Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP₅-Binding Proteins in Plants

Area of Science:

• Plant molecular biology
• Cell signaling
• Biochemistry

Background:

• Inositol pyrophosphates (PP-InsPs) are crucial eukaryotic signaling molecules.
• While some PP-InsPs regulate phosphate homeostasis via SPX proteins, the function of 4/6-PP-InsP5 is largely unknown.
• Understanding 4/6-PP-InsP5 interactions is key to elucidating its biological roles.

Purpose of the Study:

• To systematically identify proteins that bind to 4/6-PP-InsP5 in Arabidopsis thaliana.
• To investigate potential isomer-specific interactions of PP-InsPs.
• To explore novel regulatory roles of 4/6-PP-InsP5 in plant signaling.

Main Methods:

• Employed two complementary affinity-based strategies: a matrix approach and a photoaffinity probe.
• Utilized competition experiments with different PP-InsP isomers to assess specificity.
• Performed in vitro binding assays to validate candidate interactions, exemplified by AtFHA2.

Main Results:

• Identified partially overlapping sets of 4/6-PP-InsP5-binding proteins using the two methods.
• Photoaffinity enrichment captured potentially transient or weak interactions.
• Demonstrated that the FHA domain protein AtFHA2 binds 4-PP-InsP5 with higher affinity than InsP6.

Conclusions:

• Revealed a diverse protein network associated with 4/6-PP-InsP5 in plants.
• Identified FHA domain-containing proteins as potential novel interactors and targets of inositol pyrophosphate regulation.
• Established a platform for further investigation into the biological functions of 4/6-PP-InsP5 in plants and other organisms.