Assays to study phospholipase D regulation by inositol phospholipids and ADP-ribosylation factor 6

Phospholipase D (PLD) is an enzyme implicated in the regulation of both exocytic and endocytic vesicle trafficking as well as many other processes. Consistent with this, the small GTPase Arf6 and regulated changes in inositol phospholipids levels are two factors that regulate both PLD and vesicle trafficking. Here we describe three methodologies through which the activation of PLD by Arf6 and inositol phospholipids may be investigated. The first method described is an in vitro protocol that allows the analysis of purified proteins or cell lysates. Furthermore, this protocol can be used to analyze the effects of different inositol phospholipids by changing the composition of the substrate vesicle. The major advantage of this protocol lies in the ability to analyze the effects of direct interactions on PLD activation by using pure proteins and lipids. The other two methods are in vivo protocols for the analysis of PLD activation in response to extracellular stimuli. Modification of cellular composition using overexpression/deletion or knockout of specific genes can be utilized with these protocols to characterize PLD activation pathways. The first of these methods uses the detection of radiolabeled PLD products and can be used for most cell types whereas the second of these two protocols is used to measure PLD products when radiolabeling of cells is not possible, such as freshly isolated cells that will not survive long enough to attain radiochemical equilibrium.