Abstract

The essential nutrient copper is toxic in excess. Therefore, plants must tightly control copper uptake and distribution. Arabidopsis thaliana high-affinity copper transporters (COPTs) mediate copper uptake, partitioning, and redistribution. Here we show that COPT1 localizes to the plasma membrane and endoplasmic reticulum in stably transgenic plants expressing a COPT1–green fluorescent protein (GFP) fusion protein, and the fusion protein is rapidly degraded upon plant exposure to excess copper. MG132 treatment largely abolished copper-induced degradation of COPT1, implying a link between the proteasome and COPT1 activity in modulating copper uptake. Co-immunoprecipitation analyses revealed that COPT1 cannot be ubiquitinated in the presence of excess copper and MG132. Through site-directed mutagenesis, we identified Lys159 in the C-terminal cytoplasmic tail of COPT1 as critical for copper acquisition, but not for copper-mediated down-regulation of COPT1, in plants. Furthermore, pharmacological analysis showed that treatment with a vesicle trafficking inhibitor or a V-ATPase inhibitor does not alter the subcellular dynamics of COPT1–GFP, consistent with the absence of a connection between the endosomal recycling/vacuolar system and COPT1 degradation. Together, our data suggest that proteasomal degradation rather than vacuolar proteolysis is important for the regulation of copper transport to maintain copper homeostasis in plants.