title: Defective cellular localization of mutant ATP7B in Wilson's disease patients and hepatoma cell lines.
abstract: OBJECTIVE
  Wilson's disease, a hereditary disorder caused by mutations in the Wilson's disease gene (ATP7B), leads to hepatic and/or neurological pathology resulting from cellular copper overload. In vitro studies showed that ATP7B, located in the trans-Golgi network, traffics to a cytoplasmic vesicular compartment in response to increased copper concentration. Mislocalization and failed intracellular trafficking of ATP7B mutants are suggested to be among disease-causing mechanisms; however, the effect of mutations on ATP7B localization in human tissues has not been directly shown. Therefore, we characterized the subcellular localization of normal and mutant ATP7B in human livers and in hepatoma cell lines.
  
  
  METHODS
  Subcellular distribution of ATP7B in liver tissue from 3 control individuals and 3 Wilson's disease patients harboring a homozygous H1069Q-ATP7B mutation was analyzed by using immunogold electron microscopy. In addition, 14 ATP7B mutants tagged to green fluorescent protein were generated and expressed in HuH-7 and HepG2 cells; intracellular localization of these mutants was characterized by confocal microscopy.
  
  
  RESULTS
  In hepatocytes, ATP7B was localized in trans-Golgi vesicles, whereas H1069Q-ATP7B was trapped in the endoplasmic reticulum. Similar results were observed for wild-type ATP7B and H1069Q-ATP7B expressed in hepatoma cells. Most ATP7B proteins harboring missense mutations were distributed similarly to wild-type ATP7B. In contrast, truncated ATP7B mutants showed a diffuse, clustered, cytoplasmic pattern, distinct from the trans-Golgi network or endoplasmic reticulum.
  
  
  CONCLUSIONS
  These results provide a detailed demonstration of the ATP7B distribution in control and diseased human livers and indicate that several Wilson's disease mutations lead to incorrect localization of ATP7B to distinct cell compartments.
authors.affiliation: Department of Internal Medicine II, University of Leipzig, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Huster
authors.forename: Michael
authors.initials: M
authors.lastname: Hoppert
authors.forename: Svetlana
authors.initials: S
authors.lastname: Lutsenko
authors.forename: Jan
authors.initials: J
authors.lastname: Zinke
authors.forename: Claudia
authors.initials: C
authors.lastname: Lehmann
authors.forename: Joachim
authors.initials: J
authors.lastname: Mössner
authors.forename: Frieder
authors.initials: F
authors.lastname: Berr
authors.forename: Karel
authors.initials: K
authors.lastname: Caca
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000648:ultrastructure; D027682:Cation Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D049452:Green Fluorescent Proteins; D022781:Hepatocytes / Q000378:metabolism / Q000648:ultrastructure; D006527:Hepatolenticular Degeneration / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D008099:Liver / Q000378:metabolism / Q000648:ultrastructure; D008113:Liver Neoplasms / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012016:Reference Values; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D027682:Cation Transport Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases