Functional assessment of the carboxy-terminus of the Wilson disease copper-transporting ATPase, ATP7B.

The carboxy-terminus of ATP7B, the protein defective in the copper-transport disorder Wilson disease, was investigated with respect to its role in copper delivery to the ferroxidase ceruloplasmin. We use yeast as a model system to assess the functional capabilities of ATP7B variants. The yeast ferroxidase, Fet3p, acquires copper from Ccc2p and cannot function if Ccc2p is impaired; expression of wild-type ATP7B in ccc2 yeast complements the iron-deficient phenotype. Our results demonstrate that the C-terminus of ATP7B is necessary for protein stability, as removal of the nonmembranous terminus leads to reduced protein levels and cessation of growth in iron-limited medium. Growth is partially restored when an additional three amino acids are present and is near wild-type levels when only one-third of the C-terminus is present. Measurement of ferroxidase activity is a more sensitive indicator of copper transport function and allowed identification of impaired variants not detected with the growth assay.