S-acylation and tonoplast localization of the Geminivirus Rep-Interacting Kinase/SnRK1-Activating Kinase (GRIK/SnAK) proteins differentially regulate salt and energy stress responses in Arabidopsis

SnRK1 and SnRK3.11/SOS2 are key protein kinases in plant cellular energy and salt stress signaling, respectively. Using cellular assays, we confirm that the GRIK/SnAK (Geminivirus Rep-Interacting Kinase/SnRK1-Activating Kinase) proteins act as their main activating upstream kinases in Arabidopsis, catalyzing T-loop phosphorylation on the SnRK1α1 T175 and SOS2 T168 residues. Remarkably, SnRK1α1 phosphorylation on the neighbouring S176 residue competes with GRIK-mediated T175 phosphorylation to negatively regulate SnRK1 activity. Cellular assays and transgenic plants also revealed that the GRIK proteins, via N-terminal S-acylation, are predominantly localized at the tonoplast, where they interact with SnRK1α1 and SOS2. We optimized a leaf mesophyll protoplast-based Acyl PEG Exchange (APE) protocol to further explore GRIK protein S-acylation and tonoplast recruitment and identified the amino acid residues involved. GRIK1 tonoplast localization is likely mediated by initial membrane sampling via N-terminal domain hydrophobicity and local S-acylation, independently of a secretory pathway. Finally, grik1-1 grik2-1 double KO mutants complemented with a non-S-acylatable mutant GRIK1 protein exhibit increased salt sensitivity (reduced SOS2 activity) but hyperactive SnRK1 signaling, demonstrating the differential importance of GRIK subcellular localization for Arabidopsis salt and energy stress responses. ### Competing Interest Statement The authors have declared no competing interest.