Biochemical characterization of the Wilson disease protein and functional expression in the yeast Saccharomyces cerevisiae.
Wilson disease is a disorder of copper metabolism characterized by hepatic cirrhosis and neuronal degeneration due to inherited mutations in a gene encoding a putative copper-transporting P-type ATPase. Polyclonal antisera generated against the amino terminus of the Wilson protein detected a specific 165-kDa protein in HepG2 and CaCo cell lysates. Further analysis revealed that this protein is synthesized as a single-chain polypeptide and localized to the trans-Golgi network under steady state conditions. An increase in the copper concentration resulted in the rapid movement of this protein to a cytoplasmic vesicular compartment. This copper-specific cellular redistribution of the Wilson protein is a reversible process that occurs independent of a new protein synthesis. Expression of the wild-type but not mutant Wilson protein in the ccc2Delta strain of Saccharomyces cerevisiae restored copper incorporation into the multicopper oxidase Fet3p, providing direct evidence of copper transport by the Wilson protein. Taken together these data reveal a remarkable evolutionary conservation in the cellular mechanisms of copper metabolism and provide a unique model for the regulation of copper transport into the secretory pathway of eucaryotic cells.
Hung
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Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Suzuki
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Yamaguchi
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Yuan
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Klausner
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Gitlin
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D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D001693:Biological Transport, Active; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D027682:Cation Transport Proteins; D002451:Cell Compartmentation; D002460:Cell Line; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D019084:Fluorescent Antibody Technique, Indirect; D005656:Fungal Proteins / Q000378:metabolism; D005816:Genetic Complementation Test; D006056:Golgi Apparatus / Q000378:metabolism; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
C092974:CCC2 protein, S cerevisiae; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases