Regulated exocytosis in chromaffin cells. Translocation of ARF6 stimulates a plasma membrane-associated phospholipase D.
The ADP-ribosylation factor (ARF) GTP-binding proteins have been implicated in a wide range of vesicle transport and fusion steps along the secretory pathway. In chromaffin cells, ARF6 is specifically associated 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 stimulated chromaffin cells. We found that stimulation of intact chromaffin cells or direct elevation of cytosolic calcium in permeabilized cells triggered the rapid translocation of ARF6 from secretory granules to the plasma membrane and the concomitant activation of PLD in the plasma membrane. To probe the existence of an ARF6-dependent PLD in chromaffin cells, we measured the PLD activity in purified plasma membranes. PLD could be activated by a nonhydrolyzable analogue of GTP and by recombinant myristoylated ARF6 and inhibited by specific anti-ARF6 antibodies. Furthermore, a synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 inhibited both PLD activity and catecholamine secretion in calcium-stimulated chromaffin cells. The possibility 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.
Caumont
A S
AS
INSERM, U-338 Biologie de la Communication Cellulaire, Strasbourg, France.
Galas
M C
MC
Vitale
N
N
Aunis
D
D
Bader
M F
MF
D020001:1-Butanol / Q000494:pharmacology; D020727:ADP-Ribosylation Factors; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology* / Q000187:drug effects; D004789:Enzyme Activation; D000431:Ethanol / Q000494:pharmacology; D005089:Exocytosis; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*
D002352:Carrier Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000431:Ethanol; D020001:1-Butanol; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors; D002118:Calcium