A predicted amphipathic helix contributes to the efficient S-acylation of the Arabidopsis thaliana Calcineurin B-like protein 2

Abstract

Membrane targeting of the Calcineurin B-like (CBL) calcium sensor proteins through protein S-acylation is crucial for various processes in plants, like nutrient uptake, plant development, and response to abiotic and biotic stresses. Certain CBLs target specifically to the vacuolar membrane, but which factors contribute to this particular localization and to the lipid modification efficiency are not yet known. Here, we examined the structural features of the N-terminus of Arabidopsis thaliana CBL2 and show that the lipid-modified cysteines are integrated within a predicted amphipathic helix. Mutations of amino acids, which contribute to the formation of this specific domain, affect S-acylation efficiency, membrane binding and function of CBL2. Interestingly, overexpression of the protein S-acyl transferase (PAT) 10 can compensate for the binding deficiency of a CBL2 mutant variant, which harbours a helix breaker mutation. This indicates that helix formation is rather involved in the S-acylation mechanism and is less important for membrane binding. Moreover, the introduction of basic residues resulted in a partial shift of the protein from the vacuolar to the plasma membrane, indicating that the underrepresentation of positively charged amino acids contributes to the vacuolar targeting specificity. Overall, our data suggest that helix formation is potentially an initial step in the S-acylation process and provides a deeper understanding of the mechanistic interplay between PATs and tonoplast targeted CBLs.