Harada M, Sakisaka S, Terada K, Kimura R, Kawaguchi T, Koga H, Taniguchi E, Sasatomi K, Miura N, Suganuma T, Fujita H, Furuta K, Tanikawa K, Sugiyama T, Sata M
Second Department of Medicine, Kurume University School of Medicine, Kurume, Japan.
Gastroenterology. 2000 May;118(5):921-8. doi: 10.1016/s0016-5085(00)70178-8.
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.
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.
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.
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.
威尔逊病是一种遗传性疾病，其特征是由于胆汁铜排泄缺陷导致体内铜蓄积。威尔逊病基因已被克隆；然而，该基因产物（ATP7B）的确切定位及其在胆汁铜排泄中的作用尚未阐明。
我们构建了绿色荧光蛋白（GFP）与ATP7B之间的嵌合蛋白（GFP-ATP7B），并在人肝癌细胞系（Huh7）和分离的大鼠肝细胞中表达。通过荧光显微镜观察高尔基体、晚期内体、溶酶体和胆小管。使用布雷菲德菌素A和诺考达唑重新分布高尔基体蛋白。使用巴弗洛霉素A1分析GFP-ATP7B与晚期内体之间的关联。
GFP-ATP7B与罗丹明-葡聚糖和晚期内体标记物共定位，但不与高尔基体标记物、溶酶体标记物或紧密连接蛋白共定位。布雷菲德菌素A和诺考达唑重新分布了高尔基体蛋白，但它们不影响ATP7B的分布。
尽管普遍认为ATP7B位于高尔基体，但它的主要定位是在晚期内体。ATP7B似乎将铜从细胞质转运到晚期内体腔，从而通过溶酶体参与胆汁铜排泄。
