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Originally published online as doi:10.2353/ajpath.2008.071134 on October 30, 2008

Published online before print October 30, 2008
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(American Journal of Pathology. 2008;173:1783-1794.)
© 2008 American Society for Investigative Pathology
DOI: 10.2353/ajpath.2008.071134

Copper-Induced Translocation of the Wilson Disease Protein ATP7B Independent of Murr1/COMMD1 and Rab7

Karl Heinz Weiss, Javier Carbajo Lozoya, Sabine Tuma, Daniel Gotthardt, Jürgen Reichert, Robert Ehehalt, Wolfgang Stremmel and Joachim Füllekrug

From the Department of Gastroenterology, University Hospital Heidelberg, Heidelberg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Copper is a trace element in the diet, but is required as a protein co-factor for basic cellular processes and therefore essential for all living organisms. However, too much intracellular copper is cytotoxic, leading to the formation of reactive oxygen species. In mammals, intestinal copper uptake does not seem to be regulated, and homeostasis is achieved primarily by adjusting biliary excretion of copper.1,2

Wilson disease is characterized by gradual accumulation of copper in tissues, manifested by liver disease and/or neurological symptoms.3,4 This autosomal recessive disorder of copper homeostasis in humans is caused by a functional deficiency of ATP7B, the Wilson disease protein (WDP).5,6 Many different mutations distributed along the whole ATP7B gene lead to Wilson disease.7,8 The WDP is critical for biliary excretion of copper9 but also supplies copper ions for the ferroxidase ceruloplasmin,10 which is the main copper containing protein of serum.11 Copper transport defects may also lead to systemic copper deficiency when ATP7A, a related intestinal P-type ATPase, is mutated.12-14

The WDP is a copper-translocating ATPase highly expressed in hepatocytes. ATP7B features eight transmembrane domains forming a channel, and shows unidirectional ATP-dependent transport of cytoplasmic copper ions across lipid bilayers. In hepatocytes, translocated copper is secreted on the apical side into the bile or is transferred to the ferroxidase ceruloplasmin at a late Golgi compartment,3,10 and is then secreted to the basolateral side (serum).

ATP7B shows striking changes in its subcellular localization when copper concentrations are manipulated. At low copper levels, ATP7B is localized to the late Golgi compartment15,16 where presumably copper loading of ceruloplasmin takes place. However, when copper levels are high, ATP7B shifts away from the Golgi apparatus to cytoplasmically dispersed vesicles, which in polarized liver cells accumulate subapically.16-20 It is not clear how copper transported into these vesicles would finally reach the bile, but it is generally assumed that the translocation of ATP7B is a necessary precondition for copper excretion.3,21,22

Certainly other proteins than ATP7B contribute to the molecular mechanism of copper excretion, and mutations or polymorphisms of these proteins might contribute to Wilson disease, maybe explaining the highly variable clinical presentation23-25 and course of this disease. Canine copper toxicosis of Bedlington terriers26 is caused by a deficiency of Murr1/COMMD127,28 and resembles Wilson disease, although ceruloplasmin levels are not decreased29 and there are no evident neurological symptoms. Murr1/COMMD1 has been reported to interact physically with ATP7B.30-32 Based on these observations, a role of Murr1/COMMD1 in the biliary excretion of copper downstream of Golgi-localized ATP7B has been suggested.2,3,33 Consistent with this, Murr1/COMMD1 was found on endolysosomal membranes but also in the cytosol28 and in the nucleus.34 Depletion of Murr1/COMMD1 by RNA interference results in an intracellular copper accumulation.35,36 The lack of exon 2 of the Murr1 gene leads to copper toxicosis in dogs, but the same deletion is embryonically lethal in mice.37 Murr1 is part of a larger protein family sharing a C-terminal leucine-rich domain termed the copper metabolism Murr1 domain (COMMD),34,38 and there is evidence that the other members of this family also bind to ATP7B.31 Murr1 is involved in NF-{kappa}B-mediated regulation of gene transcription,39,40 which has been recently reviewed together with other possible functions of the COMMD family.38 XIAP is another protein interacting with Murr1, and enhanced degradation of XIAP mediated by copper binding sensitizes hepatocytes for apoptosis,41 providing an unexpected new angle for copper-induced cell damage.

Here, we applied confocal microscopy and RNA interference to investigate the copper-induced translocation of the WDP, testing current models2,3,33 that hypothesize an interaction of Murr1 and ATP7B during this intracellular trafficking step. We found no evidence for a role of Murr1, suggesting that this protein is involved in a different step of copper excretion. Furthermore, we analyzed the reported relationship between ATP7B and Rab7-positive endosomes.42 Although we could document a partial co-localization between Murr1 and Rab7, the WDP was not observed in Rab7-positive endosomes, even after copper-induced translocation to cytoplasmically dispersed vesicles.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Antibodies

The antibody against ATP7B was essentially prepared as described.18 Oligonucleotides were designed and used to amplify the region of the Wilson protein encoding amino acids 325 to 635. This region was amplified by polymerase chain reaction (PCR) and subcloned in the pGEX-2T vector (Amersham Pharmacia Biotech, Uppsala, Sweden). Escherichia coli BL21 cells harboring the expression plasmid were grown to an optical density of 1.5 at 600 nm at 31°C and induced with isopropyl 1-thio-β-D-galactopyranoside. Cultures were harvested by centrifugation, resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100, and lysed using a high-pressure cell crusher. The supernatant was incubated with glutathione-agarose beads. Bound glutathione S-transferase (GST) fusion protein was thrombin-cleaved to obtain the ATP7B-fragment. New Zealand White rabbits were immunized with 4 x 100 mg of this recombinant protein (immunization was performed according to standard procedures by EuroGentec, Seraing, Belgium).

Affinity purification of the antiserum was performed using AminoLink Plus columns as recommended by the manufacturer (Pierce, Rockford, IL). Recombinant protein was coupled to the columns using 0.1 mol/L sodium citrate, 0.05 mol/L sodium carbonate, pH 10. Remaining active sites were blocked by sodium cyanoborohydride solution (5 mol/L NaCNBH3 in 0.01 mol/L NaOH). An additional pre-elution step using elution buffer (0.2 mol/L glycine-HCl, pH 2.6) was performed to remove noncovalently bound recombinant protein. After washing the column with PBS (pH 7.2), the antiserum was loaded to the column. After repeated washing, the bound antibodies were eluted from the column using the elution buffer. Protein concentration of the eluate was determined using a mini Bradford assay. The eluted antibody solution was rebuffered in PBS and 5% bovine serum albumin using PD-10 columns. For long-term storage 50% glycerol was added to the rebuffered antibody solution. The monoclonal COMMD1/Murr1 antibody (M01, clone 2A12) was obtained from Abnova Corporation (Jhongli City, Taiwan). The goat anti-Rab7 antibody (Sc6563) was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Monoclonal anti β-actin antibody (clone AC-15) was obtained from Sigma (St. Louis, MO).

Cell Culture

HeLa [American Type Culture Collection (ATCC) no.: CCL-2], Ptk2 (ATCC no.: CCL-56; derived from normal kidney of Potorous tridactylis), CaCo-2 (ATCC no.: HTB-37), MDCK (ATCC no.: CCL-34) cells were cultured according to the ATCC protocols. The immortalized human keratinocyte cell line, HaCaT, and the human hepatoma cell line, HuH7, were cultured in Dulbecco’s modified Eagle’s medium (high glucose 4.5 g/L), supplemented with glutamine (2 mmol/L), penicillin (400 U/ml), streptomycin (50 ng/ml), and 10% fetal bovine serum in a humidified 5% CO2 atmosphere at 37°C. The phoenix-gp packaging cell line was cultured as previously described.43

Expression Plasmids

Rab7-GFP,44 GFP-Rab7-DN(T22N),44 Rab11-GFP,45 Rab9-YFP,46 and caveolin-1-GFP47 were provided by Marino Zerial (MPI Molecular Cell Biology and Genetics, Dresden, Germany) and Lucas Pelkmans (ETH, Zurich, Switzerland), respectively. The T2-GFP plasmid48 was supplied by Jamie White (EMBL, Heidelberg, Germany). The full-length ATP7B-cDNA5 was provided by John R. Forbes (Department of Medical Genetics, University of Alberta, Edmonton, Canada) in a yeast vector. ATP7B-cDNA was subcloned using BamHI into pcDNA3 (Invitrogen, Carlsbad, CA), resulting in a mammalian expression vector. Generation of fluorescent protein-MURR1 fusion constructs was performed as follows: the complete coding region of human MURR1 was amplified from the total cDNA of the human CaCo-2 cells by PCR using sense primer (5'-ACGTAAGCTTACCATGGCGGCGGGCGAGCTTG-3') and antisense primer (5'-CAC TGATCAGCCAGCCTAACGCGGATCCACGT-3'). The sense primer introduced a HindIII site and the consensus Kozak sequence ACC in front of the starter ATG, the antisense primer covered a BamHI restriction site and the stop codon. The HindIII to BamHI fragment of whole-length Murr1 cDNA was cloned upstream of green fluorescent protein (GFP) cDNA in the mammalian expression vector pEGFP-N1 (BD Biosciences Clontech, Mountain View, CA). The correct sequence was confirmed by bidirectional sequencing. Murr1-RFP was derived from the Murr1-GFP plasmid by replacing GFP (BamHI, NotI) with PCR-modified monomeric RFP.49 Murr1-GFP and Murr1-mRFP had identical localizations when co-expressed (see Supplemental Figure 1 at http://ajp.amjpathol.org).

Immunoblotting

Equal amounts of protein (50 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 10% gels followed by electrophoretic transfer to nitrocellulose membranes, incubated with the primary antibody and visualized using enhanced chemiluminescence detection. Immunostaining of β-actin served as a further loading control.

Transfection, Copper Exposure, and Immunofluorescence

For transient transfection cells were grown on coverslips in a six-well plate (10 cm2/well) to a density of ~80% and transfected using 4 µg of plasmid DNA and 10 µl of Lipofectamine 2000 reagent (Invitrogen) per well according to the manufacturer’s protocol. Six hours after transfection, cells were washed in prewarmed PBS (pH 7.2) and normal culture medium was added to the cells. Copper exposure experiments were started 46 hours after transfection. Before copper exposure, cells were washed twice in prewarmed PBS (pH 7.2) to remove culture medium. Cells were then either incubated with prewarmed Dulbecco’s modified Eagle’s medium containing 100 µmol/L of the copper chelator bathocuproinedisulfonic acid (BCS) or 100 µmol/L copper sulfate (CuSO4) for 2 hours.

After repeated washing in PBS, cells grown on coverslips were fixed with methanol (–20°C) for 3 minutes. With this fixation procedure Murr1-GFP and Murr1-RFP showed a clean vesicular distribution. Use of paraformaldehyde emphasized the cytosolic and nuclear localization of Murr1 (see Supplemental Figure 1, a and b, at http://ajp.amjpathol.org). Antibodies were incubated in PBS containing 0.1% saponin (Sigma), 0.5% gelatin (Teleostan gelatin, Sigma), and 5 mg/ml bovine serum albumin. Corresponding secondary antibodies (donkey anti-rabbit/mouse; Dianova, Hamburg, Germany) were used conjugated to Cy3 or fluorescein isothiocyanate. Coverslips were mounted in Prolong Gold antifade mounting medium (Molecular Probes, Leiden, The Netherlands) and confocal images were taken on a TCS SP2 microscope (objective: x63 magnification, oil immersion, NA 1.32; Leica Microsystems, Wetzlar, Germany). Double-immunofluorescence images were taken sequentially, and parameters were adjusted so that all light intensities were in the recording range. Intensity of the laser beam and photo multiplier levels were adjusted for each slide and each fluorophore (Cy3, fluorescein isothiocyanate, GFP, RFP, YFP, lysotracker); line averaging was set to 4 to optimize signal to noise ratio. Images shown were derived from representative single confocal planes and arranged with Adobe Photoshop and Adobe Illustrator (Adobe, San Jose, CA).

RNA Interference

Target sequences for human Murr1/COMMD139 and Rab750 were 5'-AAGUCUAUUGCGUCUGCAGAC-3' and 5'-CGGUUCCAGUCUCUCGGUG-3', respectively. Oligonucleotides encoding the corresponding shRNAs were designed and cloned into pSuper as described51 except that BglII and XhoI were used. For confirmation, plasmids were sequenced in both directions.

Cloning of the retroviral plasmids, transfection of the packaging cell line, collection of pseudotyped retrovirus particles, and transduction of HuH7 cells were as published.43 Briefly, the shRNA expression cassette was transferred by subcloning into the XhoI/EcoRI site of pRVH1-puro. Cells of the human phoenix gag-pol packaging cell line were grown in 10-cm dishes to near confluence. Using Lipofectamine 2000 reagent, phoenix-gp cells were co-transfected with 2.5 µg of DNA of the pVSV-G-plasmid (pseudotyping; enhanced stability and efficient infection) and 24 µg of DNA of the pRVH1-puro plasmid containing the Murr1, Rab7, or control shRNA expression cassette, respectively. Twenty-four hours after transfection, medium was changed from high glucose (4.5 g/L) to low glucose (1.0 g/L) and cells were placed at 32°C. Batches of virus-containing supernatants were collected every 24 hours for up to 6 days.

For target cell transduction, 1.5 x 105 HuH7 cells were mixed with 450 µl of virus-containing supernatant in the presence of 4 µg/ml polybrene (Sigma) and seeded into a well of a six-well plate and incubated at 32°C. Twenty-four hours after infection, the medium was changed to normal culture medium and cells were returned to 37°C. From 48 hours to 144 hours after infection, cells were grown in the presence of 2 µg/ml of puromycin (BD Biosciences) to eliminate nontransduced cells. Cells were used for experiments from 14 to 18 days after infection. The RNAi-mediated depletion of target mRNA by Murr1-shRNAi and Rab7-shRNAi was measured by reverse transcriptase (RT)-PCR and visualized by immunofluorescence staining and Western blot. Results were compared to HuH7 cells infected with the control virus containing no shRNA sequence.

Quantification of ATP7B Translocation

After copper exposure and ATP7B immunofluorescence, coverslips were analyzed with an Olympus IX50 fluorescence microscope (Olympus, Tokyo, Japan) equipped with a x60 oil immersion objective. Counting was blinded because the examiner did not know which treatment the cells had received. A cell was classified as translocated when a diffuse vesicular staining was observed. Cells showing a tight perinuclear ATP7B immunoreactivity without a vesicular pattern were classified as not translocated. Translocated cells were divided by the total number of cells (~200 cells were counted for each coverslip, translocation ratio was calculated per coverslip, two coverslips were counted per experiment). SD was calculated from three independent experiments.

Real-Time PCR

Total RNA was isolated from tissue culture cells with the RNeasy mini kit including DNase digestion. The Omniscript RT kit and random N6 primers were used for reverse transcription. All reagents and procedures for cDNA synthesis were from Qiagen, Hilden, Germany. Real-time PCR was performed using the LightCycler system (Roche, Mannheim, Germany). Transcripts were detected with SYBR Green I and normalized to β-actin as internal control. Light Cycler Relative Quantification Software Version 1.0 together with calibration plasmids [pRRL-PGK-ATP7B,52 Murr1-GFP (this study), Rab7-GFP (this study), EST-actin (RZPD IOH27856, Berlin, Germany)] was used for the calculation of ATP7B/actin and Murr1/actin ratios and Rab7/actin ratios (efficiency corrected relative quantification). Primer sequences human ATP7B: 5'-CCACATGAAGCCCCTGAC,GTACTGCTCCTCATCCCTGC-3'; human COMMD1/Murr1: 5'-GGGATTCTTAAGTCTATTGCGTCTTCT,GTCGTCAGATGTGATCCCACCTTGCTT-3'; human Rab7: 5'-TGTTGGGAAACAAGATTGACCACC,CTTCTTAAGTGCATTCCGTGCAA-3'; human β-actin: 5'-AGGATGCAGAAGGAGATCACTGCTG,GGGGGGTGTAACGCAACTAAGTCATAG-3'.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Copper-Dependent Translocation of the WDP ATP7B in Ptk2 Cells

To study the intracellular trafficking of the WDP we raised and affinity-purified an antiserum against the N-terminal domain and screened different cell lines for expression. Although high levels of ATP7B were found in hepatocyte-derived cell lines, these cells were not easy to evaluate morphologically even by confocal microscopy. HeLa cells were suitable for analysis when ATP7B was ectopically expressed (Figure 1A) . Kidney-derived Ptk2 cells are, because of their flat and extended cell body, an excellent model system to study intracellular trafficking by light microscopy, and were found to express significant amounts of endogenous WDP (Figure 1A) .


Figure 1
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  		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.

 
Copper concentrations different from standard culture medium (MEM) were achieved using either incubation with 100 µmol/L copper sulfate or the copper-chelating reagent BCS. Comparison to a Golgi-GFP marker protein48 demonstrated that the WDP showed a Golgi pattern when copper concentrations were low, but a diffuse vesicular pattern under high copper levels (Figure 1B) . This is in line with reports on primary hepatocytes or liver-derived cell lines.16-18 Withdrawal of copper leads to a return of WDP to the Golgi apparatus (not shown).

Analyzing this apparent translocation event at different time points, it became clear that this intracellular movement is not synchronized across the whole cell population. In fact, 60% of cells showed translocation after 1 hour, but an additional 20% changed their WDP localization only within the second hour (Figure 1C) . Both the copper-dependent translocation of endogenous WDP and the time scale proved to be essentially the same in hepatocyte-derived and other cell lines (HuH7, Caco-2, MDCK; data not shown). Although the physiological role of ATP7B in copper excretion has been suggested to be essential only in the liver (summarized by J.D. Gitlin3 ), we concluded that the copper-dependent trafficking step is remarkably conserved across very different cell types.

Comparison of ATP7B to Rab Marker Proteins of the Endosomal Pathway

Although the Golgi localization of the WDP under standard conditions (ie, low copper concentration) has been documented extensively,16-19 the nature of the dispersed vesicular structures after copper exposure is not clear and no co-localizing proteins have been reported to date. Based on the recent report that overexpressed GFP-ATP7B resides in late endosomes together with Rab7,42 we analyzed the distribution of endogenous WDP in relation to different rab marker proteins of the endosomal pathway, again in Ptk2 cells. However, we did not observe any overlap between Rab7 and ATP7B, regardless of the copper concentration (BCS, Figure 2A ; MEM, Figure 2B ; added copper, Figure 2C ). Rab7 and Rab9 both localize to late endosomes but occupy distinct subdomains with little co-localization,46 but again Rab9 was present in different structures when compared to ATP7B (Figure 2) . Recycling endosomes marked by Rab11 were also distinct from endogenous WDP staining (Figure 2) . In addition labeling with the lysotracker dye marking less specifically the late endosomal/lysosomal pathway did not show any overlap with ATP7B (see Supplemental Figure 2 at http://ajp.amjpathol.org). We repeated the comparison between WDP and Rab7, Rab9, and Rab11 in HeLa cells transiently expressing ectopic WDP but results were unchanged (see Supplemental Figure 3 at http://ajp.amjpathol.org). Endosomal structures termed caveosomes53 were also investigated but did not overlap with ATP7B (see Supplemental Figure 4 at http://ajp.amjpathol.org).


Figure 2
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  		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.

 
Because the copper-induced redistribution of the WDP from the trans-Golgi compartment to dispersed vesicles is of major functional importance especially in liver cells, we further evaluated the hepatoma cell line HuH7. ATP7B and Rab7 (Figure 3, a–c) , Rab9, or Rab11 (see Supplemental Figure 5 at http://ajp.amjpathol.org) remained segregated in HuH7 cells regardless of the copper concentration, consistent with the data obtained for Ptk2 and HeLa cells. An overlap between Rab7 and overexpressed ATP7B has been reported in MDCK cells.42 Therefore we repeated our localization analysis in this canine kidney cell line, but again no co-localization between endogenous WDP and Rab7-GFP could be observed (Figure 3d) .


Figure 3
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  		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.

 
Murr1/COMMD1 Localization to Intracellular Vesicles Distinct from ATP7B

Murr1 has been suggested to participate in the excretion of copper by interacting with ATP7B during or after copper-triggered translocation away from the Golgi apparatus.2,3,33 The reported vesicular localization of Murr1 in HeLa cells28 was consistent with the idea that there could be a direct interaction with translocated ATP7B on membrane structures. Therefore we constructed a Murr1-GFP fusion protein, which indeed gave a convincing punctate membrane pattern when expressed in HeLa cells. Surprisingly, there was no significant overlap with the WDP, neither under low, basal (MEM) or high copper concentrations (Figure 4A) . Moreover, although ATP7B shifted its localization in response to copper, we did not observe any change in the pattern for Murr1/COMMD1. Murr1-GFP also gave a punctate pattern in Ptk2 cells (data not shown). An antibody against Murr1 became recently available so that we were able to confirm the segregation of Murr1 and ATP7B by immunofluorescence staining of endogenous proteins in Ptk2 cells (Figure 4B) . We again confirmed with the HuH7 cells that our findings are also relevant for endogenous WDP and Murr1/COMMD1 in hepatocyte-derived cells (Figure 4C) . The proposed interaction between Murr1 and the WDP2,30,33 implies that there should be corresponding expression patterns. The relative quantification of mRNA expression levels by efficiency corrected real-time PCR of human cell lines was generally consistent with this idea (Figure 4D) , however the keratinocyte HaCaT cell line only expressed Murr1/COMMD1 but not the WDP.


Figure 4
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  		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.

 
Partial Localization of Murr1/COMMD1 to Late Endosomes

We observed significant co-localization of punctate structures between Murr1 and Rab7, which was obvious even without overlaying the images (Figure 5a) . This suggests that although neither Murr1/COMMD1 nor Rab7 overlap with the WDP, they do share some common vesicular compartment, presumably of endosomal origin. Rab7 is involved in the regulation of vesicular traffic and has been localized to late endosomes and lysosomes.44,54 Rab9, which regulates transport between late endosomes and the trans-Golgi network,55 also gave a partial overlap with Murr1 although in general less extensive (Figure 5b) . No co-localization was evident when Murr1/COMMD1 was compared to the recycling endosome marker Rab11 (Figure 5c) . We did not see any pattern changes when extracellular copper concentrations were manipulated (data not shown).


Figure 5
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  		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.

 
The Translocation of ATP7B Is Not Impaired by Knockdown of Either Murr1 or Rab7

Although we did not detect any co-localization between the WDP and either Murr1 or Rab7, this did not rule out a functional contribution of these proteins to the copper-induced trafficking of ATP7B. To directly test this, we developed human hepatoma cell lines (HuH7) depleted for Murr1 or Rab7 by RNA interference (RNAi). This was based on validated RNAi target sequences,39,50 which we designed into small hairpin RNA templates (shRNA). The shRNA coding sequences were incorporated into retroviruses that were then used for stable transduction of the target cells.43 Murr1/COMMD1 and Rab7 were efficiently depleted from HuH7 cells as confirmed by immunofluorescence, RT-PCR quantification, and Western blotting (Figure 6, A–C) . HuH7 cells depleted for Murr1 or Rab7 were treated with copper as before, stained for the WDP, and blinded before translocation efficiency was scored (Figure 6E) . An additional, independent line of experiments used the well-described dominant-negative Rab7 mutant.44 Translocation of the WDP still occurred as assessed by the localization pattern of ATP7B after copper treatment, even in cells highly expressing the dominant-negative Rab7 mutant (Figure 6D) . Summarizing, all cell lines had a translocation efficiency similar to control cells (Figure 6E) suggesting that neither Murr1 nor Rab7 are critically involved in the movement of the WDP from the Golgi apparatus to the copper-induced vesicular structures.


Figure 6
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  		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.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The WDP shows a copper-dependent subcellular localization, and it is thought that the translocation from the Golgi apparatus to subapical vesicles is a necessary step before systemic copper excretion into the bile.3,14,20-22 The nature of these vesicles still represent an enigma, and no other protein components have been identified to date.14,56 The molecular mechanism of copper-dependent translocation of ATP7B is not restricted to hepatocytes and hepatoma-derived cell lines; it appears to be a broadly conserved feature of many different cell types eg, CHO cells,21,57 HeLa, Ptk2 (this study), MDCK, Caco-2 (K.H.W. and J.F., unpublished). In some cell lines where the Golgi apparatus has a more fragmented appearance as opposed to a tight perinuclear localization, translocation of ATP7B is more difficult to assess but these problems are greatly reduced when co-evaluating a Golgi marker protein. Although the translocation itself might be easier to score in liver-derived cells, kidney-derived Ptk2 cells offer a remarkable spatial resolution and were chosen as the main model system for this study. Although the first cells showed a dispersed vesicular ATP7B staining pattern already after 15 to 20 minutes, many cells took longer times and some did not respond at all; our data correspond well to an earlier examination of HepG2 cells.16

Rab proteins are small GTPases involved in the regulation of vesicular trafficking, and they are localized to specific intracellular sites.58,59 The fact that Rab-GFP proteins faithfully mimic their endogenous counterparts has been characterized extensively.46,58,60,61 Because endosomes and lysosomes have been implicated in the pathway of copper excretion,9,42,62-64 we compared ATP7B to a subset of well-established rab marker proteins of the endolysosomal pathway. Rab7 localized to late endosomes46,54 was of special interest because a co-localization with WDP/ATP7B had been claimed.42 However we did not observe any overlap between the WDP and either Rab7, Rab9 (late endosomes but different from Rab746,54,55 ), Rab11 (recycling endosomes65-67 ), or a more general marker of the endolysosomal pathway, the lysotracker dye (Figure 2 ; and see Supplemental Figure 2 at http://ajp.amjpathol.org). This held true for basal (standard tissue culture medium) as well as low and high copper concentrations.

Rab11 had been described to give a partial overlap with ATP7B in copper-treated WIF-B cells.19 This could be attributable to the limitations of light microscopy because an overlap does not necessarily mean co-localization. With this in mind, segregation between two proteins is easier to interpret than an overlap, especially in the perinuclear region where recycling endosomes, Golgi apparatus, microtubule organizing center, and lysosomes tend to occupy the same space.

Transiently expressed GFP-ATP7B was reported to reside in late endosomes marked by Rab7, regardless of the copper concentration.42 However the cells shown in the micrographs42 display a diffuse but strong signal for Rab7 throughout the whole cell, with little subcellular resolution. This together with a perinuclear signal for GFP-ATP7B gives a yellow color in the merged picture, but is seems appropriate to exert some caution when interpreting this as the sole evidence for a co-localization. GFP-ATP7B did not overlap completely with ATP7B-DsRed in the same study,42 which might indicate that the GFP or DsRed modification could change the trafficking/localization properties of ATP7B. We were unable to see any co-localization between endogenous ATP7B and Rab7, despite trying many different conditions and cell lines. In addition to Ptk2 cells (Figure 2) , we also analyzed endogenous WDP in the hepatoma cell line HuH7 (Figure 3, a–c) , and overexpressed WDP in HeLa cells (see Supplemental Figure 3 at http://ajp.amjpathol.org). Finally, mimicking previous conditions,42 we confirmed our finding by staining endogenous WDP in MDCK cells cultured with standard medium (without any manipulation of copper concentrations; Figure 3d ).

Vast overexpression of ATP7B in HeLa cells was the only condition in which a marginal overlap could be observed (K.H.W. and J.F., unpublished). However these cells in general did not appear healthy and had lost the translocation response to elevated copper. In principle, this could suggest that ATP7B cycles through late endosomes as part of its trafficking itinerary and is made visible only under these conditions of high overexpression. A more likely explanation is simply an overflow phenomenon after saturation of internal membranes with ATP7B, compromising the targeting/sorting capacity. In line with this hypothesis, Hubbard and Braiterman68 recently pointed out that higher expression of GFP-ATP7B yielded protein aggregates that were unresponsive to changes in copper.

In an independent approach, we depleted Rab7 by RNA interference and analyzed the distribution of endogenous ATP7B in hepatoma cells (Figure 6) . Because neither the steady state localization nor the copper-induced translocation of ATP7B was impaired, this suggested that Rab7 is not functionally required for the copper-induced translocation of the WDP. Finally, overexpression of a dominant-negative Rab7 mutant protein was also without any discernible effect on the localization of ATP7B. We have to conclude that regulation of membrane traffic by the GTPase Rab7 is not relevant for the intracellular movement of the WDP in response to elevated copper concentrations.

Deficiency of Murr1 is the main cause for copper toxicosis in dogs,1,26,69,70 and co-precipitation experiments suggest a direct interaction with the WDP.30-32 In our hands, there was no co-localization between Murr1/COMMD1 on membrane vesicular structures and ATP7B (Figure 4, A–C) . However, an interaction between cytosolic Murr1/COMMD1 and the WDP cannot be excluded by our immunofluorescence data, especially if only part of the total cellular Murr1 would bind to ATP7B. Because several other interaction partners for Murr1 have already been put forward (HIF-{alpha},37 XIAP,35 RelA/NF{kappa}B,34,39,40 other COMMD proteins,34 {Delta}ENaC71 ), and Murr1 has also been localized to the nucleus,34 it seems reasonable to assume that only a fraction of Murr1 at any given time may interact with ATP7B.

We were expecting that depletion of Murr1/COMMD1 (by RNAi, Figure 6 ) would impair the first step of copper excretion from the cell, the translocation of ATP7B away from the Golgi apparatus toward the dispersed vesicles. However we could not find any difference to control cells in any parameter. In line with this, the localization of Murr1/COMMD1 was not influenced by copper treatment. The obvious conclusion is that Murr1/COMMD1 is required only at a later step of copper excretion, maybe in guiding copper-loaded subapical vesicles to the apical membrane, where hepatocytes can finally discharge copper into the bile. An earlier interference with copper transport seems unlikely because the loading of ceruloplasmin by ATP7B in the late Golgi is not affected even in knockout animals (Bedlington terriers28,29 ). In a side observation, even high overexpression of Murr1/COMMD1 in HeLa cells would neither change the steady state localization of the WDP nor the copper-dependent translocation (K.H.W. and J.F., unpublished).

Interestingly, we did find a partial overlap between Murr1 and rab marker proteins of the late endosomal pathway. It could be that this is the pool of Murr1 involved in copper excretion, but this speculation would need to be supported by future studies; eg, addressing precisely the partitioning of Murr1 between membranes, cytosol, and the nucleus and the relevance of these subpopulations for copper homeostasis. Alternatively, the influence of Murr1/COMMD1 on ubiquitination38 might solve the puzzle how exactly Murr1 is involved in copper secretion from cells.

In conclusion, our data imply that the copper-induced translocation of the WDP is a widely conserved mechanism that functions independently of Rab7-regulated late endosomal pathways. The Murr1/COMMD1 protein presumably has a function later in the pathway of copper excretion than currently considered2,3,14,33 because it is not involved in copper-mediated translocation of ATP7B.


   Acknowledgements
 
We thank Uta Merle and Karin Bents for help with the RT-PCR quantifications; Celine Johanssen and Mark Schäfer for helpful comments on the manuscript; Marino Zerial, Lucas Pelkmans, and Jamie White for the generous supply of reagents; and Günther Giese at the microscopy facility of the MPI for Medical Research, Heidelberg.


   Footnotes
 
Address reprint requests to Joachim Füllekrug, Ph.D., Department of Gastroenterology, University Hospital Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany. E-mail: joachim.fuellekrug{at}med.uni-heidelberg.de

Supported by the Dietmar Hopp Foundation (grant to W.S.), the Medical Faculty of the University of Heidelberg (young investigator grant to K.H.W.), and by the Stiftung Nephrologie (to R.E. and J.F.).

K.H.W. and J.C.L. contributed equally to this study.

Supplemental material for this article can be found on http://ajp.amjpathol.org.

Present addresses of J.C.L.: the Institute of Pharmacology and Toxicology, University of Heidelberg, Mannheim, Germany; and J.R.: the Heidelberg University Biochemistry Center, Heidelberg, Germany.

Accepted for publication September 9, 2008.


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 Introduction
 Materials and Methods
 Results
 Discussion
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