Redox state and cellular uptake of copper is regulated by N-terminus of human Copper Transporter-1

Copper(I) is essential for all life forms. Though Cu(II) is the most abundant state in environment, its reduction to Cu(I) is a prerequisite for bio-utilization, the mechanism of which is still uncharacterized. We show that in the human Copper Transporter-1, two amino-terminal methionine-histidine clusters and neighbouring aspartates distinctly binds Cu(II) to Cu(I) preceding its import. The endocytosis of hCTR1 from the basolateral membrane of polarized epithelia to Common-Recycling-Endosomes is dependent on copper reduction and coordination of Cu(I) by methionine residues. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by aspartates also acts as another crucial determinant of hCTR1 endocytosis. Mutating 7Met-Gly-Met9 and Asp13 abrogates copper uptake and endocytosis, which was corrected by reduced and non-reoxidizable Cu(I). Histidine motifs, on the other hand, are crucial for hCTR1 functioning at limiting copper. Finally, we show that the two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. This study proposes a model where His-Met-Asp residues of hCTR1 amino-terminal not only coordinate copper but also maintains its reduced state crucial for intracellular uptake.