Biogenesis of regulated exocytotic carriers in neuroendocrine cells

Ca2+-triggered exocytosis is a hallmark of neurosecretory granules, but the cellular pathway leading to the assembly of these regulated exocytotic car riers is poorly understood. Here we used the pituitary AtT-20 cell line to study the biogenesis of regulated exocytotic carriers involved in peptide h ormone secretion. We show that immature secretory granules (ISGs) freshly b udded from the trans-Golgi network (TGN) exhibit characteristics of unregul ated exocytotic carriers. During a subsequent maturation period they underg o an important switch to become regulated exocytotic carriers. We have iden tified a novel sorting pathway responsible for this transition. The SNARE p roteins, 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 t his sorting pathway with BFA arrested the maturing granules in a state that responded poorly to stimuli, suggesting that the transition to regulated e xocytotic carriers requires the removal of a putative inhibitor. In support of this, we found that overexpression of Syt IV reduced the stimulus-respo nsiveness of maturing granules. We conclude that secretory granules undergo a switch from unregulated to regulated secretory carriers during biogenesi s. The existence of such a switch may provide a mechanism for cells to modu late their secretory activities under different physiological conditions.