Basic–liver, pancreas, and biliary tractATP7B Mediates Vesicular Sequestration of Copper: Insight Into Biliary Copper Excretion
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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2021, Biochimica et Biophysica Acta - General SubjectsCitation Excerpt :On the contrary, it appears more likely that the function of ATP7B in intestinal enterocytes is one of copper storage prior to copper export by ATP7A. In Chinese hamster ovary (CHO-K1) and Caco-2 cells, elevated copper levels cause ATP7B to redistribute from the Golgi to dispersed vesicles in the cytosol, indicative of a role for ATP7B in the sequestration of copper in intracellular vesicles [52,53]. In addition, in CHO-K1 cells that endogenously express Atp7a, stable expression of ATP7B led to increased cellular copper levels compared to WT when cells were treated with 100 CuCl2 for 24 h [53].
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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.