EVIDENCE THAT PHOSPHOLIPASE-D MEDIATES ADP-RIBOSYLATION FACTOR-DEPENDENT FORMATION GOLGI COATED VESICLES

Formation of coatomer-coated vesicles from Golgi-enriched membranes re quires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi-enriched memb ranes. It has been proposed that ARF, which is recruited onto membrane s from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles. We present evidence that cytosolic ARF is not necessary for initiating coat asse mbly on Golgi membranes from cell lines with high constitutive PLD act ivity. Conditions are also described under which ARF is at most a mino r component relative to coatomer in coated vesicles from all cell line s tested, including Chinese hamster ovary cells. Formation of coated v esicles was sensitive to ethanol at concentrations that inhibit the pr oduction of phosphatidic acid (PA) by PLD. When PA was produced in Gol gi membranes by an exogenous bacterial PLD, rather than with ARF and e ndogenous PLD, coatomer bound to Golgi membranes. Purified coatomer al so bound selectively to artificial lipid vesicles that contained PA an d phosphatidylinositol (4,5)-bisphosphate (PIP2). We propose that acti vation of PLD and the subsequent production of PA are key early events for the formation of coatomer-coated vesicles.