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

Copper(I) is essential for all life forms. Though Cu(II) is most abundant state in environment, its reduction to Cu(I) is prerequisite for bio-utilization, by a mechanism that is uncharacterized. We show that in human Copper Transporter-1, two amino-terminal methionine-histidine clusters and neighbouring aspartates distinctly binds Cu(II) and Cu(I) preceding its import. The endocytosis of hCTR1 from basolateral membrane of polarized epithelia to Common-Recycling-Endosomes is dependent on copper reduction and Cu(I) coordination by methionines. The transient binding of both Cu(II) and Cu(I) during the reduction process facilitated by aspartates acts as another crucial determinant of hCTR1 endocytosis. Mutating 7Met-Gly-Met9 and Asp13 abrogates copper uptake and endocytosis that is correctable by reduced and non-reoxidizable Cu(I). Histidines clusters are crucial for hCTR1 functioning at limiting copper. Finally, we show that two N-terminal His-Met-Asp clusters exhibit functional complementarity in regulating Cu(I)-induced hCTR1 endocytosis. We propose a mechanistic model where His-Met-Asp residues of amino-terminal hCTR1 coordinates copper and maintains its reduced state crucial for uptake.