Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP5-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-InsP5 remain largely unknown. Here, we employed 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 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-InsP5 in vitro with substantially higher affinity than InsP6. 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.