Enhanced AtAAP1 endocytosis is correlated with calcium induced-proline hyper-sensitivity in Arabidopsis

The calcium (Ca²⁺) could enhance the toxicity of exogenous proline and the production of reactive oxygen species (ROS). However, the molecular mechanism underlying calcium-enhanced proline toxicity (CEPT) is still elusive. Here, we find that CEPT depends on the presence of the amino acid permease 1 in Arabidopsis (AtAAP1), since CEPT was significantly attenuated in ataap1 mutant than in wild-type plants. Notably, the role of AtAAP1 in CEPT is not related to its primary function as an amino acid transporter, neither was the expression of AtAAP1 affected under calcium or proline treatment. Further analysis revealed that treatment with 15 mM calcium, but not with proline, significantly induced the endocytosis of AtAAP1 protein through both clathrin and membrane micro-domain pathways, which was not blocked by translation inhibitor cycloheximide. After being internalized into cells, AtAAP1 protein go through the early endosome and late endosome, as evidenced by the co-localization of AtAAP1-GFP with the markers for the early (TGN) or late endosome (PVC). These results suggested that internalized AtAAP1 protein was not destined for degradation in vacuole, because the AtAAP1-GFP were not co-localized with the marker of PVC/vacuole. Collectively, our results indicated that Ca²⁺ can promote the endocytosis of AtAAP1, playing a role in relaying a signal of high amino acid levels in the surrounding environment.