ARF1(2-17) DOES NOT SPECIFICALLY INTERACT WITH ARF1-DEPENDENT PATHWAYS - INHIBITION BY PEPTIDE OF PHOSPHOLIPASE-C-BETA, PHOSPHOLIPASE-D ANDEXOCYTOSIS IN HL-60 CELLS

The small GTP-binding protein ARF has been shown recently to regulate phospholipase D (PLD). In order to investigate the role of ARF protein s in regulated exocytosis, we have used the N-terminal peptide ARF1(2- 17) of the ARF1 protein. ARF1 reconstituted PLD activity in cytosol-de pleted HL60 cells was inhibited by ARF1(2-17). In the presence of endo genous cytosol, ARF1(2-17) also inhibited GTP-gamma-S-stimulated PLD a ctivity and exocytosis. Mastoparan Politses jadwagae and mastoparan Ve spula lewisii which exhibit similar structural properties to ARF1(2-17 ) also inhibited GTP-gamma-S-stimulated PLD and exocytosis. GTP-gamma- S-stimulated phospholipase C-beta (PLC-beta) was also inhibited by ARF (2-17) and mastoparan. In cytosol-depleted HL60 cells, the ARF(2-17) i nhibited the reconstitution of GTP-gamma-S-stimulated PLC-beta activit y with exogenously-added PLC-beta 1 and phosphatidylinositol transfer protein. We conclude that the widely-used ARF1(2-17) peptide inhibits both ARF-independent (i.e. PLC-B) and ARF-dependent pathways (i.e. PLD ) and therefore cannot be regarded as a specific inhibitor of ARF func tion.