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 markedly higher affinity than InsP6, while no reliable binding parameters could be obtained for its the enantiomeric isomer 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.

INSTRUMENT(S):

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell

SUBMITTER: Sara Christina Stolze  

LAB HEAD: Hirofumi Nakagami

PROVIDER: PXD073760 | Pride | 2026-06-08

REPOSITORIES: Pride