Arabidopsis Phospholipase Dα1 Interacts with the Heterotrimeric G-protein α-Subunit through a Motif Analogous to the DRY Motif in G-protein-coupled Receptors

Abstract

Phospholipase D (PLD) and heterotrimeric G-protein both play important, diverse roles in cellular regulation and signal transduction. Here we have determined the physical interaction between plant PLD and the only canonical ␣-subunit (G␣) of the G-protein in Arabidopsis thaliana and the molecular basis for the interaction. PLD␣1 expressed in either Escherichia coli or Arabidopsis was co-precipitated with G␣. PLD␣1 contains a sequence motif analogous to the G␣-interacting DRY motif normally conserved in G-protein-coupled receptors. Mutation of the central Lys residue PLD K564A of this motif abolished the PLD␣1-G␣ binding, whereas mutation of the two flanking residues PLD E563A and PLD F565A decreased the binding. Addition of G␣ to PLD␣1 inhibited PLD␣1 activity, whereas the PLD K564A mutation that disrupted the G␣-PLD␣1 binding abolished the inhibition. GTP relieved the G␣ inhibition of PLD␣1 activity and also inhibited the binding between PLD␣1 and G␣. Meanwhile, the PLD␣1-G␣ interaction stimulated the intrinsic GTPase activity of G␣. Therefore, these results have demonstrated the direct binding between G␣ and PLD␣1, identified the DRY motif on PLD␣1 as the site for the interaction, and indicated that the interaction modulates reciprocally the activities of PLD␣1 and G␣.Phospholipase D (PLD), 1 which hydrolyzes phospholipids to phosphatidic acid and a head group, plays diverse roles in cellular metabolism and regulation. Plant PLD comprises a family of enzymes with different regulatory properties (1). Several Arabidopsis PLDs have been shown to display different requirements for Ca 2ϩ , polyphosphoinositides, and free fatty acids as well as varied substrate selectivity. Arabidopsis has at least 12 PLDs, of which PLD␣1 is most prevalent and responsible for the common plant PLD activity (1, 2). PLD␣1 produces a majority of the phosphatic acid under several stress conditions, such as freezing and wounding (3, 4). Suppression of PLD␣1 delayed abscisic acid (ABA)-promoted senescence (5), decreased wound-induced accumulation of jasmonic acid (4) and reactive oxygen generation (6), and increased freezing tolerance and water loss (3, 7). These results show that the common plant PLD has multifaceted functions, including roles in metabolism and cell signaling, dependent on the nature and severity of the stress conditions.The G␣ subunit of heterotrimeric G-proteins plays an important role in signal transduction. In animal systems, G␣ interacts with the upstream transmembrane G-protein-coupled receptors (GPCRs) and with the ␤-subunit. The binding of a ligand to a cognate receptor promotes the exchange of GDP for GTP on G␣, and the GTP-bound G␣ activates the downstream effector proteins (8). In addition, G␣ may interact with nonreceptor proteins to mediate signaling (9). Mammalian cells contain a number of G␣s that mediate many distinctive cellular functions (8). In contrast, the number of G␣s is very limited in plants; Arabidopsis has only a single canonical G␣ gene, GPA1 (10, 11). Arabidopsis G␣-null mutants are impaired i...

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