TRANSPORT VIA THE REGULATED SECRETORY PATHWAY IN SEMIINTACT PC12 CELLS - ROLE OF INTRA-CISTERNAL CALCIUM AND PH IN THE TRANSPORT AND SORTING OF SECRETOGRANIN-II

To gain insight into the mechanisms governing protein sorting, we have developed a system that reconstitutes both the formation of immature secretory granules and their fusion with the plasma membrane. Semi-int act PC12 cells were incubated with ATP and cytosol for 15 min to allow immature granules to form, and then in a buffer containing 30 mu M [C a2+](free) to induce exocytosis. Transport via the regulated pathway, as assayed by the release of secretogranin II (SgII) labeled in the TG N, was inhibited by depletion of ATP, or by the inclusion of 100 mu M GTP gamma S, 50 mu M AlF3-5 or 5 mu g/ml BFA. When added after immatur e granules had formed, GTP gamma S stimulated rather than inhibited ex ocytosis. Thus, exocytosis of immature granules in this system resembl es the characteristics of fully matured granules. Transport of SgII vi a the regulated pathway occurred at a fourfold higher efficiency than glycosaminoglycan chains, indicating that SgII is sorted to some exten t upon exit from the TGN. Addition of A23187 to release Ca2+ from the TGN had no significant effect on sorting of SgII into immature granule s. In contrast, depletion of lumenal calcium inhibited the endoproteol ytic cleavage of POMC and proinsulin. These results establish the impo rtance of intra-cisternal Ca2+ in prohormone processing, but raise the question whether lumenal calcium is required for proper sorting of Sg II into immature granules. Disruption of organelle pH gradients with a n ionophore or a weak base resulted in the inhibition of transport via both the constitutive and the regulated pathways.