Gastroenterology

Gastroenterology

Volume 118, Issue 5, May 2000, Pages 921-928
Gastroenterology

Liver, Pancreas, and Biliary Tract
Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes,☆☆

https://doi.org/10.1016/S0016-5085(00)70178-8Get rights and content

Abstract

Background & Aims: Wilson's disease is a genetic disorder characterized by the accumulation of copper in the body caused by a defect of biliary copper excretion. The Wilson's disease gene has been cloned; however, the precise localization of the gene product (ATP7B) and its role in biliary copper excretion have not been clarified. Methods: We constructed a chimeric protein between green fluorescent protein (GFP) and ATP7B (GFP-ATP7B) and expressed it in a human hepatoma cell line (Huh7) and isolated rat hepatocytes. The Golgi apparatus, late endosomes, lysosomes, and bile canaliculus were visualized by fluorescence microscopy. Brefeldin A and nocodazole were used to redistribute the Golgi proteins. Bafilomycin A1 was used to analyze the association between GFP-ATP7B and the late endosomes. Results: GFP-ATP7B colocalized with rhodamine-dextran and late endosome markers but not with the Golgi markers, lysosome markers, or a tight junction protein. Brefeldin A and nocodazole redistributed the Golgi proteins, but they did not affect the distribution of ATP7B. Conclusions: Although it is widely believed that ATP7B is located at the Golgi apparatus, its main localization is in late endosomes. ATP7B seems to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes.

GASTROENTEROLOGY 2000;118:921-928

Section snippets

Cells

Monolayer Huh7 cells were cultured in RPMI 1640 (Life Technologies, New York, NY) supplemented with 10% fetal calf serum (Wako Pure Chemical Industries, Ltd., Osaka, Japan), penicillin (100 U/mL, crystalline penicillin G Meiji; Meiji Seika Kaisya, Tokyo, Japan), and streptomycin (0.1 mg/mL; Meiji Seika Kaisya).

IRHs were obtained from male Sprague–Dawley rats weighing 200–250 g (8–10 weeks of age). Rats received humane care in accordance with the guidelines of Kurume University (Kurume, Japan,

Results

The immunofluorescent signals of endogenous and transfected ATP7B showed juxtanuclear patterns in Huh 7 cells (Figure 1A and B).

. Confocal laser scanning microscopic images of Huh 7 cells and IRHs. (A) Nontransfected and (B) ATP7B-transfected Huh 7 cells were labeled with anti-ATP7B antibody and visualized with fluorescein isothiocyanate–secondary antibody. GFP-ATP7B was transfected in (C) Huh 7 cells and (D) IRHs. (E ) pEGFP-C2 was transfected in Huh 7 cells. ATP7B and GFP-ATP7B distributed in

Discussion

This study shows that ATP7B, a copper transporter, localizes in the endocytic compartments, especially in the late endosomes in hepatocytes.

We constructed a chimeric GFP-ATP7B protein, yielding bright green fluorescence without any cofactors, for examining the intracellular localization. We did this for the following reasons. First, the intensity of fluorescent signals obtained from endogenous ATP7B in Huh7 cells by indirect immunofluorescent study is not sufficient in comparison with those of

Acknowledgements

The authors thank Dr. B. Hoflack for providing the anti-MPR polyclonal antibody, Dr. J. T. August for providing the anti-lamp1 and anti-lamp2 antibodies, and M. Inayoshi and K. Maeda for expert technical assistance.

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      This localization is consistent with their function in cuproenzyme biosynthesis, as several cuproenzymes are synthesized within the secretory pathway. Some controversy exists about the localization of ATP7B, as it has also been suggested that this protein resides in an endosomal compartment [145,146]. In addition, a smaller isoform of ATP7B exists, which has been localized to mitochondria [155].

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    Address requests for reprints to: Masaru Harada, M.D., Second Department of Medicine, Kurume University School of Medicine, 67 Asahi-Machi, Kurume 830-0011, Japan.

    ☆☆

    Supported in part by a grant from the alumni association of Kurume University School of Medicine (to M.H.).

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