Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP₅-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-InsP₅ remain largely unknown. Here, we employ two complementary affinity-based strategies-a matrix approach and a photoaffinity probe-to systematically identify 4/6-PP-InsP₅-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-InsP₅ in vitro with markedly higher affinity than InsP₆, while no reliable binding parameters could be obtained for its enantiomer 6-PP-InsP₅. 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-InsP₅ and establish a versatile platform for dissecting its biological roles in plants and other organisms.