Spatio-temporal dynamics of AHA2 reveals that RAFL1 induces its endocytosis and vacuolar degradation.

Plasma membrane H⁺-ATPases (AHAs) are master enzymes of plant biology, driving the absorption of cellular nutrients and ion transport across the cell membrane. Here, we analyzed the diffusion dynamics of GFP-AHA2 in Arabidopsis thaliana by combining single-particle tracking with variable-angle total internal reflection fluorescence microscopy. Treatment with RALF1, which caused extracellular alkalinization, markedly inhibited AHA2 activity and decreased the velocity of GFP-AHA2. RALF1 promoted the internalization and degradation of GFP-AHA2 via both clathrin-mediated endocytosis (CME) and clathrin-independent endocytosis (CIE). Moreover, Single-particle tracking revealed that phosphorylation affected AHA2 spatiotemporal dynamics. Our findings identify a previously unreported role for RALF1 in promoting AHA2 internalization and degradation by synergistic endocytosis under RALF1 treatment, providing insights that can broadly impact research into plant signaling, environmental responses, and protein endocytosis.