IONIC MILIEU CONTROLS THE COMPARTMENT-SPECIFIC ACTIVATION OF PROOPIOMELANOCORTIN PROCESSING IN ATT-20 CELLS

Newly synthesized prohormones and their processing enzymes transit thr ough the same compartments before being packaged into regulated secret ory granules. Despite this coordinated intracellular transport, prohor mone processing does not occur until late in the secretory pathway. In the mouse pituitary AtT-20 cell line, conversion of proopiomelanocort in (POMC) to mature adrenocorticotropic hormone involves the prohormon e convertase PC1. The mechanism by which this proteolytic processing i s restricted to late secretory compartments is unknown; PC1 activity c ould be regulated by compartment-specific activators/inhibitors, or th rough changes in the ionic milieu that influence its activity. By arre sting transport in a semi-intact cell system, we have addressed whethe r metabolically labeled POMC trapped in early secretory compartments c an be induced to undergo conversion if the ionic milieu in these compa rtments is experimentally manipulated. Prolonged incubation of labeled POMC trapped in the endoplasmic reticulum or Golgi/trans-Golgi networ k did not result in processing, thereby supporting the theory that pro cessing is normally a post-Golgi/trans-Golgi network event. However, a cidification of these compartments allowed effective processing of POM C to the intermediate and mature forms. The observed processing increa sed sharply at a pH below 6.0 and required millimolar calcium, regardl ess of the compartment in which labeled POMC resided. These conditions also resulted in the coordinate conversion of PC1 from the 84/87 kDa into the 74-kDa and 66-kDa forms. We propose that POMC processing is p redominantly restricted to acidifying secretory granules, and that a c hange in FH within these granules is both necessary and sufficient to activate POMC processing.