CALCIUM-DEPENDENT AND PH-DEPENDENT AGGREGATION AND MEMBRANE ASSOCIATION OF THE PRECURSOR OF THE PROHORMONE CONVERTASE PC2

PC2 is a subtilisin-like protease which is thought to be involved in t he processing of prohormones and proneuropeptides in neuroendocrine ce lls. The mature 68-kDa enzyme is generated by intracellular proteolyti c processing of a 75-KDa pro-PC2 polypeptide. Neuroendocrine cells con tain at least two secretory pathways: the regulated pathway whereby se creted products are concentrated, stored in granules, and released in response to external stimulation of the cell, and the constitutive pat hway, whereby secretory and plasma membrane proteins are continuously transported to the cell surface without prior concentration or storage . An important step in the sorting of proteins into these pathways is thought to involve the aggregation of proteins destined for storage gr anules. To define the mechanisms in the intracellular sorting of PC2 t o secretory vesicles, the present study was undertaken to investigate the aggregation and membrane association properties of precursor and m ature forms of PC2. Using material expressed in microinjected Xenopus oocytes, it was demonstrated that the 75-kDa pro-PC2 polypeptide under goes calcium- and acid pH-dependent aggregation. Calcium exhibited an effect on the aggregation of pro-PC2 at pH 7.0 and 6.5, whereas below 6.5 aggregation was independent of calcium. Association of pro-PC2 wit h membranes was observed at pH 5.5, but not at pH 7.0, 6.5, nor 6.0. T he mature 68-kDa PC2 polypeptide remained soluble under conditions tha t caused aggregation and membrane association of the 75-kDa propolypep tide. Deletion of COOH-terminal sequences had no effect on the associa tion of pro-PC2 with membranes, whereas a peptide corresponding to ami no acids 57-85 of the propeptide was able to partially compete the mem brane associated pro-PC2 away from the membranes.