Copper-induced translocation of the Wilson disease protein ATP7B independent of Murr1/COMMD1 and Rab7

Abstract

Wilson disease is a genetic disorder of copper metabolism. Impaired biliary excretion results in a gradual accumulation of copper, which leads to severe disease. The specific gene defect lies in the Wilson disease protein, ATP7B, a copper-transporting ATPase that is highly active in hepatocytes. The two major functions of ATP7B in the liver are the copper loading of ceruloplasmin in the Golgi apparatus, and the excretion of excess copper into the bile. In response to elevated copper levels, ATP7B shows a unique intracellular trafficking pattern that is required for copper excretion from the Golgi apparatus into dispersed vesicles. We analyzed the translocation of ATP7B by both confocal microscopy and RNA interference, testing current models that suggest the involvement of Murr1/COMMD1 and Rab7 in this pathway. We found that although the ATP7B translocation is conserved among nonhepatic cell lines, there is no co-localization with Murr1/COMMD1 or the Rab marker proteins of the endolysosomal system. Consistent with this finding, the translocation of ATP7B was not impaired by the depletion of either Murr1/COMMD1 or Rab7, or by a dominant-negative Rab7 mutant. In conclusion, our data suggest that the translocation of ATP7B takes place independently of Rab7-regulated endosomal traffic events. Murr1/COMMD1 plays a role in a later step of the copper excretion pathway but is not involved in the translocation of the Wilson disease protein.

Figure 1

A: Endogenous expression of the WDP ATP7B in Ptk2 cells. Cell lysates (50 μg of protein) were analyzed by Western blotting with the affinity-purified rabbit antibody against ATP7B (molecular weight of ATP7B: 157 kDa). Kidney-derived Ptk2 cells express endogenous ATP7B (lane 1) comparable to the human hepatoma cell line HuH7 (lane 4). The level of endogenous ATP7B in HeLa cells was low (lane 2), and transient expression of the WDP was necessary for analysis (lane 3, Hela+). Molecular weight standards are indicated in kDa. B: Copper-dependent translocation of endogenous ATP7B in Ptk2 cells. Ptk2 cells expressing the Golgi marker protein T2-GFP were incubated for 2 hours either with the copper-chelating agent BCS (a), in standard culture medium (MEM, b), or with 100 μmol/L CuSO₄ (+Cu, c). Cells were fixed and endogenous ATP7B stained by indirect immunofluorescence. Overlap between ATP7B (WDP, red) and the Golgi marker protein in the basal and copper-chelated state (a and b, overlay) is indicated by arrowheads. No co-localization was observed after copper treatment (c). C: Time course of translocation of endogenous ATP7B in Ptk2 cells. After an initial treatment with the copper chelator BC for 2 hours, Ptk2 cells were exposed to 100 μmol/L copper ions for variable amounts of time. Microscopy evaluation was done after indirect immunofluorescence staining of ATP7B, scoring Golgi pattern (not translocated) versus dispersed staining (translocated). Data are expressed as mean with SD. The percentage of translocated ATP7B for cells in basal medium was 21% versus 18% for BCS-treated cells. This difference was not statistically significant. Scale bars = 10 μm.

Figure 2

ATP7B and endolysosomal rab marker proteins remain segregated after copper-dependent translocation. Ptk2 cells were transiently transfected with Rab7-GFP (late endosomes, lysosomes), Rab9-YFP (late endosomes), or Rab11-GFP (recycling endosomes) as indicated in the figures. Two days after transfection, cells were either treated with BCS (A) or cultured in basal medium (B) or incubated with copper (C) for 2 hours, fixed, and processed for indirect immunofluorescence staining of ATP7B (WDP, shown in red). Scale bars = 10 μm.

Figure 3

ATP7B and Rab7 are segregated in the hepatoma cell line HuH7 and in MDCK cells. HuH7 cells (a–c) and MDCK cells (d) were transiently transfected with Rab7-GFP. Two days after transfection cells were either treated with BCS (a) or cultured in basal medium (b, d) or treated with copper (c) for 2 hours, fixed, and processed for indirect immunofluorescence staining of ATP7B (WDP, shown in red). Scale bars = 10 μm.

Figure 4

A: ATP7B/WDP and Murr1/COMMD1 do not co-localize in HeLa cells. HeLa cells were transiently transfected with Murr1-GFP and ATP7B and processed for indirect immunofluorescence of ATP7B (WDP, shown in red). Before fixation with methanol, cells were either treated with BCS (a), cultured in basal medium (b), or treated with copper (c) for 2 hours. No co-localization was observed. B: No overlap between endogenous ATP7B/WDP and Murr1/COMMD1 in Ptk2 cells. Ptk2 cells were processed for indirect immunofluorescence of endogenous Murr1/COMMD1 (red) and ATP7B (WDP, green). Before fixation with methanol, cells were either treated with BCS (a), cultured in basal medium (b), or treated with copper (c) for 2 hours. No co-localization was observed. C: No co-localization between endogenous ATP7B/WDP and Murr1/COMMD1 in HuH7 cells. HuH7 cells cultured in standard medium were processed for indirect immunofluorescence of endogenous Murr1/COMMD1 (red) and ATP7B (WDP, green). D: RT-PCR quantification of ATP7B and Murr1 in human cell lines. HuH7 (hepatoma), Caco-2 (intestine), HeLa (cervical), and HaCaT (keratinocytes) cells were evaluated for their mRNA content of ATP7B/WDP and Murr1/COMMD1. *HaCaT cells did not show any signal above background for ATP7B. The results confirmed a low expression of ATP7B in HeLa cells. Arbitrary units were obtained by normalizing the signal to the internal control (values shown are 10⁻³ * signal ATP7B, Murr1/signal actin). Scale bars = 10 μm.

Figure 5

Murr1 is partially localized to late endosomes. HeLa cells co-expressing Murr1/COMMD1-mRFP and either Rab7-GFP (a; late endosomes, lysosomes), Rab9-YFP (b; late endosomes, trans-Golgi network), or Rab11-GFP (c; recycling endosomes) were fixed with methanol and analyzed by confocal microscopy. Overlapping structures are marked by arrowheads. Scale bars = 10 μm.

Figure 6

Copper-dependent translocation of ATP7B functions independent of Murr1 and Rab7. A: Immunofluorescence of RNAi knockdown cells. HuH7 cells were stably transduced with a control retrovirus (a, c) or the shRNAi retrovirus specific for Murr1 (b) or specific for Rab7 (d). Endogenous Murr1 (a, b) and Rab7 (c, d) were stained by indirect immunofluorescence (red). Microscopy exposure times and other settings were identical when comparing a and b and c and d, respectively. All cells were also stained with Hoechst dye to visualize cell nuclei. B: RT-PCR quantification of Murr1 and Rab7 in stably transduced HuH7-RNAi knockdown cells. Arbitrary units were obtained by normalizing the signal to the internal control (values shown are 10⁻⁴ * signal Murr1/signal actin and 10⁻¹ * signal Rab7/signal actin, respectively). C: Western blot of Murr1 and Rab7 in stably transduced HuH7-RNAi knockdown cells. Protein lysates were analyzed by gel electrophoresis and Western blotting. Blots were stripped and reprobed with β-actin. D: Dominant-negative Rab7 does not impair the translocation of the WDP. HuH7 cells transfected with a dominant-negative Rab7 mutant (GFP-Rab7DN) were treated for 2 hours either with BCS or copper ions and processed for indirect immunofluorescence staining for ATP7B. ATP7B (red) showed a perinuclear Golgi pattern when copper concentrations were low, but a diffuse vesicular pattern with high copper levels. E: Translocation is not impaired after Murr1 or Rab7 knockdown. Stably transduced RNAi-HuH7 cells or HuH7 cells expressing the dominant-negative Rab7 mutant (Rab7-DN) were treated for 2 hours either with BCS, basal medium, or copper ions. After fixation and indirect immunofluorescence staining for ATP7B, slides were blinded and scored for translocation. Scale bars = 20 μm.