The Arabidopsis SPFH protein HIR2 modulates receptor signaling and plasma membrane organization.

Plant plasma membrane proteins are organized into distinct domains; however, the principles that govern this spatial organization are not fully understood. HYPERSENSITIVE-INDUCED REACTION (HIR) proteins are plant-specific members of the stomatin/prohibitin/ flotillin/HflK/C (SPFH) family implicated in membrane organization. Arabidopsis thaliana HIR2 interacts with multiple plasma membrane proteins, including receptor kinases such as BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE (BAK1)-INTERACTING RECEPTORS 2 and 3 (BIR2 and 3) and BAK1. These interactions connect HIR2 to BAK1-mediated signaling pathways, as evidenced by impaired growth and immunity phenotypes in hir2 mutants. HIR2 is anchored to the inner leaflet of the plasma membrane through a hydrophobic domain and S-acylation. Single-particle-tracking photoactivated localization microscopy (sptPALM) revealed that HIR2 affects BAK1 mobility and clustering, exemplifying a role in regulating plasma membrane dynamics. Structural modeling with AlphaFold 3 predicts a multimeric cup-like assembly for HIR2, consistent with high molecular weight complexes identified through blue-native PAGE. These findings indicate that HIR2 contributes to the formation of membrane sub-compartments, providing a potential structural framework for spatial membrane organization that influences the dynamics and function of membrane-resident receptors.