Copper-dependent trafficking of Wilson disease mutant ATP7B proteins

We have previously developed a functional assay in yeast for the copper tra nsporter, ATP7B, defective in Wilson disease (WND). Analysis of WND variant ATP7B proteins revealed that several were able to completely, or nearly co mpletely, complement a mutant yeast strain in which the ATP7B ortholog CCC2 was disrupted, indicating that these ATP7B proteins retained copper transp ort activity. We analyzed the intracellular localization of these active WN D ATP7B variant proteins using transient transfection of Chinese hamster ov ary cells and triple-label immunofluorescence microscopy, as a second possi ble aspect of defective function. Two ATP7B variants, Asp765Asn and Leu776V al, which have normal copper transport activity in yeast, retained partial normal Golgi network localization, but were predominantly mislocalized thro ughout the cell. Asp765Asn and Leu776Val proteins were capable of only part ial copper-dependent redistribution. WND variant protein Arg778Leu, which h as defective function in yeast, was extensively mislocalized, presumably to the endoplasmic reticulum. ATP7B variant proteins Gly943Ser, which has nea rly normal function in yeast, and CysProCys/Ser (mutation of the conserved CysProCys motif to SerProSer), inactive in yeast, were localized normally b ut were unable to redistribute in response to copper. Localization data fro m this study, combined with functional data from our yeast studies, provide a biochemical mechanism that can explain in part the variable biochemical features of WND, in particular the normal holoceruloplasmin levels observed in some patients. Our data have direct implications for WND diagnosis, ind icating that decreased serum ceruloplasmin concentration is not likely to b e observed with certain genetic variants of WND.