Role of ATP7B in biliary copper excretion in a human hepatoma cell line and normal rat hepatocytes.
OBJECTIVE
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.
Harada
M
M
Second Department of Medicine, Kurume University School of Medicine, Kurume, Japan.
Sakisaka
S
S
Terada
K
K
Kimura
R
R
Kawaguchi
T
T
Koga
H
H
Taniguchi
E
E
Sasatomi
K
K
Miura
N
N
Suganuma
T
T
Fujita
H
H
Furuta
K
K
Tanikawa
K
K
Sugiyama
T
T
Sata
M
M
D033982:Adaptor Protein Complex gamma Subunits; D000251:Adenosine Triphosphatases / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001648:Bile Canaliculi / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular; D002352:Carrier Proteins / Q000378:metabolism* / Q000502:physiology; D002402:Cathepsin D / Q000378:metabolism; D027682:Cation Transport Proteins; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D011992:Endosomes / Q000378:metabolism; D005700:Galactosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D008360:Mannosephosphates / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D015739:Nocodazole / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D051381:Rats; D051116:Receptors, Scavenger; D012795:Sialoglycoproteins / Q000378:metabolism; D014407:Tumor Cells, Cultured; D062826:Zonula Occludens-1 Protein
D033982:Adaptor Protein Complex gamma Subunits; D015703:Antigens, CD; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D051907:Lysosomal Membrane Proteins; D008360:Mannosephosphates; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D051116:Receptors, Scavenger; C074299:SCARB2 protein, human; D012795:Sialoglycoproteins; C569042:TJP1 protein, human; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D020126:Brefeldin A; C027693:mannose-6-phosphate; D003300:Copper; D005700:Galactosyltransferases; D002402:Cathepsin D; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D015739:Nocodazole