DISTINCT MOLECULAR EVENTS DURING SECRETORY GRANULE BIOGENESIS REVEALED BY SENSITIVITIES TO BREFELDIN-A

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their tempor al order and interrelationships, we have developed a pulse-chase proto col that follows the synthesis and packaging of a sulfated hormone, pr o-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated in to POMC predominantly on N-linked endoglycosidase H-resistant oligosac charides. Subcellular fractionation and pharmacological studies confir m that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sul fation, POMC undergoes a number of molecular events before final stora ge in dense-core granules. The first step involves the transfer of POM C from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic co nversion to mature adrenocorticotropic hormone. Proteolytic cleavage p roducts were found in vesicular fractions corresponding to ISGs, sugge sting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little un regulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage e vent within the maturing granule. The second step of granule biogenesi s thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregul ated release to one that favors intracellular storage. By using BFA, w e show that the two processes occurring in ISGs may be uncoupled: alth ough the unregulated secretion from ISGs is impaired by BFA, proteolyt ic processing of POMC within this organelle proceeds unaffected. The f inding that BFA impairs constitutive secretion from both the TGN and I SGs also suggests that these secretory processes may be related in mec hanism. Finally, our data indicate that the unusually high levels of u nregulated secretion often associated with endocrine tumors may result , at least in part, from inefficient storage of secretory products at the level of ISGs.