TRIMERIC G-PROTEINS CONTROL REGULATED EXOCYTOSIS IN BOVINE CHROMAFFINCELLS - SEQUENTIAL INVOLVEMENT OF GO ASSOCIATED WITH SECRETORY GRANULES AND GI(3) BOUND TO THE PLASMA-MEMBRANE

Regulated secretion requires both calcium and MgATP. Studies in divers e secretory Systems indicate that ATP is required to prime the exocyto tic apparatus whereas Ca2+ triggers the final ATP-independent fusion e vent. In this paper, we examine the possible role of trimeric G protei ns in these two steps of exocytosis in chromaffin cells. We show that in the presence of low concentrations of Mg2+, mastoparan selectively stimulates G proteins associated with purified chromaffin granule memb ranes. Under similar conditions in permeabilized chromaffin cells, mas toparan inhibits ATP-dependent secretion but is unable to trigger ATP- independent release. This inhibitory effect of mastoparan on secretion was specifically reversed by anti-G alpha o antibodies and a syntheti c peptide corresponding to the carboxyl terminus of G alpha o. In cont rast, mastoparan required millimolar Mg2+ for the activation of plasma membrane-bound G proteins and stimulation of ATP-independent secretio n in permeabilized chromaffin cells. The latter effect was completely inhibited by anti-G alpha i(3) antibodies and a synthetic peptide corr esponding to the carboxyl terminus of G alpha i(3). By confocal immuno fluorescence and immunoreplica analysis, we provide evidence that in c hromaffin cells Go is preferentially associated with secretory granule s, while Gi(3) is essentially present on the plasma membrane. Our find ings suggest that these two trimeric G proteins act in series in the e xocytotic pathway in chromaffin cells: a secretory granule-associated Go protein controls the ATP-dependent priming reaction, whereas a plas ma membrane-bound Gi(3) protein is involved in the late calcium-depend ent fusion step, which does not require ATP.