Biochemical Analysis of the Interaction Between Phospholipase D1 and GTP-Binding Protein -Subunit from Arabidopsis thaliana

Phospholipase Ds (PLDs) play diverse roles in plant lipid metabolism and cellular signaling processes. The sole canonical G-protein α-subunit (Gα) in Arabidopsis also plays multiple roles in plant growth and cellular signaling processes. Interestingly, overlapping functions of PLD and Gα have been indicated in many cellular processes, including abscisic acid (ABA)-mediated stomata movement and water loss, gibberellic acid (GA)-regulated seed germination, and auxin signaling. This interaction between PLD and Gα has also been suggested in biochemical and physiological studies. Here we described the methods used for studying the interaction between the major PLD form PLDα1 and Gα. From pulldown experiments with purified bacterially expressed PLDα1 and Gα, co-immunoprecipitation of plant protein extract, and yeast two-hybrid assay, we showed that there is a physical interaction between PLDα1 and Gα, and identified a key DRY motif in PLDα1, which is an essential element for the interaction. The interaction of PLDα1 and Gα can be affected by factors like GTP or GDP, but it also affected PLD phospholipase activity and Gα GTPase activity in turn.