Optimized small‐molecule pull‐downs defineMLBP 1 as an acyl‐lipid‐binding protein
| Autor: | Assaf Mosquna, Maayan Gal, Yelena Sterlin, Jiorgos Kourelis, Sean R. Cutler, Julius Ben-Ari, Gil Zimran, Sang-Youl Park, Inge Verstraeten, Oded Pri-Tal |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Linolenic Acids Mutant Arabidopsis Nicotiana benthamiana Context (language use) Plant Science Fatty Acid-Binding Proteins Ligands 01 natural sciences 03 medical and health sciences Plant Growth Regulators Affinity chromatography Genetics Binding site biology Arabidopsis Proteins fungi Intracellular Signaling Peptides and Proteins food and beverages Cell Biology Plants Genetically Modified biology.organism_classification Small molecule 030104 developmental biology Biochemistry Biotinylation Abscisic Acid Protein Binding Signal Transduction 010606 plant biology & botany |
| Zdroj: | The Plant Journal. 98:928-941 |
| ISSN: | 1365-313X 0960-7412 |
| DOI: | 10.1111/tpj.14272 |
| Popis: | Abscisic acid (ABA) receptors belong to the START domain superfamily, which encompasses ligand-binding proteins present in all kingdoms of life. START domain 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 characterized START domain proteins are the 14 PYR/PYL/RCAR ABA receptors, while the other members of the superfamily do not have assigned ligands. To address this, we used affinity purification of biotinylated proteins expressed transiently in Nicotiana benthamiana coupled to untargeted LC-MS to identify candidate binding ligands. We optimized this method using ABA-PYL interactions and show that ABA co-purifies with wild-type PYL5 but not a binding site mutant. The Kd of PYL5 for ABA is 1.1 μm, which suggests that the method has sufficient sensitivity for many ligand-protein interactions. Using this method, we surveyed a set of 37 START domain-related proteins, which resulted in the identification of ligands that co-purified with MLBP1 (At4G01883) or MLP165 (At1G35260). Metabolite identification and the use of authentic standards revealed that MLBP1 binds to monolinolenin, which we confirmed using recombinant MLBP1. Monolinolenin also co-purified with MLBP1 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. |
| Databáze: | OpenAIRE |
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