Roles of transmembrane domains in Arabidopsis COPT1 activity: more than just copper coordination

Copper uptake at the plasma membrane and subsequent delivery to intracellular copper chaperons and copper-binding proteins are essential for various biological processes. In Arabidopsis, COPT1 protein functions in high affinity copper uptake at the plasma membrane. However, little is known about the mechanisms by which COPT1 modulates high affinity copper uptake. In this study, we show that residues of methionine of the ¹²¹MxxxM¹²⁵ motif in TMD2, and histidine or glycine of ¹³⁸GHxxG¹⁴² motif in TMD3 are required for copper uptake in growth complementation experiments. Fluorescence observation and protein interaction experiments demonstrate that the MxxxM motif is not essential for assembly of COPT1 homomerization unless the GxxxG motif is disrupted, but the two motifs are simultaneously required for plasma membrane-targeting pattern of COPT1. Furthermore, mutation of Met125, His139 or the GxxxG motif completely inhibited copper-mediated degradation of COPT1. Together, these results suggest that COPT1 has evolved locally different solutions to finetuning oligomerization, spatial localization, degradation and subsequent copper transport.