Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / metabolism
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Amino Acid Sequence
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Biological Transport
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Cation Transport Proteins / chemistry*
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Cation Transport Proteins / genetics
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Cation Transport Proteins / metabolism
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Ceruloplasmin / genetics
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Ceruloplasmin / metabolism
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Computer Simulation
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Copper / metabolism
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Copper Transport Proteins
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Copper-Transporting ATPases
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Culture Media / metabolism
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Genetic Complementation Test
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Humans
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Models, Molecular
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Molecular Sequence Data
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Mutagenesis, Site-Directed
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Plasmids
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins / genetics
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Transformation, Genetic
Substances
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CCC2 protein, S cerevisiae
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Cation Transport Proteins
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Copper Transport Proteins
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Culture Media
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Recombinant Fusion Proteins
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Saccharomyces cerevisiae Proteins
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Copper
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Ceruloplasmin
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FET3 protein, S cerevisiae
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Adenosine Triphosphatases
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ATP7B protein, human
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Copper-Transporting ATPases