Abstract
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.
Highlights
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
S. cerevisiae was instrumental in the discovery of copper transporter 1 (Ctr1),3 a homotrimeric plasma membranespanning protein expressed in eukaryotes and responsible for the uptake of extracellular copper [8, 9]
In an effort to understand the origins of mammalian Ctr2, 125 Ctr protein sequences from a wide variety of eukaryotic organisms were analyzed via a molecular phylogenetic approach
Summary
DuPont Pioneer, Johnston, IA 50131. 2 To whom correspondence should be addressed: Dept. of Pharmacology and Cancer Biology, Duke University School of Medicine, Box 3813, Durham, NC 27710. Ctr contains a large extracellular amino-terminal metal-binding domain rich in methionine residues that facilitates high-affinity Cuϩ transport through the transmembrane pore [10]. Lining the transmembrane pore is an M-X3-M motif present on transmembrane domain two that is proposed to function as a Cuϩ selectivity filter [11, 12]. Mutation of these residues to leucine results in a protein incapable of Cuϩ transport. The third transmembrane domain contains a glycine zipper motif that is required for multimerization of Ctr monomers into functional Cuϩ-transporting homotrimers [13, 14]. Other fungi possess redundant high-affinity Cuϩ transporters, such as Ctr and Ctr from Schizosaccharo-
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