Analysis of constitutive and constitutive-like secretion in semi-intact pituitary cells.
To study biosynthetic transport through the constitutive and regulated secretory pathways, we have designed a semi-intact mammalian cell system that restores the transport of secretory proteins from the trans-Golgi/trans-Golgi network (TGN) to the cell surface. The mouse pituitary AtT-20 cell line is a suitable model to biochemically analyze molecular sorting in the secretory pathway. The prohormone proopiomelanocortin is sulfated on N-linked carbohydrate chains in the trans-Golgi prior to proteolytic processing in the secretory granule. Radiolabeling with [35S]sulfate therefore provides a convenient tool to selectively follow molecular events in the regulated secretory pathway without interference from earlier steps. Likewise, transport through the constitutive secretory pathway may be monitored using sulfate-labeled glycosaminoglycan chains. We show that export from the TGN is efficiently reconstituted in cells made semi-intact with streptolysin O, and is dependent on temperature, ATP and GTP hydrolysis, and cytosol. Packaging of proopiomelanocortin into immature secretory granules also activates the proteolytic processing machinery which eventually converts the prohormone to its bioactive mature product, adrenocorticotropic hormone. In addition, a large fraction of incompletely processed proopiomelanocortin is secreted as the processing intermediates from immature secretory granules. This process of constitutive-like secretion can be clearly distinguished from direct constitutive secretion from the trans-Golgi network by kinetic and compositional criteria. Furthermore, we have found that specific inhibitors of different protein phosphatases and kinases are potent blockers of constitutive and constitutive-like secretion. This experimental model should provide a valuable system to elucidate the molecular mechanism regulating post-Golgi traffic during secretory granule biogenesis.
Dumermuth
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Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California, 94720-3200, USA.
Moore
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HP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins; D001692:Biological Transport; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D003594:Cytoplasmic Granules / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D051379:Mice; D008954:Models, Biological; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D011333:Pro-Opiomelanocortin / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011499:Protein Processing, Post-Translational; D013301:Streptolysins / Q000494:pharmacology
D001426:Bacterial Proteins; D006025:Glycosaminoglycans; D047428:Protein Kinase Inhibitors; D013301:Streptolysins; C025964:streptolysin O; C013786:A73025; D011333:Pro-Opiomelanocortin; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases