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

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 employed 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 yielded partially overlapping protein sets, with photoaffinity enrichment likely capturing additional transient and/or weak interactions. Moreover, competition experiments with different isomers were applied to obtain information about potential isomer-specific interactions. As a proof-of-concept, one candidate interactor (FHA domain-containing protein AtFHA2) was shown to bind 4-PP-InsP ₅ in vitro with substantially higher affinity than InsP ₆ . 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.