Molecular heterogeneity of phospholipase D (PLD) - Cloning of PLD gamma and regulation of plant PLD gamma, -beta, and -alpha by polyphosphoinositides and calcium

Phospholipase D (PLD) has emerged as an important enzyme involved in signal transduction, Vesicle trafficking, and membrane metabolism. This report describes the cloning and expression of a new Arabidopsis PLD cDNA, designated PLD gamma, and the regulation of PLD gamma, -beta, and -alpha by phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+, The PLD gamma cDNA is 3.3 kilobases in length and codes for an 855-amino acid protein of 95,462 Da with a pi of 6.9. PLD gamma shares a 66% amino acid sequence identity with PLD beta, but only a 41% identity with PLD alpha. A potential N-terminal myristoylation site is found in PLD gamma, but not in PLD alpha and -beta. Catalytically active PLD gamma was expressed in Escherichia coli, and its activity requires polyphosphoinositides. Both PLD gamma and -beta are most active at mu M Ca2+ concentrations, whereas the optimal PLD alpha activity requires mM Ca2+ concentrations. Binding studies showed that the PLDs bound PIP2 in the order of PLD beta > PLD gamma > PLD alpha. This binding ability correlates with the degree of conservation of a basic PIP2-binding motif located near the putative catalytic site. The binding of [H-3]PIP, was saturable and could be competitively decreased by addition of unlabeled PIP2. Neomycin inhibited the activities of PLD gamma and -beta, but not PLD alpha. These results demonstrate that PLD is ancoded by a heterogeneous gene family and that direct polyphosphoinositide binding is required for the activities of PLD gamma and -beta, but not PLD alpha. The different structural and biochemical properties suggest that PLD alpha, -beta, and -gamma are regulated differently and may mediate unique cellular functions.