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

Genomics. 2004 Mar;83(3):473-81. doi: 10.1016/j.ygeno.2003.08.022.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Sequence
  • Biological Transport
  • Cation Transport Proteins / chemistry*
  • Cation Transport Proteins / genetics
  • Cation Transport Proteins / metabolism
  • Ceruloplasmin / genetics
  • Ceruloplasmin / metabolism
  • Computer Simulation
  • Copper / metabolism
  • Copper Transport Proteins
  • Copper-Transporting ATPases
  • Culture Media / metabolism
  • Genetic Complementation Test
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Plasmids
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics
  • Transformation, Genetic

Substances

  • CCC2 protein, S cerevisiae
  • Cation Transport Proteins
  • Copper Transport Proteins
  • Culture Media
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Copper
  • Ceruloplasmin
  • FET3 protein, S cerevisiae
  • Adenosine Triphosphatases
  • ATP7B protein, human
  • Copper-Transporting ATPases