Gastroenterology

Gastroenterology

Volume 130, Issue 2, February 2006, Pages 493-506
Gastroenterology

Basic–liver, pancreas, and biliary tract
ATP7B Mediates Vesicular Sequestration of Copper: Insight Into Biliary Copper Excretion

https://doi.org/10.1053/j.gastro.2005.10.054Get rights and content

Background & Aims: 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.

Section snippets

Cells and Antibodies

The human HepG2 hepatoma cell line was cultured at 37°C in Dulbecco’s modified Eagle medium (high glucose) (Trace BioSciences, Nobel Park, Victoria, Australia) supplemented with 0.2 mmol/L proline, 5% fetal calf serum (FCS), 2 mmol/L L-glutamine, 0.6 mmol/L NaHCO3, 20 mmol/L HEPES, 200 μg/mL penicillin, and 200 μg/mL streptomycin (Commonwealth Serum Laboratories, Broadmedows, Victoria, Australia). Chinese hamster ovary (CHO-K1) cells were cultured at 37°C as monolayers in Eagle basal medium

ATP7B Traffics From the TGN to Pericanalicular Vesicles and not to the Canalicular Membrane in HepG2 Cells

In HepG2 cells, the trafficking of ATP7B from the TGN has previously been shown to be stimulated by physiologically relevant levels of copper (1–20 μmol/L), with the degree of redistribution copper-dose dependent.10, 17 To demonstrate that ATP7B resides at the TGN under low copper conditions as previously shown,11, 17 the subcellular localization of ATP7B was compared with that of the TGN resident protein p230. The p230 protein localizes to the cytosolic-facing peripheral membrane of the TGN

Discussion

There have been contradictory reports in the literature regarding the subcellular localization of ATP7B and the effect of copper on its localization.11, 12, 17, 36 Clarification of this issue is essential to elucidate the mechanism of copper efflux from hepatocytes into the biliary canaliculus (bile). Here, we studied the copper-dependent subcellular localization of endogenous ATP7B in the HepG2 human hepatoma cell line as a model of hepatocytes. HepG2 cells retain the biosynthetic capability

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    Supported by a National Health and Medical Research Council R. Douglas Wright Fellowship (to S.L.F.) and in part by the National Health and Medical Research Council of Australia, the Australian Research Council, and the International Copper Association.

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