Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes.
Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.
Lörinczi
Eva
E
Max-Planck-Institute of Biophysics, D-60438 Frankfurt am Main, Germany.
Tsivkovskii
Ruslan
R
Haase
Winfried
W
Bamberg
Ernst
E
Lutsenko
Svetlana
S
Friedrich
Thomas
T
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D000073840:Copper-Transporting ATPases; D005614:Freeze Fracturing; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008024:Ligands; D008954:Models, Biological; D009865:Oocytes / Q000201:enzymology* / Q000648:ultrastructure; D010446:Peptide Fragments; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D014981:Xenopus
D027682:Cation Transport Proteins; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008024:Ligands; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C475510:tyrosyl-prolyl-tyrosyl-aspartyl-valyl-prolyl-aspartyl-tyrosyl-alanine; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases