Molecular Cloning and Functional Analysis of Polyphosphoinositide-dependent Phospholipase D, PLDβ, from Arabidopsis

Abstract

A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLD␤ for this PIP 2 -dependent PLD and PLD␣ for the previously characterized PIP 2 -independent PLD that requires millimolar Ca 2؉ for optimal activity. The PLD␤ cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLD␤ cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLD␤, but not PLD␣, activity. The activity of the expressed PLD␤ is dependent on PIP 2 and submicromolar amounts of Ca 2؉ , inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLD␤ is evolutionarily divergent from PLD␣ and that its N terminus contains a regulatory Ca 2؉ -dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.Phospholipase D (PLD; EC 3.1.4.4) 1 -catalyzed hydrolysis of glycerophospholipids produces phosphatidic acid (PA) and a hydrophilic constituent. This activity was first identified in plants and since has been found in animals and microorganisms. PLD in plants was originally proposed to be important in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress injuries (1, 2). Recent studies in plants, animals, and yeast indicate that PLD hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation (3-9). Activation of PLD has been proposed to mediate many cellular processes including cell proliferation, membrane trafficking, meiosis, and responses to external and internal stimuli. It has been suggested that multiple forms of PLD are involved in these diverse cellular processes since several studies have shown the presence of PLD variants that are expressed differently (9 -12). In castor bean (9) and rice (12), one PLD variant is constitutive whereas the appearance of other variants is associated with specific conditions such as rapid growth, wounding, and senescence. A distinct property shared by these variants is their in vitro requirement of millimolar Ca 2ϩ concentrations for optimal activity. Further analyses of the castor bean PLD variants have led to the suggestion that the catalytic activity of these variants results from the same gene product (9 -11).A recent study has provided important evidence for the presence of two plant PLDs that are derived from different gene products and regulated distinctly (13). One PLD requires polyphosphoinositides and submicromolar concentrations of Ca 2ϩ for activity and the other is PIP 2 -independent and is most active in the presence of millimolar amounts of Ca 2ϩ . The latter is the prevalent form of PLD that has been purified and cha...

Molecular Heterogeneity of Phospholipase D (PLD)

“…After BamHI digestion, the insert was ligated into pBluescript SK or pGEX-2T, and the recombinant plasmids were transformed into E. coli JM109. The expression of PLD␥ was induced using the same procedure as described for PLD␣ and -␤ (3,5). The cells were lysed by sonication in the resuspension buffer, and cell debris was removed by centrifugation at 10,000 ϫ g for 5 min.…”

“…The peptide was conjugated to keyhole limpet hemocyanin and used as an antigen to raise antibodies in rabbits (5). For immunoblot analysis, proteins were separated on 8% SDS-polyacrylamide gels, transferred onto a polyvinylidene difluoride membrane, and incubated with antiserum that contained JM109 lysate to remove nonspecific reactive bacterial proteins.…”

“…For immunoblot analysis, proteins were separated on 8% SDS-polyacrylamide gels, transferred onto a polyvinylidene difluoride membrane, and incubated with antiserum that contained JM109 lysate to remove nonspecific reactive bacterial proteins. The immunoblot analysis was performed as described (5).…”

“…Recent advances in the molecular understanding of PLD have brought new insights into the control and cellular roles of PLD. An intracellular PLD was first cloned from castor bean (3), and the cloning of PLDs has since been reported from Arabidopsis (4,5), rice, maize (6), yeast (7), human (8), and mouse (9). Analysis of the PLD sequences has led to the identification of probable catalytic and regulatory domains of PLD (10,11).…”

“…Analysis of the PLD sequences has led to the identification of probable catalytic and regulatory domains of PLD (10,11). Two PLDs with distinct regulatory properties have been cloned from Arabidopsis: one is the conventional mM Ca 2ϩ -requiring PLD, designated PLD␣, and the other, named PLD␤, requires M Ca 2ϩ and polyphosphoinositides for activity (4,5,12).…”