Biogenesis of regulated exocytotic carriers in neuroendocrine cells.
Ca(2+)-triggered exocytosis is a hallmark of neurosecretory granules, but the cellular pathway leading to the assembly of these regulated exocytotic carriers is poorly understood. Here we used the pituitary AtT-20 cell line to study the biogenesis of regulated exocytotic carriers involved in peptide hormone secretion. We show that immature secretory granules (ISGs) freshly budded from the trans-Golgi network (TGN) exhibit characteristics of unregulated exocytotic carriers. During a subsequent maturation period they undergo an important switch to become regulated exocytotic carriers. We have identified a novel sorting pathway responsible for this transition. The SNARE proteins, VAMP4 and synaptotagmin IV (Syt IV), enter ISGs initially but are sorted away during maturation. Sorting is achieved by vesicle budding from the ISGs, because it can be inhibited by brefeldin A (BFA). Inhibition of this sorting pathway with BFA arrested the maturing granules in a state that responded poorly to stimuli, suggesting that the transition to regulated exocytotic carriers requires the removal of a putative inhibitor. In support of this, we found that overexpression of Syt IV reduced the stimulus-responsiveness of maturing granules. We conclude that secretory granules undergo a switch from unregulated to regulated secretory carriers during biogenesis. The existence of such a switch may provide a mechanism for cells to modulate their secretory activities under different physiological conditions.
Eaton
B A
BA
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3200, USA.
Haugwitz
M
M
Lau
D
D
Moore
H P
HP
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins / Q000096:biosynthesis*; D002460:Cell Line; D005089:Exocytosis; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009490:Neurosecretory Systems / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011233:Precipitin Tests; D011333:Pro-Opiomelanocortin / Q000378:metabolism / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D050857:Synaptotagmins; D033921:Vesicular Transport Proteins
D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D020030:Nitric Oxide Donors; D050683:R-SNARE Proteins; D050600:SNARE Proteins; C494293:Syt4 protein, mouse; C494294:Syt4 protein, rat; D033921:Vesicular Transport Proteins; D050857:Synaptotagmins; D011333:Pro-Opiomelanocortin; D000324:Adrenocorticotropic Hormone; D002118:Calcium