NH2-terminal signals in ATP7B Cu-ATPase mediate its Cu-dependent anterograde traffic in polarized hepatic cells

Y. Guo, L. Nyasae, L. T. Braiterman, and A. L. Hubbard

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 8 June 2005 ; accepted in final form 23 June 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Cu is an essential cofactor of cellular proteins but is toxic in its free state. The hepatic Cu-ATPase ATP7B has two functions in Cu homeostasis: it loads Cu+ onto newly synthesized apoceruloplasmin in the secretory pathway, thereby activating the plasma protein; and it participates in the excretion of excess Cu+ into the bile. To carry out these two functions, the membrane protein responds to changes in intracellular Cu levels by cycling between the Golgi and apical region. We used polarized hepatic WIF-B cells and high-resolution confocal microscopy to map the itinerary of endogenous and exogenous ATP7B under different Cu conditions. In Cu-depleted cells, ATP7B resided in a post-trans-Golgi network compartment that also contained syntaxin 6, whereas in Cu-loaded cells, the protein relocated to unique vesicles very near to the apical plasma membrane as well as the membrane itself. To determine the role of ATP7B's cytoplasmic NH2 terminus in regulating its intracellular movements, we generated seven mutations/deletions in this large [~650 amino acid (AA)] domain and analyzed the Cu-dependent behavior of the mutant ATP7B proteins in WIF-B cells. Truncation of the ATP7B NH2 terminus up to the fifth copper-binding domain (CBD5) yielded an active ATPase that was insensitive to cellular Cu levels and constitutively trafficked to the opposite (basolateral) plasma membrane domain. Fusion of the NH2-terminal 63 AA of ATP7B to the truncated protein restored both its Cu responsiveness and correct intracellular targeting. These results indicate that important targeting information is contained in this relatively short sequence, which is absent from the related CuATPase, ATP7A.

ATP7B; WIF-B; copper; trafficking; hepatocyte


COPPER IS AN ESSENTIAL cofactor of many proteins, including mitochondrial cytochrome-c oxidase, cytosolic superoxide dismutase, and secretory pathway enzymes, such as lysyl oxidase, tyrosinase, and ceruloplasmin (22). Due to its oxidizing capacity, free Cu is toxic; consequently, its intracellular levels are tightly regulated. Among the proteins known to be important in the regulation of Cu levels in vertebrates are two Cu-transporting membrane ATPases, ATP7A (Menkes protein) and ATP7B (Wilson protein). They belong to a large family of cation-transporting P-type ATPases, which translocate ions across cell membranes using the energy of ATP hydrolysis (34, 42). ATP7A is expressed in most tissues except the liver, where ATP7B is expressed. The proteins are ~60% identical in sequence with six copper binding domains (CBDs) in their ~650-amino acid (AA) cytosolic NH2 termini followed by either transmembrane domains (TMDs) containing cytoplasmic loops characteristic of P-type ATPases and ~80-AA cytosolic COOH termini. Mutations in the genes encoding these proteins cause two human diseases. Menkes disease is an often-fatal condition of Cu deficiency, due to mutations in ATP7A and failure of dietary Cu accumulated in the small intestine to be delivered to the circulation for distribution to the rest of the body (51). Wilson disease is a treatable condition of Cu excess, due to mutations in ATP7B and failure to excrete Cu that has accumulated in the liver (33). The presentations of these diseases indicate that both Cu-ATPases function in the cellular efflux of Cu in addition to their roles of transporting Cu into the secretory pathways of cells.

The importance of ATP7A and ATP7B in disease has spurred extensive research of the genes and proteins (22, 34). Results of studies aimed at understanding the cellular basis of the proteins' dual functions showed that ATP7A and ATP7B proteins relocate in response to changes in Cu levels (39, 47). Under low-Cu conditions, both proteins are concentrated in the trans-Golgi network (TGN) region; in elevated Cu, their steady-state distributions shift to the plasma membrane (ATP7A) or small cytoplasmic vesicles (ATP7B) (7, 14, 23, 31, 35, 36). However, the two proteins have been studied predominantly in nonpolarized cells [but see ATP7B (41) and ATP7A (18)], whereas the phenotypes of the human diseases point to polarized epithelia as crucial sites where Cu efflux is dysfunctional. Importantly, Cu efflux by ATP7A in the intestine is into the basolateral space (i.e., interstitium), whereas that of ATP7B in hepatocytes is into the apical space (i.e., bile) (35, 41). Therefore, the two Cu-ATPases must traffic to opposite plasma membrane (PM) domains to carry out their efflux functions.

Structural signals involved in the Cu-dependent trafficking of the Cu-ATPases have been identified using deletion/mutagenesis approaches. For example, retrieval of ATP7A from the cell surface requires a dileucine motif at the COOH terminus (15), although it is still unclear whether ATP7A internalization occurs via a clathrin-mediated pathway (8, 32). The same AAs serve as a basolateral targeting signal when ATP7A is expressed in polarized epithelial cells (7). Truncation or inactivation of the six CBDs of ATP7A in various combinations has shown that CBD 5 or 6 alone is sufficient for both transport and trafficking of the protein and that CBDs 1–4 will not substitute (35, 46; but see also Ref. 17 for a different result). Again, most studies have used nonpolarized cells with limited analysis of the membrane compartments harboring the Cu-ATPases.

In hepatocytes, ATP7B transports Cu into the secretory pathway for loading onto newly synthesized apoceruloplasmin, and it excretes excess Cu out of the cell via the bile (9). ATP7B's biosynthetic function takes place in the TGN region. However, the cellular site for its efflux function remains controversial.

In the present study, we used the polarized, rat-human hepatic WIF-B cell line (6, 27) to follow the itinerary of endogenous and exogenous ATP7B under different Cu conditions and determine the roles played by selected sequences at the NH2 terminus in regulating the protein's intracellular movements. Our results demonstrate that 1) endogenous ATP7B redistributes among at least three intracellular compartments in a Cu-responsive fashion, and modestly overexpressed exogenous ATP7B behaves similarly; 2) CBDs 5 and 6 in ATP7B are not sufficient for ATP7B's Cu-sensing/trafficking response; and 3) the first 63 AA contain necessary information for ATP7B's Cu-induced exit from its post-Golgi location.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Materials

The following cDNAs were gifts: GST-hATP7B encoding AA 1–653 (the NH2 terminus) from Dr. T. Sugiyama (Akia University School of Medicine), full-length ATP7B cDNA from Dr. J. Gitlin (Washington University School of Medicine, St. Louis, MO), and pcTYR, which encodes human tyrosinase in the pcDNA 3.1 A (–)myc/His, from Dr. D. Hebert (University of Massachusetts). Antibodies were obtained from the following sources: rabbit anti-ATP7B (Novus Biologicals, Littleton, CO); rabbit anti-green fluorescent protein (GFP; Molecular Probes, Eugene, OR); mouse anti-TGN38, anti-rab11, and anti-syntaxin 6 (Transduction Labs, Newington, NH); rat anti-human ATP7B (S. Lutsenko, University of Oregon Health Center, Portland, OR); rabbit anti-mannose-6-phosphate receptor (M6PR) (F. Gorelick, Yale Medical School, New Haven, CT); rabbit anti-syntaxin 16 (W. Hong, Institute of Molecular and Cell Biology, Singapore); and rabbit anti-cathepsin D (A. Haslik, Marburg, Germany). Rabbit anti-asialoglycoprotein receptor (ASGPR) and anti-aminopeptidase N (APN) were described by us (1) as was mouse monoclonal anti-endolyn78 and anti-5'-nucleotidase (26). Secondary antibodies conjugated to Cy3 or Cy5 were from Jackson ImmunoResearch Laboratories (West Grove, PA), and those conjugated to Alexa 488 and Alexa 568 were from Molecular Probes (Eugene, OR). Bathocuproine disulfonic acid (BCS) and cycloheximide were purchased from Sigma (St. Louis, MO). All other chemicals were of the highest purity and obtained from sources identified in previous publications.

Plasmid Constructions

Three GFP-hATP7B plasmids (pRH20, nt 1–4012; pRH19, nt 1326–4012; pRH16, nt 1618–4012) lacking the COOH terminus were cloned into the recombinant adenoviral vector, pAdLox, using multiple steps (details furnished on request). Briefly, the hATP7B NH2 terminus (from a GST-ATP7B plasmid, AA 1–653) was inserted into pEGFP-C1 (Clontech) to give pRH9. Two overlapping cDNA clones (pRH7, nt 1298–2716 and pRH11, nt 2577–3571) were obtained by RNA ligase-mediated rapid amplification (GeneRacer Kit, Invitrogen) using HepG2 mRNA prepared with Oligotex (Qiagen). Then, pRH11 was modified at the 3'-end by joining an image clone (image 3852352, nt 3820–4012) to it. GFP-wtATP7B (pYG7, nt 1–4398) was obtained by joining the ATP7B COOH terminus (nt 1326–4398 from a ATP7B full-length cDNA) to pRH20. To generate GFP-CBD1–4C/S ATP7B (pYG41, nt 1–4398), in which both of the cysteines (C) within each of the 1–4 copper binding domains in GFP-wtATP7B were mutated to serines (S), multisite-directed mutagenesis of GFP-wtATP7B was carried out using four primers (nAH250, -251, -252, and -253). Mutagenesis procedures were performed essentially according to the manufacturer's description using the QuikChange Multisite-Directed Mutagenesis kit (Stratagene). GFP-{Delta}CBD5–6 ATP7B (pYG33), which has an in-frame deletion (AA 445–651), was constructed by cloning an EcoRI/AgeI-digested PCR product (nt 1954–2920 of ATP7B) amplified with the forward primer encoding a 5' EcoRI site and the reverse primer into EcoRI/AgeI sites of pYG7. GFP-{Delta}CBD1–4 ATP7B (pYG10, nt1327–4398) and GFP-{Delta}CDB1–5 ATP7B (pYG9, nt1618–4398) were obtained by joining the 5'-end of pRH19 (HindIII/EcoRV fragment, nt 1326–2762) and pRH16 (SphI/AgeI fragment, nt 1618–2762) into digested pYG7, respectively. GFP-{Delta}CDB1–4 ATP7B or GFP-{Delta}CDB1–5 ATP7B was digested with XbaI/BspEI and then filled in and religated to generate {Delta}CDB1–4 ATP7B (pYG30) or {Delta}CDB1–5 ATP7B (pYG29). A PCR product (nt 1–189 of ATP7B) was amplified from pYG7 with the primer pair nAH219/nAH220 (a BstBI site was engineered at primer ends) and cloned into the BstBI site of pYG10 to generate GFP-1–63{Delta}CBD1–4 ATP7B (pYG36).

Sequences of all plasmids were verified. The full-length wtATP7B cDNA generated in this study has five nucleotide changes that translate into different AAs from those in the NCBI database (GI: 55743070). Three AA changes, A406S, V456L, and V1140A, represent single-nucleotide polymorphisms with cluster report numbers of rs1801243, rs1801244, and rs1801249, respectively. We propose that the two others, D96G and R875G, are also alleles found in the general population, because they were identified in human ATP7B ests, GI: 19689152 and GI: 19191518, respectively. All primer sequences not listed are available on request.

Cell Culture

WIF-B cells were seeded at 2 x 104 cells/cm2 on glass coverslips (22 x 22 mm), cultured as described (6, 27), and used ~10–13 days later, when maximal polarity had been achieved. Skin fibroblasts from a Menkes disease patient (line 84–1350) and the patient's mother were obtained from Dr. G. Thomas (Kennedy-Kreiger Institute, Johns Hopkins University, Baltimore, MD) and maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum supplemented with penicillin/streptomycin. They were immortalized with SV40 T antigen as described (12) and used for transfection. CRE8 cells [from Hardy et al. (21)] and HEK293A cells (from Qbiogene, Irvine, CA) were cultured as recommended by the suppliers and used in the preparation of recombinant adenoviruses.

Expression of ATP7B Constructs in WIF-B Cells

Because WIF-B cells are difficult to transfect, the constructs were expressed by recombinant adenovirus infection. Each ATP7B construct was packaged into recombinant adenovirus using the CRE-lox system and purified as described (2). For infection, viral dilutions were made in Opti-MEM (Invitrogen).

Fully polarized WIF-B cells were rinsed in PBS with 1 mM MgCl2, and 200 µl of the virus were applied to a coverslip. Parallel cells were treated with Opti-MEM as control. After incubation at 37°C for 50 min, the virus was aspirated, fresh medium was added, and the cells were returned to 37°C for 24 h before analysis. High expression in >90% of the cells was obtained with 3 x 109 viral particles (v.p.)/coverslip and low-to-modest expression in ~40% of the cells with 0.5 x 109 v.p./coverslip.

Cycloheximide Treatment

Infected cells were treated for 2 h with the protein synthesis inhibitor cycloheximide (50 µg/ml) and then treated an additional 4 h with cycloheximide plus 200 µM CuCl2.

Analysis of ATP7B Expression

Immunofluorescence microscopy In general, infected and control cells were rinsed briefly in PBS, placed on ice, fixed with chilled PBS containing 4% paraformaldehyde for 1 min, and permeabilized with ice-cold methanol for 10 min. Cells that were stained with rat anti-ATP7B, rabbit anti-syntaxin16, mouse anti-syntaxin6, or mouse anti-rab11 were first permeabilized in 0.2% Triton X-100 in 60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.8 (2 min at room temperature), and then fixed (30 min at room temperature) in PBS containing 4% paraformaldehyde. Cells were further processed for indirect immunofluorescence according to previously published methods (26) with the following primary antibodies: rat anti-ATP7B (1:200), anti-rab11 (1:50), anti-TGN38 (1:1,000), anti-APN (1:500), anti-ASGPR (1:200), anti-M6PR (1:400), anti-cathepsin D (1:200), anti-endolyn78 (1:300), and rabbit anti-ATP7B (1:100). Alexa 488-, Alexa 568-, or Cy5-conjugated secondary antibodies were used. Images taken on an epifluorescence microscope (Zeiss) were processed with IPLab and Photoshop 6.0 software, whereas those taken on the LSM 510 META (Zeiss) were collected with a x63 PLAN-APO, 1.4 numerical aperture, oil-immersion objective and processed using Zeiss software.

To compare the levels of exogenous vs. endogenous ATP7B protein expression, we infected WIF-B cells with virus-encoding untagged wtATP7B (~0.5x109 v.p./coverslip), cultured them overnight as described above, treated them with 200 µM BCS for 4 h, and then fixed and stained the cells with rat anti-ATP7B antibody. Images taken on the LSM 510 META were processed with Volocity 3.5.0 software (Improvision England). Approximately 50 cells per field were visually inspected and classified into one of three classes: those with faint anti-ATP7B labeling in the TGN region were classified as uninfected and the signal as representing endogenous ATP7B; those with brighter but still modest anti-ATP7B labeling were classified as low overexpressors; and those with bright labeling were classified as high overexpressors. Voxel intensities of the TGN regions from 15 cells per class were measured and averaged.

Western blot analysis. Infected WIF-B cells on coverslips were briefly rinsed with PBS, scraped into 200 µl SDS-PAGE sample buffer (20 mM Tris·HCl, pH 8.8, 5% SDS, 0.1 M DTT, 15% sucrose, 2 mM EDTA, and 5 M urea), and placed on ice. The cell lysates were boiled (5 min) and centrifuged (13,000 rpm, 5 min). Fifteen microliters of the supernatants were loaded onto 7% SDS-polyacrylamide gels and electrophoresed at 25 mA for 4 h. The resolved proteins were transferred to nitrocellulose membrane (Millipore, Billerica, MA), and the membrane was blocked for 1 h at room temperature with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBST). The membrane was subsequently incubated for 1 h or overnight with anti-GFP (1:5,000 in 1% BSA/PBS) or rabbit anti-ATP7B (1:1,000 in 1% BSA/PBS), probed with horseradish peroxidase (HRP)-conjugated donkey anti-rabbit antibody (1:5,000 in 5% nonfat milk/PBST) for 30 min, and developed using ECL (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol.

Tyrosinase activation assay. Immortalized MNK fibroblasts grown on glass coverslips were cotransfected with 1 µg each pcTYR and an ATP7B expression plasmid using LipofectAMINE. Twenty-four hours later, cells were washed twice in PBS and fixed for 30 s in acetone-methanol (1:1) at –20°C. These fixation conditions do not destroy tyrosinase activity (50). The cells were then incubated for ~2–3 h at 37°C in phosphate buffer (0.1 M, pH 6.8) containing 0.15% (wt/vol) levo-3,4-dihydroxy-L-phenylalanine (L-DOPA) to allow formation of the brown DOPA-chrome pigment. Coverslips were mounted on slides and cells were examined by phase and fluorescence microscopy.


    RESULTS
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 MATERIALS AND METHODS
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Map of Endogenous ATP7B Locations in Low and High Extracellular Cu

Before studying exogenous ATP7B, we determined the dynamics of endogenous ATP7B in polarized hepatic cells. To do this, we first tested several ATP7B antibodies using livers from Long-Evans Aguoti (LEA) and Long-Evans Cinnamon (LEC) rats as positive and negative controls, respectively. The LEC rat is a well-established model for Wilson Disease, because it fails to express endogenous ATP7B (49). We found that a rat anti-human antibody raised against the NH2-terminal 650 AA of ATP7B reproducibly immunoprecipitated a polypeptide of ~180 kDa from LEA but not LEC liver extracts and immunostained a Cu-sensitive ~180-kDa protein in mouse livers (unpublished observation) and WIF-B cells. We tested other ATP7B antibodies and found that they specifically immunoprecipitated and/or immunoblotted a similar protein only in LEA rat livers, yet by immunofluorescence they stained a juxtanuclear compartment in the LEC livers, indicating that they recognized a protein other than ATP7B (data not shown). Therefore, we used only rat anti-human ATP7B for immunofluorescence detection of the endogenous protein.

When we treated polarized WIF-B cells with the Cu chelator BCS under conditions similar to those reported by others (14, 28, 40, 43) and fixed and labeled them with rat anti-human ATP7B, we found the endogenous protein concentrated in a juxtanuclear region, which in polarized cells faced the apical surface (Fig. 1). Because other researchers had reported overlap with TGN markers (24, 41), we first double labeled Cu-depleted cells with anti-TGN38. Careful examination of single confocal images showed that the TGN38 and ATP7B patterns were not coincident, even in the fully Cu-depleted condition (data not shown). This finding prompted us to perform extensive marker analysis using confocal microscopy. By double staining Cu-depleted cells with antibodies to ATP7B and markers of organelles known to localize in polarized WIF-B cells between the nucleus and apical membrane domain (26), we saw substantial overlap of ATP7B and a post-TGN t-SNARE, syntaxin 6 (Fig. 1). M6PR and endolyn 78, markers of late endosomes and endosomes/lysosomes, respectively, were also in the juxtanuclear region of polarized WIF-B cells, but endogenous ATP7B showed relatively little (M6PR) or no (endolyn 78) overlap with them (Fig. 1).



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Fig. 1. In Cu-depleted WIF-B cells, endogenous ATP7B distribution partially overlaps with post-trans-Golgi network (TGN) and endosomal but not lysosomal markers. WIF-B cells were depleted of Cu for 4 h, fixed, stained with antibodies against ATP7B (A-C) and syntaxin 6 (A), M6PR (B), or endolyn (C), and then analyzed by confocal microscopy. Insets are enlarged 2.5x. Scale bar = 10 µm. *Apical space; n, nucleus.

 
When we incubated WIF-B cells with extracellular Cu concentrations reported by others to induce the redistribution of ATP7B (7, 35), we found that endogenous ATP7B's intracellular localization had changed dramatically (Fig. 2). The protein was still in the nuclear-apical region but was no longer in the compartment marked by syntaxin 6. Rather, it was even closer to the apical membrane and in structures that partially overlapped with the apical membrane marker APN, and the small GTPase rab 11, but not with endosome or lysosome markers.



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Fig. 2. In Cu-loaded cells, endogenous ATP7B distribution partially overlaps with the apical marker, aminopeptidase N (APN), and rab11 but not with markers of endosomes or lysosomes. WIF-B cells were loaded with Cu for 4 h, fixed, and stained with antibodies against ATP7B (A-E) and syntaxin 6 (Syn6; A), APN (B), rab11 (C), endolyn (D), or cathepsin D (E). Insets are enlarged 2.5x. Scale bar = 10 µm. *Apical space.

 
Characterization of the ATP7B Constructs

Having established the localizations of endogenous ATP7B in WIF-B cells in low and high Cu, we next examined the behavior of exogenous wtATP7B under similar conditions. The ATP7B constructs we generated are shown in Fig. 3A, together with results of their Cu transport activities in a tyrosinase activation assay. Tyrosinase, a Cu-dependent endosomal/lysosomal membrane enzyme normally expressed in melanocytes, was used as a reporter of Cu-ATPase activity (40). Immortalized fibroblasts from a Menkes patient expressed neither detectable ATP7A protein by Western blot analysis nor ATP7B, which is not normally expressed in fibroblasts (data not shown). When pcTYR-myc alone was transfected into them, no tyrosinase activity was detected, although the protein was clearly present when stained with anti-myc antibody (Fig. 3B). However, when GFP-wtATP7B was cotransfected with pc-TYR-myc, we detected both a GFP signal and a black reaction product indicative of GFP-ATP7B and tyrosinase activity, respectively (Fig. 3B). All constructs in this study except GFP-{Delta}CBD5–6 ATP7B, which was retained in the endoplasmic reticulum, exhibited Cu transport activity in this qualitative assay (Fig. 3A).



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Fig. 3. Characterization of ATP7B constructs. A: schematic drawing of the NH2 termini of the ATP7B constructs used in this study and their ability to transport Cu as assessed by the tryosinase activation assay (see MATERIALS AND METHODS). Copper binding domains (CBD) are numbered 1–6 and either unfilled (active) or shaded gray (C-S mutation); TMD1, transmembrane domain 1; stippled circle, GFP tag. B: illustration of the tyrosinase assay. Top row, immunofluorescence with myc antibody and GFP fluorescence; bottom row, phase-contrast images after the DOPA reaction. First column shows a control cell expressing myc-Tyr but no ATP7B and therefore lacking L-DOPA reaction product. Middle and right columns show cells expressing myc-TYR and either GFP-wtATP7B (middle) or GFP-{Delta}BD1–4 (right), with black reaction product (arrowheads) indicating active tyrosinase in both cases. C: Western blot analysis of infected WIF-B cells expressing exogenous ATP7B proteins indicated.

 
We used Western blot analysis with anti-GFP or rabbit anti-ATP7B antibodies to determine whether infected WIF-B cells expressed ATP7B constructs of the correct size (Fig. 3C). All of the GFP-tagged ATP7B proteins migrated as single bands using either anti-GFP or the COOH-terminal-specific anti-ATP7B, demonstrating no breakdown of the intact proteins. However, the two untagged ATP7B constructs, {Delta}CDB1–5 ATP7B and {Delta}CDB1–4 ATP7B, exhibited multiple immunoreactive band(s) above their predicted sizes, suggesting aggregation (Fig. 3, C2). In addition, the apparent size of monomeric {Delta}CDB1–5 ATP7B was smaller than expected (75 vs. 101 kDa, Fig. 3, C2), suggesting degradation and prompting use of only the {Delta}CDB1–4 ATP7B construct in the experiments.

Copper Induces Relocation of GFP-wtATP7B from the TGN Region to Vesicles and the Apical Membrane in WIF-B Cells

WIF-B cells were first infected with GFP-wtATP7B virus, fixed, and then labeled with markers of Golgi (TGN38) and apical membranes (APN). In basal medium (0.02 µM Cu2+), GFP-wtATP7B was localized between the nucleus and the apical membrane (data not shown). When cells were depleted of Cu, GFP-wtATP7B became concentrated in the TGN region (Fig. 4, BCS). In contrast, when cells were cultured in CuCl2, GFP-wtATP7B was present both in vesicular structures outside of the TGN region and the apical membrane (Fig. 4, Cu). These results are consistent with the redistribution of endogenous ATP7B in WIF-B cells. We occasionally observed low levels of exogenous GFP-wtATP7B at the basolateral membrane (Fig. 4, Cu, arrow). The overlap of the GFP-ATP7B and APN signals seen in high Cu suggested apparent apical membrane localization for GFP-ATP7B (Fig. 4, yellow, Cu), although the GFP-wtATP7B labeling pattern was more punctate than that of APN. When Cu-loaded cells were rinsed and then recultured in Cu-depleting conditions, GFP-ATP7B was no longer localized near/in the apical membrane but had returned to the TGN region (Fig. 4, Cu->BCS). This result indicated that the redistribution of ATP7B was reversible and dependent upon Cu levels. We observed similar results at external Cu concentrations as low as 10 µM, although the time course of GFP-wtATP7B's redistribution was longer. We also confirmed that the relocation of GFP-wtATP7B was not dependent on new protein synthesis by treating infected cells with cycloheximide before and during exposure to CuCl2. Under these conditions, the GFP-wtATP7B signal showed no dramatic decline and redistribution still occurred (data not shown).



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Fig. 4. GFP-wtATP7B redistributes in response to Cu levels in polarized WIF-B cells. WIF-B cells were infected with GFP-wtATP7B adenovirus, cultured overnight, and then depleted of Cu for 4 h (BCS; A), loaded for 4 h with Cu (Cu; B) or first loaded and then depleted of Cu (Cu->BCS; C). They were fixed and stained with antibodies to markers of the TGN (TGN38; blue) and the apical plasma membrane (PM; APN; red) and then analyzed by confocal microscopy. Scale bar = 10 µm. Dashed circle, nuclear region; *apical space; arrow, basolateral plasma membrane (PM); arrowhead, apical PM.

 
GFP-wtATP7B's Intracellular Distribution Overlaps Partially with Known Organellar Markers in Cu-Depleted WIF-B Cells but not in Cu-Loaded Cells

Given the initial results described above for endogenous ATP7B, we performed comprehensive marker analysis to map the localizations of exogenous GFP-wtATP7B under low and high Cu conditions. Our results are from at least three separate experiments in which both BCS and Cu treatments were applied and ~15 cells were examined per experiment. We scored infected cells that expressed no more than threefold GFP-wtATP7B over the endogenous protein, as assessed by immunolabeling with the rat anti-human antibody. In Cu-depleted conditions and in agreement with the localization of endogenous ATP7B, GFP-wtATP7B overlapped most extensively with syntaxins 6 (Fig. 5, A and B) and 16 (data not shown). Additionally, GFP-wtATP7B was located very close to but overlapped only partially with M6PR (Fig. 5, C and D), much less with endolyn (Fig. 5, E and F) and not at all with cathepsin D (Fig. 5, E and F). Other markers showing no overlap included rab7, VAMP8, syntaxin8, and {gamma}-adaptin (data not shown). In Cu-loaded conditions, the intracellular vesicles containing GFP-wtATP7B did not stain with antibodies to any of the markers (Fig. 6). Thus these results are consistent with those obtained for endogenous ATP7B and indicate that GFP-wtATP7B trafficks to a unique compartment in Cu-loaded cells.



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Fig. 5. In Cu-depleted cells, GFP-wtATP7B distribution partially overlaps with post-TGN and endosomal but not lysosomal markers. WIF-B cells infected with GFP-wtATP7B were depleted of Cu for 4 h, fixed, stained with antibodies against syntaxin6, M6PR, cathepsin D, and endolyn, and then analyzed by confocal microscopy. Boxed regions were enlarged 3.5x. *Apical space.

 


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Fig. 6. In Cu-loaded cells, GFP-ATP7B distribution does not overlap with markers of endosomes or lysosomes. WIF-B cells were infected with GFP-wtATP7B were loaded with Cu for 4 h, fixed, and stained with antibodies against endolyn and cathepsin D. Boxed regions were enlarged 3.5x. Scale bar = 10 µm. *Apical space.

 
Inactivation of CBD1–4s Has no Discernable Effect on the Cu Transporting and Trafficking Activities of Mutant ATP7B in WIF-B Cells

Having established the Cu-dependent trafficking pattern of GFP-wtATP7B in WIF-B cells, we next explored the role(s) played by the NH2-terminal ~600-AA domain in the protein's cycling. We generated a GFP-ATP7B construct in which the essential cysteines of the first four CBDs were mutated to serines (CBD1–4C/S, Fig. 3) and expressed it in WIF-B cells. Previous studies of ATP7A (35) and ATP7B (7) showed that mutation of the two critical cysteines to serines within the sequence motifs GMXCXXC in all six CBDs (CBD1–6C/S) abrogated the copper-induced trafficking of ATP7B in nonpolarized cells. However, if the signature motif in either CBD5 or CBD6 was intact, the mutant protein retained wild-type Cu-transporting activity and Cu-responsive trafficking behavior in nonpolarized cells. Our results in polarized hepatic cells confirmed that GFP-CBD1–4C/S ATP7B behaved similarly to GFP-wtATP7B in both low and high Cu conditions (Fig. 7). GFP-CBD1–4C/S ATP7B also exhibited Cu-transporting activity in the tyrosinase activation assay (Fig. 3A).



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Fig. 7. GFP-CBD1–4C/S ATP7B redistributes in response to changes in Cu levels in WIF-B cells. WIF-B cells were infected with GFP-CBD1–4C/S ATP7B adenovirus and treated as described in Fig. 4. Scale bar = 10 µm. Apical marker, APN (red); dashed circle, nuclear region; *apical space; arrowhead, apical PM.

 
CBD 5 + 6 or 6 Alone Is Not Sufficient for the Cu-Induced Trafficking of ATP7B

Because inactivation of CBDs 1–4 in the context of an otherwise wild-type ATP7B had no discernable effect on the behavior of Cu-ATPase, we next deleted the first four or five CBDs and all intervening sequences, thus generating GFP-{Delta}CBD1–4 ATP7B and GFP-{Delta}CBD1–5 ATP7B (Fig. 3). Similar truncations in ATP7A reportedly had no effect on the resulting mutant protein's behavior in fibroblasts (35, 46), suggesting that the entire NH2 terminal to CBD5 was dispensable for the Cu-responsive behavior, at least in nonpolarized cells. Both truncated proteins transported Cu (Fig. 3A). However, when we infected WIF-B cells with viruses encoding these two constructs and Cu depleted or Cu loaded the cells for 4 h, the two truncated proteins behaved very differently from GFP-wtATP7B (Fig. 8). In polarized WIF-B cells, they were localized in the TGN region and the basolateral membrane irregardless of the Cu levels (Fig. 8, middle). Furthermore, we never detected the mutant proteins at or near the apical PM. These results indicated that the truncated ATP7B proteins were insensitive to Cu levels and they were targeted to the wrong PM domain relative to GFP-wtATP7B.



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Fig. 8. ATP7B containing only CBDs 5 and 6 in its NH2 terminus targets constitutively to the wrong PM domain. Infected WIF-B cells were incubated 4 h in depleted (+BCS) or high-Cu (+Cu) media before fixation. Top and middle: GFP-wt ATP7B (top) and GFP-{Delta}CBD1–4 ATP7B (middle) were detected through their GFP tags. {Delta}CBD1–4 ATP7B was stained for immunofluorescence using rabbit-anti-ATP7B and Alexa 488 secondary antibody (bottom). *Apical space; arrow, basolateral PM.

 
To test whether the GFP tag in the NH2 terminus of ATP7B was responsible for the aberrant localization and trafficking of these truncated proteins, we expressed untagged constructs in WIF-B cells. As shown in Fig. 8, bottom, {Delta}CBD1–4 ATP7B behaved similarly to its GFP-tagged counterpart, suggesting that the loss of Cu sensitivity and mistargeting of the truncated ATP7B proteins were due to loss of trafficking signal(s) within the first 442 AA.

Addition of the NH2-Terminal 63 AA to {Delta}CBD1–4 ATP7B Restores Cu-Responsive Trafficking in WIF-B Cells

We were initially puzzled by our results with GFP-{Delta}CBD1–4 ATP7B and GFP-{Delta}CBD1–5 ATP7B, because they were at variance with published work reporting that only CBD5 or CBD6 was needed for a wild-type ATP7B phenotype (7). However, close inspection of the sequence of a similar ATP7B mutant generated by Cater et al. (7) revealed that the authors had made an internal deletion of AA 64–539, leaving intact the first 63 AA, to which an antibody had been raised. Therefore, we restored the first 63 AA to {Delta}CBD1–4 ATP7B, generating the mutant GFP-1–63{Delta}CBD1–4 ATP7B (Fig. 3). In Chinese hamster ovary (CHO) cells the mutant protein exhibited a Cu-sensitive trafficking response that was very similar to that of GFP-wtATP7B (Fig. 9). Likewise, in infected, polarized WIF-B cells, the protein showed Cu responsiveness; that is, it moved from the TGN region to the apical membrane and dispersed vesicles (Fig. 10).



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Fig. 9. Addition of the NH2-terminal 63-amino acid (AA) to mutant ATP7B restores Cu sensing and trafficking in Chinese hamster ovary (CHO) cells. CHO cells were transfected with the indicated plasmids (all tagged with GFP at their NH2 termini), cultured overnight, treated with BCS or CuCl2 for 4 h, and then fixed and imaged by epifluorescence microscopy. Arrows, GFP signal at the PM.

 


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Fig. 10. GFP-1–63{Delta}CBD1–4ATP7B exhibits Cu responsiveness and redistributes like wtATP7B in WIF-B cells. WIF-B cells were infected with the indicated GFP construct and then treated as described in Fig. 2. Apical marker, APN (red); dashed circle, nuclear region; *apical space; arrowhead, apical PM.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
Using a recombinant protein approach coupled with extensive organelle marker analysis, we have documented for the first time that the hepatic Cu-ATPase ATP7B requires the first 63 AA plus CBD 5 and/or 6 to traffic in a Cu-responsive and polarized fashion in hepatic cells. We expressed GFP-wtATP7B in polarized WIF-B cells and carefully compared its itinerary to that of the endogenous protein under different Cu conditions. We then expressed 7 ATP7B mutant proteins in WIF-B cells and found that when CBDs 5 and/or 6 alone constituted the NH2 terminus of ATP7B, the mutant protein was unable to sense Cu levels and constitutively trafficked to the wrong plasma membrane domain. By fusing AA 1–63 to AA 443, which precedes CBD5, we restored Cu sensitivity and corrected the mutant protein's trafficking. These results are consistent with the presence of information in the first 63 AA of ATP7B for both Cu responsiveness and correct targeting.

Our study has several important implications. First, exogenous ATP7B overexpressed at modest levels retains an itinerary similar to that of the endogenous protein in WIF-B cells. Second, ATP7B has basolateral targeting information that is revealed when the NH2 terminus and the first four or five CBDs are deleted. Third, the 63-AA NH2 terminus of human ATP7B, which is 53 AA longer than that of human ATP7A, must be recognized by cellular machinery that either retains the protein in a post-TGN compartment in Cu-depleted conditions or targets the protein to its two destinations (vesicles and, perhaps, the apical PM) in Cu-loaded conditions.

The Cu-Dependent Itinerary of ATP7B in Polarized WIF-B Cells

Our results in Cu-depleted WIF-B cells confirm earlier reports showing that endogenous ATP7B resides primarily in the Golgi region of nonpolarized and polarized cells (14, 35, 41). However, we are the first to show such extensive overlap with the post-TGN t-SNARE syntaxin 6 (3). Syntaxin 6 has been implicated in the retrograde trafficking of several interesting proteins, including the glucose tranporter 4 in 3T3-L1 adipocytes (38). However, its varied interactions with multiple t- and v-SNAREs have led to the idea that syntaxin 6's function is cell-type specific (55). Although immuno-EM localization will be necessary to definitively localize ATP7B with syntaxin 6, the fact that the membrane Cu-ATPase also recycles between the Golgi and dispersed vesicles near to/at the apical PM makes it tempting to speculate that syntaxin 6 may play a functional role in the retrograde movement of the Cu-ATPase.

On elevation of intracellular Cu in vivo, endogenous ATP7B in hepatocytes relocates out of the TGN to as-yet-unidentified vesicular compartments but not to the apical plasma membrane (41, 43). In nonpolarized cells in vitro (14, 31), ATP7B was found in small vesicles distributed widely throughout the cytoplasm, whereas in polarized HepG2 (41) and WIF-B cells (this study) the protein trafficked to both the apical membrane and vesicular structures outside of the TGN region. How can these results be reconciled? Perhaps the protein's regulation in polarized hepatic cells is different than that in vivo. Alternatively, the punctate nature of the label seen in cultured cells and at the apical PM in polarized hepatic cells may reflect the protein's residence in a unique a subapical compartment. The overlap between endogenous ATP7B and rab 11, which has been localized to apical recycling compartments in polarized cells (54), supports the latter possibility. Future studies will be required to answer this question.

How do we account for our finding that in low Cu exogenous wtATP7B resides in an M6PR compartment to a greater extent than the endogenous protein? First, both syntaxin 6 and M6PR are capable of moving among the TGN and endosomal subcompartments (16, 55), and exogenous wtATP7B may behave similarly. Second, we found that the expression levels of GFP-wtATP7B had an impact on Cu-dependent localization and trafficking. Approximately 2.5x overexpression of a protein resulted in behavior most consistent with that of the endogenous protein. However, when a construct was expressed at levels greater than 10x over the endogenous protein levels, we detected GFP protein in puncta throughout the cell. Furthermore, elevation of Cu did not recruit protein from these structures, because they were still present after 4 h in 200 µM Cu (data not shown). Harada et al. (19, 20) reported that GFP-wtATP7B expressed in either primary rat hepatocytes or Huh-7 hepatoma cells (neither polarized) showed overlap with lysosomal/endosomal markers in low Cu. These authors also observed no change in exogenous GFP-wtATP7B's distribution when intracellular Cu levels were elevated (19, 20). A possible explanation for such a finding is that ATP7B was overexpressed in those studies. Such accumulation in cells transiently expressing exogenous ATP7B suggests that additional cellular proteins may be required for appropriate targeting of this Cu-ATPase. In contrast, >50-fold overexpression of ATP7A through gene amplification, which was achieved by selection of CHO cells that were resistant to gradually increased levels of extracellular Cu (5), gave trafficking behavior kinetically identical to that in CHO cells with much lower ATP7A overexpression (~10-fold) (37). An intriguing question is whether additional cellular proteins required for wild-type ATP7A behavior are also overexpressed in these Cu-resistant and stable cell lines. Clearly, further study is warranted.

What route does ATP7B take to reach the apically located vesicles and apical PM when intracellular Cu is elevated? At present we do not know. It is well established that two classes (single TMD and GPI anchored) of newly synthesized hepatic apical PM proteins take an indirect, transcytotic route to the apical PM via the basolateral surface (reviewed in Ref. 25), whereas multi-TMD apical PM proteins, such the bile salt export pump (ABC11), are directly targeted there without an intervening stop at the opposite PM (reviewed in Ref. 30). WIF-B cells examined at early times after exposure to high Cu did not show transient accumulation of endogenous or exogenous wtATP7B at the basolateral PM (data not shown). Unfortunately, lack of antibodies to the relatively short ectodomains of the protein prohibited us from using an antibody-trafficking approach to test the possibility of indirect targeting. By analogy to other hepatic multi-TMD proteins, we anticipate that the route is direct but might include an intracellular intermediate, perhaps the subapical compartment (51).

NH2-Terminal 63 AA of ATP7B Play a Dominant Role in the Protein's Cu-Induced Trafficking Behavior

In this study, we identified the first 63 AA of ATP7B as playing a crucial role in changing the trafficking of a truncated ATP7B protein with only CBDs 5 and/or 6 in its NH2 terminus. Without this NH2-terminal sequence, the {Delta}CBD1–4 and 1–5 ATP7B proteins were targeted constitutively to the basolateral surface. Three questions arise from this result. First, what might be the basolateral targeting information in ATP7B? Second, where within the first 63 AA is/are the crucial targeting/retention signal(s)? Third, what cellular machinery recognizes the signals in a Cu-dependent fashion?

A possible answer to the first question comes from results of a recent report in which the dileucine "retrieval" signal in the COOH terminus of ATP7A was found to serve also as a basolateral targeting signal in polarized MDCK cells (18). Because ATP7B has a comparable trileucine sequence, it might serve an equivalent function in the absence of the dominant apical signal in the NH2 terminus.

Alignment of the ATP7B NH2-terminal sequences preceding CBD1 from four species reveals possible clues to answering the second question (Fig. 11). Two highly conserved regions in the sequence are reasonable candidates for containing Cu-dependent trafficking information. There are also four conserved aromatic residues, which are similar to those shown to be important in the trafficking of the cation-dependent M6PR (44). In contrast, the NH2 termini of ATP7A from three species are only 10 AA long and not homologous to ATP7B (Fig. 11).



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Fig. 11. Alignment of ATP7B and ATP7A NH2 termini. Residues in the boxes are highly conserved in ATP7B among the 4 species, as are the 4 aromatic amino acids underlined. Sequences are from the following: human 7B, GI:4502322; rat 7B, GI:6978560; mouse 7B, GI:6680757; sheep 7B, GI:2739169; human 7A, GI: 53986562; rat 7A, GI: 16258816; and mouse 7A, GI: 31982811.

 
Identification of the cellular machinery recognizing the NH2-terminal targeting signal in ATP7B is an intriguing issue arising from our study. One possible candidate is MURR1, the gene that was recently identified as defective in Cu-induced hepatoxicity of inbred dog terriers (53). This gene encodes a small cytosolic protein with no identifiable motifs but several interesting binding partners, including the NH2-terminal ~653 AA of ATP7B (48). MURR1 binding is Cu dependent and specific to ATP7B, although other recent work suggests that MURR1 levels influence Cu efflux in cells expressing only ATP7A (4).

Are There Additional Cu-Induced Trafficking Signals in the NH2 Terminus of ATP7B?

In this study, we have identified structural information for Cu-induced trafficking in noncontiguous sequences of the ATP7B NH2 terminus. Although we assert that the first 63 AA confer Cu-dependent trafficking to ATP7B, others have clearly shown that one of the last two CBDs must be functional to permit protein trafficking (7). Results of structural studies have shown that isolated CBDs undergo a conformational change on binding Cu (11). At present, the possible relationship between these two findings is unclear. However, evidence from studies of MNK patients suggests that the 27-AA sequence linking CBD6 to TMD1 of ATP7A is also important in the protein's function (10, 29). The ATP7A genes of these patients were reported to harbor splice-junction or missense mutations in the 77-bp exon 8 encoding the linker region. Expression of one such mutant protein in MNK fibroblasts showed Cu transport competence but failure to traffic out of the Golgi in high Cu levels (29). Three Wilson Disease missense mutations have also been mapped to this region [G626A, D642H, and M645R (13, 45)].

In conclusion, Cu homeostasis relies on many protein components, one of which is ATP7B. We have identified Cu-sensitive targeting signals in the ATP7B NH2 terminus that are required for Cu-induced trafficking to an apical destination. The future challenge will be to characterize the ATP7B-positive compartment and understand its contribution to the apical efflux of Cu from polarized hepatic cells.


    GRANT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANT
 REFERENCES
 
This work was supported by National Institutes of Health Grants GM-064645, DK-064388 (to A. L. Hubbard), and CHD-024061 (to G. Thomas, who donated MNK-null fibroblasts).


    ACKNOWLEDGMENTS
 
We thank colleagues who shared reagents and M. Donowitz and C. Machamer for helpful comments on the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Dept. of Cell Biology, Johns Hopkins Univ. School of Medicine, 725 N. Wolfe St., Baltimore, MD (e-mail: alh{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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