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Summary Abscisic acid (ABA) receptors belong to theSTARTdomain superfamily, which encompasses ligand‐binding proteins present in all kingdoms of life.STARTdomain proteins contain a central binding pocket that, depending on the protein, can couple ligand binding to catalytic, transport or signaling functions. In Arabidopsis, the best characterizedSTARTdomain proteins are the 14PYR/PYL/RCAR ABAreceptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently inNicotiana benthamianacoupled to untargetedLC‐MSto identify candidate binding ligands. We optimized this method usingABA–PYLinteractions and show thatABAco‐purifies with wild‐typePYL5 but not a binding site mutant. TheK_dofPYL5 forABAis 1.1 μm, which suggests that the method has sufficient sensitivity for many ligand–protein interactions. Using this method, we surveyed a set of 37STARTdomain‐related proteins, which resulted in the identification of ligands that co‐purified withMLBP1 (At4G01883) orMLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed thatMLBP1 binds to monolinolenin, which we confirmed using recombinantMLBP1. Monolinolenin also co‐purified withMLBP1 purified from transgenic Arabidopsis, demonstrating that the interaction occurs in a native context. Thus, deployment of this relatively simple method allowed us to define a protein–metabolite interaction and better understand protein–ligand interactions in plants.