Spatiotemporal dynamics of AHA2 reveal that RALF1 induces its endocytosis and vacuolar degradation

Arabidopsis thaliana H⁺-ATPases (AHAs) are key plasma membrane enzymes that drive nutrient uptake and ion transport across the membrane. RAPID ALKALINIZATION FACTOR 1 (RALF1) induces phosphorylation of AHA2 and inhibits its activity. While the phosphorylation of AHA2 induced by RALF1 has been well described, the ultimate fate of AHA2 following phosphorylation remains unclear. Here, we analyzed the diffusion dynamics of GFP-AHA2 in Arabidopsis thaliana using single-particle tracking combined with variable-angle total internal reflection fluorescence microscopy. Treatment with RALF1, which causes extracellular alkalinization, markedly inhibited AHA2 activity and reduced the velocity of GFP-AHA2. RALF1 promotes the internalization and degradation of GFP-AHA2 through both clathrin-mediated and clathrin-independent endocytosis. In addition, single-particle tracking showed that phosphorylation influences the spatiotemporal dynamics of AHA2. These findings reveal a previously unreported role for RALF1 in promoting AHA2 internalization and degradation via synergistic endocytosis, offering new insights into plant signaling, environmental responses, and protein endocytosis.