohormone convertase 1; AtT-20 cells expressing high levels of integral membrane or soluble peptidylglycine alpha-amidating monooxygenase were generated by stable transfection. Cells were incubated with [35S]methionine and chased at 4 degrees C, 15 degrees C, 20 degrees C or 37 degrees C. The endoproteolytic processing of peptidylglycine alpha-amidating mono-oxygenase, prohormone convertase 1, and proopiomelanocortin was compared following immunoprecipitation. Endoproteolytic processing of integral membrane and soluble peptidylglycine alpha-amidating monooxygenase proteins was completely blocked by incubation of cells at 20 degrees C. In contrast, prohormone convertase 1 processing from the 87 kDa precursor to the 81 kDa intermediate proceeded to completion at both 15 degrees C and 20 degrees C, while cleavage to generate the 63 kDa prohormone convertase 1 protein was completely blocked at 20 degrees C. In AtT-20 cells and neurointermediate pituitary cultures, generation of beta-lipotropin from proopiomelanocortin continued at a slow but significant rate at 20 degrees C, while processing of beta-lipotropin to beta-endorphin was blocked. Thus prohormone convertase 1 processing begins in the endoplasmic reticulum and is not completed until after the trans-Golgi network, while peptidylglycine alpha-amidating monooxygenase processing begins after the trans-Golgi network. Selected proopiomelanocortin cleavages begin before entry into immature granules.
Milgram
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Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Mains
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D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism*; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000378:metabolism*; D051379:Mice; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009097:Multienzyme Complexes; D010911:Pituitary Neoplasms; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D013696:Temperature; D014162:Transfection; D014407:Tumor Cells, Cultured
D009097:Multienzyme Complexes; D011333:Pro-Opiomelanocortin; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D043484:Proprotein Convertases; D016282:Aspartic Acid Endopeptidases