Tsung-Chang Sung

Activation of phosphatidylcholine-specific phospholipase D (PLD) has been implicated as a critical step in numerous cellular pathways, including signal transduction, membrane trafficking, and the regulation of mitosis. We report here the identification of the first human PLD cDNA, which defines a new and highly conserved gene family. Characterization of recombinant human PLD1 reveals that it is membrane-associated, selective for phosphatidylcholine, stimulated by phosphatidylinositol 4,5-bisphosphate, activated by the monomeric G-protein ADP-ribosylation factor-1, and inhibited by oleate. PLD1 likely encodes the gene product responsible for the most widely studied endogenous PLD activity.

Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited 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 vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.

Mammalian ADP-ribosylation factor 1 (mARF1) is a small GTP-binding protein that is activated by a Golgi guanine nucleotide exchange factor. Once bound to the Golgi membranes in the GTP form, mARF1 initiates the recruitment of the adaptor protein 1 (AP-1) complex and coatomer (COPI) onto these membranes and activates phospholipase D1 (PLD1). To map the domains of mARF1 that are important for these activities, we constructed chimeras between mARF1 and Saccharomyces cerevisiae ARF2, which functions poorly in all of these processes except COPI recruitment. The carboxyl half of mARF1 (amino acids 95-181) was essential for activation by the Golgi guanine nucleotide exchange factor, whereas a separate domain (residues 35-94) was required to effectively activate PLD1 and to promote efficient AP-1 recruitment. Since residues 35-94 of mARF1 are critical for optimal activity in both PLD1 activation and AP-1 recruitment, we hypothesize that this region of ARF contains residues that interact with effector molecules.