The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.

Intracellular copper (Cu) in eukaryotic organisms is regulated by homeostatic systems, which rely on the activities of soluble metallochaperones that participate in Cu exchange through highly tuned protein-protein interactions. Recently, the human enzyme glutaredoxin-1 (hGrx1) has been shown to possess Cu metallochaperone activity. The aim of this study was to ascertain whether hGrx1 can act in Cu delivery to the metal binding domains (MBDs) of the P 1B-type ATPase ATP7B and to determine the thermodynamic factors that underpin this activity. hGrx1 can transfer Cu to the metallochaperone Atox1 and to the MBDs 5-6 of ATP7B (WLN5-6). This exchange is irreversible. In a mixture of the three proteins, Cu is delivered to the WLN5-6 preferentially, despite the presence of Atox1. This preferential Cu exchange appears to be driven by both the thermodynamics of the interactions between the proteins pairs and of the proteins with Cu(I). Crucially, protein-protein interactions between hGrx1, Atox1 and WLN5-6 were detected by NMR spectroscopy both in the presence and absence of Cu at a common interface. This study augments the possible activities of hGrx1 in intracellular Cu homeostasis and suggests a potential redundancy in this system, where hGrx1 has the potential to act under cellular conditions where the activity of Atox1 in Cu regulation is attenuated. Copper (Cu) is a redox active metal and an essential trace element for human health 1,2. In biological systems, Cu is found in two oxidation states, reduced Cu(I) (cuprous) and oxidized Cu(II) (cupric). These reversible states, which allow Cu to serve as both an oxidant and reductant, render it an exceptional cofactor for redox enzymes known as oxidoreductases 1,2 , however excess intracellular concentrations of Cu are toxic and lead to cellular oxidative damage 3,4. In particular, the reaction of hydrogen peroxide with Cu(I) and the reduction of Cu(II) by superoxide, produce hydroxyl radicals that damage proteins, nucleic acids and lipids 5,6. In humans, the dysregulation of Cu metabolism is associated with diseases such as Menkes syndrome, Wilson's disease, prion diseases, Alzheimer's disease and fatal motor neuron diseases such as familial amyotrophic lateral sclerosis 7-12. To maintain the fine balance between cellular Cu requirements and toxicity, organisms have evolved sophisticated metalloregulatory systems for the coordination of metals such as Cu to proteins within the cell, and the controlled transfer of these metals between protein partners. Soluble proteins that are responsible for Cu binding and transfer are termed 'metallochaperones' 3 , which transport Cu in its reduced state (Cu(I)), often via coordination with cysteine residues 13,14. In eukaryotes, including humans, Cu concentrations are regulated through control of the levels of the Cu import protein, copper transporter 1 (Ctr1) 15,16 at the plasma membrane and the trafficking and/or activity of the Cu export proteins, ATP7A or ATP7B 17-19. In humans, ATP7A (Menkes disease protein, MNK) and ATP7B (Wilso...

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