Significance Copper is essential for normal growth and development because it serves roles in catalysis, signaling, and structure. Cells acquire copper through the copper transporter 1 (Ctr1) protein, a copper transporter that localizes to the cell membrane and intracellular vesicles. Both copper and the anticancer drug cisplatin are imported by Ctr1 by virtue of an extracellular domain rich in metal-binding amino acids. In this report we demonstrate that a protein structurally related to Ctr1, called Ctr2, plays a role in the generation or stability of a truncated form of Ctr1 lacking a large portion of the extracellular domain. Retention of this domain in mice or cells lacking Ctr2 enhances copper and cisplatin uptake, thereby establishing Ctr2 as a regulator of Ctr1 function.

Copper is an essential trace element that functions in a diverse array of biochemical processes that include mitochondrial respiration, neurotransmitter biogenesis, connective tissue maturation, and reactive oxygen chemistry. The Ctr1 protein is a high-affinity Cu ؉ importer that is structurally and functionally

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Copper is an essential element for proper organismal development and is involved in a range of processes, including oxidative phosphorylation, neuropeptide biogenesis, and connective tissue maturation. The copper transporter (Ctr) family of integral membrane proteins is ubiquitously found in eukaryotes and mediates the high-affinity transport of Cu across both the plasma membrane and endomembranes. Although mammalian Ctr1 functions as a Cu transporter for Cu acquisition and is essential for embryonic development, a homologous protein, Ctr2, has been proposed to function as a low-affinity Cu transporter, a lysosomal Cu exporter, or a regulator of Ctr1 activity, but its functional and evolutionary relationship to Ctr1 is unclear. Here we report a biochemical, genetic, and phylogenetic comparison of metazoan Ctr1 and Ctr2, suggesting that Ctr2 arose over 550 million years ago as a result of a gene duplication event followed by loss of Cu transport activity. Using a random mutagenesis and growth selection approach, we identified amino acid substitutions in human and mouse Ctr2 proteins that support copper-dependent growth in yeast and enhance copper accumulation in Ctr1 mouse embryonic fibroblasts. These mutations revert Ctr2 to a more ancestral Ctr1-like state while maintaining endogenous functions, such as stimulating Ctr1 cleavage. We suggest key structural aspects of metazoan Ctr1 and Ctr2 that discriminate between their biological roles, providing mechanistic insights into the evolutionary, biochemical, and functional relationships between these two related proteins.