Calcium (Ca2+) signaling is a universal mechanism of signal transduction and involves Ca2+ signal formation and decoding of information by Ca2+ binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca2+ binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca2+ signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca2+ signaling specificity.
Protein S-acylation is important for many biological processes. It confers proteins with the ability to attach to the plasma membrane and the membranes confining the ER and Golgi compartments. Yet, the contribution of S-acylation to regulating and targeting lysosomal/vacuolar proteins remains largely enigmatic. Here, we report that vacuolar targeting of the calcium sensor calcineurin B-like protein 6 (CBL6) from Arabidopsis thaliana is brought about by S-acylation of N-terminal cysteine residues. Our results suggest distinctions in mechanisms and efficiency of targeting between CBL6 and the previously characterized vacuolar-targeted CBL2 protein. Moreover, we define which CBL-interacting protein kinases (CIPKs) could interact with CBL6 and observe a remarkable temperature dependence of CBL6/CIPK complex formation. Collectively, these findings indicate a common S-acyla tion-dependent vacuolar membrane targeting pathway for proteins.
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.
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