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

Abstract

Phosphoinositides are both substrates for second messenger-generating enzymes and spatially localized membrane signals that mediate vital steps in signal transduction, cytoskeletal regulation and membrane trafficking. Phosphatidylcholine-specific phospholipase D (PLD) activity is stimulated by phosphoinositides, but the mechanism and physiological requirement for such stimulation to promote PLD-dependent cellular processes is not known. To address these issues, we have identified a site at which phosphoinositides interact with PLD and have assessed the role of this region in PLD function. This interacting motif contains critical basic amino acid residues that are required for stimulation of PLD activity by phosphoinositides. Although PLD alleles mutated at this site fail to bind to phosphoinositides in vitro, they are membrane-associated and properly localized within the cell but are inactive against cellular lipid substrates. Analogous mutations of this site in yeast PLD, Spo14p, result in enzymes that localize normally, but with catalytic activity that has dramatically reduced responsiveness to phosphoinositides. The level of responsiveness to phosphoinositides in vitro correlated with the ability of PLD to function in vivo. Taken together, these results provide the first evidence that phosphoinositide regulation of PLD activity observed in vitro is physiologically important in cellular processes in vivo including membrane trafficking and secretion.

Roles of Phosphoinositides and of Spo14p (phospholipase D)-generated Phosphatidic Acid during Yeast Sporulation

“…Likewise, PtdIns(4,5)P 2 stimulates Spo14p in vitro (Rose et al, 1995), and genetic analyses indicate that PtdIns(4,5)P 2 is essential for activating the function of Spo14p in vivo (Sciorra et al, 1999(Sciorra et al, , 2002. These observations suggest that PtdIns(4,5)P 2 generation is necessary to activate Spo14p for its role in prospore membrane formation.…”

“…Given the effects of the sec14(K66A K239A) and pik1 mutations on spore formation and Spo14p activity, it seemed possible that PtdIns and/or PtdIns(4)P, in addition to PtdIns(4,5)P 2 (Rose et al, 1995;Sciorra et al, 1999Sciorra et al, , 2002, might directly modulate Spo14p activity. However, when epitope-tagged Spo14p was expressed in sporulating cells, immunoprecipitated, and assayed for PLD activity, only PtdIns(4,5)P 2 had a significant effect on PLD activity (Table 5).…”

Identification of Novel Membrane-binding Domains in Multiple Yeast Cdc42 Effectors

“…Phospholipids in chloroform were purchased from Avanti Polar Lipids (Alabaster, AL). To prepare sucrose-laden liposomes (Sciorra et al, 1999), phospholipid mixtures were dried in glass test tubes under a stream of nitrogen, then resuspended in 20 mM HEPES (pH 7.5) with 20 mM NaCl and 0.2 M sucrose, vortexed (ϳ1 min), and sonicated in a water bath sonicator (four times for 1 min). This suspension (200 l) was mixed with 700 l of buffer A (20 mM NaPO 4 , pH 7.5, and 200 mM NaCl) and centrifuged at 200,000 ϫ g for 15 min at 4°C.…”

Evidence for and Characterization of Ca2+ Binding to the Catalytic Region of Arabidopsis thaliana Phospholipase Dβ

“…PIP 2 stimulates the activity of all other A. thaliana PLDs examined (6 -8) and also mammalian and yeast PLDs (29,30). Both PH and PX domains are known to interact with polyphosphoinositides.…”

“…The PLD family in plants is much more diverse than that in other organisms. The A. thaliana PLD family has 12 PLD genes (8,27), whereas only two PLD genes are known in mammals and one in yeast (29,30). Furthermore, 10 of the 12 A. thaliana PLDs contain the C2 domain, which are unique to plant PLDs.…”