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.