ATP7B mediates vesicular sequestration of copper: insight into biliary copper excretion.
OBJECTIVE
The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper.


METHODS
Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins.


RESULTS
In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane.


CONCLUSIONS
This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.
Cater
Michael A
MA
Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Deakin University, Burwood, Australia.
La Fontaine
Sharon
S
Shield
Kristy
K
Deal
Yolanda
Y
Mercer
Julian F B
JF
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001646:Bile / Q000378:metabolism*; D001648:Bile Canaliculi / Q000378:metabolism; D016466:CHO Cells; D006528:Carcinoma, Hepatocellular; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D006224:Cricetinae; D005089:Exocytosis; D006801:Humans; D008113:Liver Neoplasms; D016296:Mutagenesis; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection
D027682:Cation Transport Proteins; D011994:Recombinant Proteins; D003300:Copper; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases