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. 1998 Sep 1;95(18):10854-9.
doi: 10.1073/pnas.95.18.10854.

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

A S Payne  1 , E J Kelly, J D Gitlin
Affiliations

Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

A S Payne et al. Proc Natl Acad Sci U S A. .

Abstract

Wilson disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. To elucidate the function of the Wilson protein, wild-type and mutant Wilson cDNAs were expressed in a Menkes copper transporter-deficient mottled fibroblast cell line defective in copper export. Expression of the wild-type cDNA demonstrated trans-Golgi network localization and copper-dependent trafficking of the Wilson protein identical to previous observations for the endogenously expressed protein in hepatocytes. Furthermore, expression of the Wilson cDNA rescued the mottled phenotype as evidenced by a reduction in copper accumulation and restoration of cell viability. In contrast, expression of an H1069Q mutant Wilson cDNA did not rescue the mottled phenotype, and immunofluorescence studies showed that this mutant Wilson protein was localized in the endoplasmic reticulum. Consistent with these findings, pulse-chase analysis demonstrated a 5-fold decrease in the half-life of the H1069Q mutant as compared with the wild-type protein. Maintenance of these transfected cell lines at 28 degreesC resulted in localization of the H1069Q protein in the trans-Golgi network, suggesting that a temperature-sensitive defect in protein folding followed by degradation constitutes the molecular basis of Wilson disease in patients harboring the H1069Q mutation. Taken together, these studies describe a tractable expression system for elucidating the function and localization of the copper-transporting ATPases in mammalian cells and provide compelling evidence that the Wilson protein can functionally substitute for the Menkes protein, supporting the concept that these proteins use common biochemical mechanisms to effect cellular copper homeostasis.

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Figures

Figure 1
Figure 1
(A) Immunoblot of lysates from mottled fibroblasts (802–1) stably transfected with the wild-type Wilson cDNA. Protein (125 μg) from the parental cell line (lane 1) or two independent clones (lanes 2 and 3) with HepG2 lysate as a positive control (lane 4) were separated by SDS/7.5% PAGE, transferred to nitrocellulose, incubated with Wilson antiserum, and developed with chemiluminescence. (B) Indirect immunofluorescence localization of the wild-type Wilson protein (WD) and ARF with or without copper treatment in the stably transfected mottled fibroblast clone 4, with the parental 802–1 cell line as a negative control. (×600) (C) 64Cu retention by mottled fibroblasts (802–1 Mo −/Y), wild-type fibroblasts (802–5 Mo +/Y), and two independent clones of mottled fibroblasts stably transfected with the Wilson cDNA (802–1 WD 4 and 802–1 WD 13). Cells were incubated with 64Cu for 72 hr and processed as described in Materials and Methods. (D) Copper toxicity profile of mottled fibroblasts (802–1), wild-type fibroblasts (802–5), and mottled fibroblast clone 4 stably transfected with the Wilson cDNA (802–1 WD). Cells were grown in media supplemented with increasing concentrations of copper and assessed for viability at 72 hr by using an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide-based assay as described in Materials and Methods.
Figure 2
Figure 2
(A) Immunoblot of lysates from mottled fibroblast clones stably transfected with Wilson cDNA mutants H1069Q and H1069A. Protein (125 μg) from cells transfected with vector alone (lane 1), wild-type Wilson cDNA (lane 2), or Wilson cDNA mutants (lanes 3 and 4) were separated by SDS/7.5% PAGE, transferred to nitrocellulose, and analyzed by chemiluminescence. (B) Copper toxicity profile of mottled fibroblast clones stably transfected with the wild-type Wilson cDNA or mutants H1069Q and H1069A.
Figure 3
Figure 3
(A) Immunofluorescence on mottled fibroblast clones stably transfected with Wilson cDNA mutants H1069Q and H1069A without copper treatment. Cells were processed for indirect immunofluorescence by using antibodies against the WD protein or protein disulfide isomerase. (×600.) (B) Pulse–chase labeling of Wilson protein endogenously expressed in HepG2 cells or stably expressed in mottled fibroblast clones as the wild-type protein or mutants H1069Q and H1069A. Cultures were pulsed for 30 min with [35S]methionine and [35S]cysteine and then chased for the indicated times in media containing excess methionine and cysteine. Immunoprecipitates of cell lysates were analyzed by fluorography after SDS/7.5% PAGE and were quantified by using a Phosphorimager (Molecular Dynamics).
Figure 4
Figure 4
(A) Immunoblot analysis of lysates from mottled fibroblast clones maintained at 28°C. Protein (125 μg) from cells stably transfected with vector alone (lane 1), wild-type Wilson cDNA (lane 2), or Wilson cDNA mutants (lanes 3 and 4) were separated by SDS/7.5% PAGE, transferred to nitrocellulose, and analyzed by chemiluminescence. (B) Immunofluorescence on wild-type Wilson clone 4 with and without copper treatment by using the Wilson protein antibody. (×600.) (C) Immunofluorescence on a Wilson H1069Q clone with and without copper treatment by using antibodies against the Wilson protein or ARF. Arrows indicate trans-Golgi network localization in corresponding cells. (×600.) (D) Immunofluorescence on a Wilson H1069A clone with and without copper treatment by using antibodies against the Wilson protein or ARF. Arrows indicate trans-Golgi network localization in corresponding cells. (×600.)
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