Lipid-protein interactions for ECA1 an N-ANTH domain protein involved in stress signaling in plants.

Epsin-like Clathrin Adaptor 1 (ECA1/ PICALM1A) is an A/ENTH domain protein that acts as an adaptor protein in clathrin-mediated endocytosis. ECA1 is recruited to the membrane during salt stress signaling in plants in a phosphatidic acid (PA)-dependent manner. PA is a lipid second messenger that rapidly and transiently increases in concentration under stress stimuli. Upon an increase in PA concentration another lipid, diacylglycerol pyrophosphate (DGPP), starts to accumulate. The accumulation of DGPP is suggested to be a cue for attenuating PA signaling during stress in plants. We showed in vitro that ECA1-PA binding is modulated as a function of membrane curvature stress and charge. In this work, we investigate ECA1 binding to DGPP in comparison with PA. We show that ECA1 has more affinity for the less charged PA, and this binding is pH dependent. Additionally, plant PA binding proteins SnRK2.10, TGD2C, and PDK1-PH2 were investigated for their interaction with DGPP, since no known DGPP binding proteins are available in the literature to date. Our results shed further light on DGPP and its interactions with membrane proteins which brings us closer toward understanding the complexity of protein interactions with anionic lipids, especially the enigmatic anionic lipid DGPP.