Clathrin-mediated trafficking regulates copper tolerance by modulating the localization of HEAVY METAL ATPase 5 in Arabidopsis root cells

Abstract

Plant clathrin and its adaptor protein complexes—adaptor protein complex-1 (AP-1) at the trans-Golgi network/early endosome (TGN/EE) and the adaptor protein complex-2 (AP-2) at the plasma membrane (PM)—function in clathrin-mediated trafficking (CMT). This study reports the role of CMT in regulating copper (Cu) tolerance in plants. We found that high concentrations of exogenous Cu treatment increase the abundance of clathrin and adaptor protein complexes at the TGN/EE and/or the PM. We further found that a CMT-deficient mutants ap2μ2, clc2 clc3 exhibits hypersensitivity to Cu stress, similar to a mutant lacking the Cu transporter HEAVY METAL ATPase 5 (HMA5). As previously reported, HMA5 relocates from the endoplasmic reticulum (ER) to the PM on the soil side, where it excretes excess Cu from the root cell, which is crucial for Cu tolerance. Our protein interaction assays showed that the AP-1 and AP-2 σ subunits depend on the YXXΦ sorting motif of HMA5 for recognition. Defective AP-1 hinders HMA5 translocation to the PM after its transfer from the ER to the TGN/EE following Cu stress, while impaired AP-2 function inhibits HMA5 endocytosis at the PM. These results demonstrate that CMT mediates the endocytic recycling of HMA5 between the TGN/EE and the PM, thereby regulating Cu efflux from root cells. Our findings highlight a function of CMT in maintaining Cu homeostasis.