Affinity‐Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6‐PP‐InsP 5 ‐Binding Proteins in Plants

ABSTRACT Inositol pyrophosphates (PP‐InsPs) are central regulators of eukaryotic signaling events. While certain PP‐InsP isomers have been conclusively linked to the regulation of phosphate homeostasis through interaction with SPX domain‐containing proteins in plants, the functions of the recently discovered isomer 4/6‐PP‐InsP 5 remain largely unknown. Here, we employ two complementary affinity‐based strategies—a matrix approach and a photoaffinity probe—to systematically identify 4/6‐PP‐InsP 5 ‐binding proteins in Arabidopsis thaliana . The two methods yield partially overlapping protein sets, with photoaffinity enrichment likely capturing additional transient and/or weak interactions. Moreover, competition experiments with different isomers are applied to obtain information about potential isomer‐specific interactions. As a proof‐of‐concept, one candidate interactor (FHA domain‐containing protein AtFHA2) is shown to bind 4‐PP‐InsP 5 in vitro with markedly higher affinity than InsP 6 , while no reliable binding parameters could be obtained for its enantiomer 6‐PP‐InsP 5 . Thus, besides the SPX domain, FHA domain‐containing proteins, of which 18 exist in Arabidopsis, are potentially regulated by inositol pyrophosphates. More generally, our findings reveal a diverse protein network associated with 4/6‐PP‐InsP 5 and establish a versatile platform for dissecting its biological roles in plants and other organisms.