ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I

The small GTP-binding protein, ADP-ribosylation factor (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part o f the signal transduction pathway that can lead to secretion. In this study , substitution and deletion mutants of ARF1 were tested for their ability t o activate PLD, These map the PLD effector region of ARF1 to the alpha 2 he lix, part of the beta 2-strand and the N-terminal helix and its ensuing loo p. ARF mutants with an increased or decreased ability to activate PLD showe d similar characteristics when tested for their ability to stimulate secret ion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndell7), did not support PLD activity or secretion, and neither did it in hibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndell7 effectively competed with wild-type myrARF1 to prevent coatomer bind ing to membranes, This appears to define a structural role for Ndell7, as i t can bind a high-molecular mass complex in cytosol. In addition, ethanol h as no effect on recruitment of coatomer to membrane. We conclude that the f unction of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of AR F1-mediated coatomer binding.