Identification of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes

Phosphoinositides are both substrates for second messenger-generating enzym es and spatially localized membrane signals that mediate vital steps in sig nal transduction, cytoskeletal regulation and membrane trafficking. Phospha tidylcholine-specific phospholipase D (PLD) activity is stimulated by phosp hoinositides, but the mechanism and physiological requirement for such stim ulation to promote PLD-dependent cellular processes is not known, To addres s these issues, we have identified a site at which phosphoinositides intera ct with PLD and have assessed the role of this region in PLD function, This interacting motif contains critical basic amino acid residues that are req uired for stimulation of PLD activity by phosphoinositides, Although PLD al leles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are ina ctive against cellular lipid substrates, Analogous mutations of this site i n yeast PLD, Spo14p, result in enzymes that localize normally, but with cat alytic activity that has dramatically reduced responsiveness to phosphoinos itides. The level of responsiveness to phosphoinositides in vitro correlate d with the ability of PLD to function in vivo. Taken together, these result s provide the first evidence that phosphoinositide regulation of PLD activi ty observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.