PHOSPHOLIPASE-D STIMULATES RELEASE OF NASCENT SECRETORY VESICLES FROMTHE TRANS-GOLGI NETWORK

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activ ation has been implicated in mediating signal transduction pathways, c ell growth, and membrane trafficking in mammalian cells. Several labor atories have demonstrated that small GTP-binding proteins including AD P-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes, Since A RF-1 has also been shown to enhance release of nascent secretory vesic les from the TGN of endocrine cells, we hypothesized that this reactio n occurred via PLD activation. Using a permeabilized cell system deriv ed from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascen t secretory vesicle budding from the TGN approximately twofold. In con trast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when adde d to the permeabilized cells. The release of nascent secretory vesicle s from the TGN was sensitive to 1% l-butanol, a concentration that inh ibited PLD-catalyzed formation of phosphatidic acid, Furthermore, ARF- 1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesic le budding. Our results suggest that ARF regulation of PLD activity pl ays an important role in the release of nascent secretory vesicles fro m the TGN.