4.8 Article

Optimized small-molecule pull-downs define MLBP1 as an acyl-lipid-binding protein

期刊

PLANT JOURNAL
卷 98, 期 5, 页码 928-941

出版社

WILEY
DOI: 10.1111/tpj.14272

关键词

abscisic acid; ABA signaling; PYR; PYL; RCAR; protein-metabolite interactions; in planta; START domain; Bet v 1; technical advance; polyketide cyclase-like proteins; technical advance

资金

  1. ISF Israel Science Foundation [1069/14]
  2. BARD [IS-4919-16 R]
  3. NSF [1022378, 1656890]
  4. Direct For Biological Sciences [1656890, 1022378] Funding Source: National Science Foundation
  5. Division Of Integrative Organismal Systems [1656890] Funding Source: National Science Foundation
  6. Div Of Molecular and Cellular Bioscience [1022378] Funding Source: National Science Foundation

向作者/读者索取更多资源

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 K-d of PYL5 for ABA is 1.1 mu m, 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.

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