Repurposing a Lipid Droplet-Associated Algal Protein as a Biosensor for Phosphatidic Acid

Abstract

Phosphatidic acid (PA) is an essential signaling lipid in eukaryotic cells. Despite its importance, biosensors recognizing PA-rich membranes are in short supply, partly because there is no known consensus motif for detecting PA. Here, we demonstrate that a lipid droplet-associated protein domain from Chlamydomonas reinhardtii can be repurposed as a biosensor for PA-rich lipid membranes. We found that the lipid-binding domain of Delayed in TAG Hydrolysis 1 (DTH1^LBD) of Chlamydomonas reinhardtii binds to PA-rich phospholipid bilayers in vitro. Cysteine mutagenesis coupled with covalent incorporation of various environment-sensitive fluorophores and fluorescent confocal microscopy imaging of PA-containing giant unilamellar vesicles showed that the N-terminal amphipathic helices of DTH1^LBD are sufficient to recognize PA-containing membranes. Accordingly, we observed that binding of N-terminal amphipathic helices to PA-enriched membranes is sensitive to PA-induced membrane lipid packing defects. Our findings demonstrate that the N-terminal amphipathic helices of DTH1^LBD recognize PA-rich membranes through lipid headgroup and membrane packing defects imparted by the unique structure of PA. Thus, the amino acid sequences forming the N-terminal amphipathic helices of DTH1^LBD could potentially inform the design of next-generation PA-binding peptides.