Evolutionarily conserved BON1 regulates the basal cytosolic Ca2+ level by calmodulin-independent activation of Ca2+ pumps in Arabidopsis

Plasma membrane-localized autoinhibited Ca2+ pumps are essential for maintaining basal cytosolic Ca2+ levels for regulating growth processes and environmental responses. These pumps are known to be activated by calmodulins to maintain Ca2+ homeostasis in plants and animals. Here, we demonstrate that the evolutionarily conserved copine protein BON1 is critical for maintaining low cytosolic Ca2+ concentrations by directly regulating two plasma membrane-localized Ca2+ pumps ACA8 and ACA10 inArabidopsis. BON1 interacts with a region within the N-terminal domain of ACA8 and ACA10, preceding the calmodulin binding sites, and stimulates ACA8 activity. This activation can occur without calmodulin binding, indicating that BON1 and calmodulin independently regulate the Ca2+ pump. Loss of BON1 function results in elevated basal cytosolic Ca2+ concentrations, which can be partially rescued by overexpressing hyperactive ACA8 or ACA10. Furthermore, we show that BON1 has one high-affinity Ca2+ binding in the VWA domain that is critical for activation of ACA8 as well as for BON1 function, suggesting a feedback mechanism for Ca2+ homeostasis at resting concentrations. Our findings suggest that this Ca2+ responsive regulatory mechanism extends beyond Arabidopsis, as we show interactions between ACA and BON proteins from algae flowering plants, pointing to an ancient regulatory mechanism for maintaining basal cytosolic Ca2+. Notably, a human plasma membrane-localized autoinhibited Ca2+ pump can also be activated by a human BON protein in a yeast functional assay system, suggesting evolutionary conservation in Ca2+ regulation across species.