title: Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B).
abstract: The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hepatocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular trafficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mutation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the constitutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis.
authors.affiliation: Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, 221 Burwood Highway, Burwood, VIC 3125, Australia.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Cater
authors.forename: Sharon
authors.initials: S
authors.lastname: La Fontaine
authors.forename: Julian F B
authors.initials: JF
authors.lastname: Mercer
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D003545:Cysteine; D017931:DNA Primers; D018698:Glutamic Acid / Q000378:metabolism; D005998:Glycine; D006527:Hepatolenticular Degeneration / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D013912:Threonine
chemical_list: D027682:Cation Transport Proteins; D017931:DNA Primers; D009842:Oligopeptides; D013912:Threonine; D018698:Glutamic Acid; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D003545:Cysteine; D005998:Glycine