REGULATED EXOCYTOSIS IN CHROMAFFIN CELLS - TRANSLOCATION OF ARF6 STLMULATES A PLASMA MEMBRANE-ASSOCIATED PHOSPHOLIPASE-D

The ADP-ribosylation factor (ARF) GTP-binding proteins have been impli cated in a wide range of vesicle transport and fusion steps along the secretory pathway. In chromaffin cells, ARF6 is specifically associate d with the membrane of secretory chromaffin granules, Since ARF6 is an established regulator of phospholipase D (PLD), we have examined the intracellular distribution of ARF6 and PLD activity in resting and sti mulated chromaffin cells. We found that stimulation of intact chromaff in cells or direct elevation of cytosolic calcium in permeabilized cel ls triggered the rapid translocation of ARF6 from secretory granules t o the plasma membrane and the concomitant activation of PLD in the pla sma membrane. To probe the existence of an ARF6-dependent PLD in chrom affin cells, we measured the PLD activity in purified plasma membranes . PLD could be activated by a nonhydrolyzable analogue of GTP and by r ecombinant myristoylated ARF6 and inhibited by specific anti-ARF6 anti bodies, Furthermore, a synthetic myristoylated peptide corresponding t o the N-terminal domain of ARF6 inhibited both PLD activity and catech olamine secretion in calcium-stimulated chromaffin cells. The possibil ity that ARF6 participates in the exocytotic reaction by controlling a plasma membrane-bound PLD and thereby generating fusogenic lipids at the exocytotic sites is discussed.