Role of the Copper-binding Domain in the Copper Transport Function of ATP7B, the P-type ATPase Defective in Wilson Disease*

John R. ForbesDagger , Gloria Hsi§, and Diane W. Cox

From the Department of Medical Genetics, University of Alberta, Edmonton, T6G 2H7 Alberta, Canada

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have analyzed the functional effect of site-directed mutations and deletions in the copper-binding domain of ATP7B (the copper transporting P-type ATPase defective in Wilson disease) using a yeast complementation assay. We have shown that the sixth copper-binding motif alone is sufficient, but not essential, for normal ATP7B function. The N-terminal two or three copper-binding motifs alone are not sufficient for ATP7B function. The first two or three N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs when placed in the same sequence position with respect to the transmembrane channel. From our data, we propose that the copper-binding motifs closest to the channel are required for the copper-transport function of ATP7B. We propose that cooperative copper binding to the copper-binding domain of ATP7B is not critical for copper transport function, but that cooperative copper binding involving the N-terminal two or three copper-binding motifs may be involved in initiating copper-dependent intracellular trafficking. Our data also suggest a functional difference between the copper-binding domains of ATP7A and ATP7B.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Heavy metal transporting P-type ATPases (designated CPx-type ATPases) are distinguished from other P-type ATPases, by the presence of a large N-terminal metal-binding domain (1). This domain contains repeats of a GMXCXXCXXXIE motif evolutionarily conserved in heavy metal-binding proteins. Metal binding to these motifs occurs via the cysteine (C) residues (2-4). The number of motifs varies between proteins. The copper-binding domains of ATP7A (human Menkes disease protein) and ATP7B (human Wilson disease protein) contain six repeats of the copper-binding motif, whereas Ccc2p, the yeast orthologue of ATP7B/ATP7A, has an N-terminal domain containing two motifs (5-10). The copper-binding domains of ATP7A and ATP7B expressed and purified from bacteria have been shown to bind at least six atoms of copper (2, 3). These domains bind copper presented as either Cu(I) or Cu(II). However once bound, copper is found only as Cu(I). Copper binding to the N-terminal domain of ATP7B may be cooperative, based on results from competitive zinc binding experiments (3).

The solution structure of the fourth copper-binding motif of ATP7A has been solved by NMR (4). The structure of this motif was solved with silver(I) bound, which was assumed to be similar to the copper bound form. The fourth ATP7A motif folded independently, and the structure closely resembled the overall fold of the MerP mercury-binding protein which has a mercury-binding domain well conserved at the sequence level with the copper-binding motifs found in CPx-type ATPases. The structure of the fourth ATP7A motif likely represents the prototypical fold of copper-binding motifs found in CPx-type ATPases. Each motif may fold individually, and then fold again with respect to the other motifs to form the complete copper-binding domain.

There have been several hypotheses presented regarding the role of copper binding to the copper-binding domain in the overall function of CPx-type ATPases. The copper-binding domain has been proposed to remove copper from cytosolic ligands and transiently bind copper prior to transport (5, 11). A recent paper substantiated this hypothesis (12). Atx1p is a yeast copper chaperone protein required upstream of Ccc2p for iron and copper homeostasis in yeast (13). Yeast two-hybrid analysis demonstrated that Atx1p, which contains one copper-binding motif, was able to directly interact with the putative copper-binding domain of Ccc2p, but no other predicted domain of Ccc2p (12). This interaction was dependent on copper ions and suggested that Atx1p could donate copper to Ccc2p by direct interaction and copper exchange between homologous GMXCXXCXXXIE motifs (12). Since a human orthologue of Atx1p exists (ATOX1: originally designated HAH1) and was able to functionally replace Atx1p (14), likely ATP7B also requires a copper chaperone, probably ATOX1, as its major source of copper for transport. These data support the hypothesis that copper removed from a cytosolic chaperone protein and transiently bound to the ATP7B copper-binding domain is the source of copper for subsequent transport.

Another proposed role for the copper-binding domain is that of a copper sensor (3, 5, 15). When mammalian cells were exposed to high concentrations of copper, ATP7A and ATP7B underwent a reversible copper-regulated trafficking event, from the trans-Golgi network to the plasma membrane or a post-Golgi vesicular compartment, respectively (15, 16). This trafficking may represent a change in physiologic function of ATP7B from cupro-enzyme biosynthesis in the Golgi apparatus, to a copper efflux function in the plasma membrane or secretory vesicles. The observed trafficking event may be triggered by conformation changes induced by cooperative copper binding to the N-terminal domains of these proteins (3).

The metal binding properties of the ATP7B copper-binding domain are beginning to be understood. However, the functional significance of copper binding to the copper-binding domain of ATP7B is not yet well characterized. In this study we describe the functional consequences of mutations and deletions in the copper-binding domain of ATP7B in an attempt to further understand the role of this domain in the overall copper transport function of ATP7B.

The assay used in our study is based on complementation of ccc2 mutant yeast by ATP7B, an assay we have previously used to determine the effect of Wilson disease missense mutations on ATP7B function (17). In yeast Saccharomyces cerevisiae, the plasma membrane protein Ctr1p transports copper into the cytoplasm, where it is carried by the copper chaperone Atx1p to Ccc2p (13, 18). Copper is supplied by Ccc2p, across the membrane of a post-Golgi vesicular compartment (19), to the multi-copper containing oxidase Fet3p. Fet3p functions, at the plasma membrane, together with the high-affinity iron transporter Ftr1p to import iron (20). When yeast cells lack Ccc2p, copper is not incorporated into Fet3p, and subsequently the cells lack high-affinity iron uptake leaving them unable to grow on iron-limited medium (10, 20). We have shown that ATP7B is able to complement the yeast mutant ccc2, delivering copper to Fet3p, thereby restoring the ability of ccc2 mutant yeast cells to grow on iron-limited medium, providing a sensitive assay for the copper transporter function of ATP7B (17).

Similar yeast complementation assays have recently been used to study the functional effect of mutations in the copper-binding domain of ATP7A (21) and the effect of deletions in the copper-binding domain of ATP7B (22). Our study represents the most comprehensive analysis to date of the copper-binding domain of ATP7B. Additionally, our data suggest a functional difference between the copper-binding domains of ATP7B, and that reported for ATP7A.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains-- The wild-type yeast strain used in all experiments was the protease-deficient S. cerevisiae strain BJ2168 (MATa pep4-3 prc1-407 prb1-1122 ura3-52 trp1 leu2) (23). The ccc2 mutant yeast stain was made as described previously (17). Transformations were performed using a modified lithium acetate method (24).

Expression Constructs-- The full-length 4.5-kilobase ATP7B cDNA was constructed as described previously (17). Site-directed mutagenesis (QuickChange, Stratagene) of the ATP7B cDNA was carried out using synthesized oligonucleotides which carried the desired codon changes. Both of the cysteines (C) within each of the six GMXCXXCXXXIE heavy metal binding motifs were mutated on separate cDNAs to serine (S). Mutant cDNA fragments were restriction enzyme digested using natural restriction sites found in the ATP7B coding region, gel purified, then re-ligated, in different combinations to create the full-length ATP7B mutant constructs. The copper-binding domain deletion (Cudel) was created by polymerase chain reaction amplification of two fragments. A 5'-fragment containing nucleotides 1-189 and a 3'-fragment containing nucleotides 1797-2847 were joined at an artificial XhoI site, deleting nucleotides 190-1796 which encode the six metal-binding motifs. Construct Cu1-5del was made by ligating the 5' 189-base pair fragment to the natural XhoI site (nucleotide position 1621) of ATP7B deleting copper motifs 1-5. The constructs Cu3-6del and Cu4-6Del were made by polymerase chain reaction amplifying nucleotides 1-555 and 1-897, respectively, incorporating an XhoI site at the 3' end of each fragment. These were ligated onto the artificial XhoI site of Cudel to create the final constructs. Cu3-5del was constructed by ligating the 555-base pair fragment to the natural XhoI site of ATP7B. All mutated or deleted constructs were sequenced to confirm that there were no secondary mutations. For expression of ATP7B in yeast, cDNAs were cloned into a multicopy 2-µm replication origin vector pG3 (25) and a single copy integrating vector pG4 (17). These vectors transcribe from the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. They utilize a phosphoglycerate kinase terminator and polyadenylation sequence, and a tryptophan selectable marker. Expression vectors were transformed into yeast and tryptophan auxotrophy was used to select transformants. Genomic DNA isolated from yeast strains carrying pG4 constructs was analyzed by Southern blotting to confirm that the constructs were correctly integrated as a single copy.

Complementation Assay and Oxidase Assay-- The complementation and oxidase assays were performed as described previously in detail (17). In brief, for the complementation assay, an equal number of cells from each strain was plated onto iron-limited medium (-Fe; iron limited by addition of the iron chelator ferrozine) to assay high-affinity iron uptake deficient phenotype of ccc2 mutant yeast expressing ATP7B mutant constructs. The mutant yeast could be rescued by addition of excess iron or copper to the iron-limited medium. Iron-sufficient medium (+Fe) or copper-sufficient medium (+Cu) served as controls for cell viability. Cells were grown on assay media for 48 h at 30 °C prior to photography. Growth rates of yeast strains in iron-limited medium were used in some instances to quantitate the relative ability of mutant ATP7B constructs to complement ccc2 mutant yeast. To measure growth curves, saturated yeast cultures grown in SD medium (standard synthetic dextrose medium lacking tryptophan) were resuspended in iron-limited medium and grown overnight. These cultures were used to innoculate fresh iron-limited medium to a cell density of OD600 = 0.1. Growth at 30 °C was monitored spectroscopically for 24 h and measurements were taken at 0, 3, 6, 12, and 24 h. Growth rates were calculated from the linear exponential growth phase between 3 and 12 h from triplicate experiments. For the oxidase assay, yeast membrane proteins were prepared by glass bead homogenization of yeast cells followed by differential centrifugation. Holo-Fet3p (Fet3p copper loaded in vivo by Ccc2p or ATP7B) oxidase activity was detected by preparing yeast membrane proteins in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent artifactual copper loading of the protein during homogenization. Total-Fet3p (apo- and holo-Fet3p activity combined) oxidase activity was measured in yeast membrane protein extracts prepared in copper buffer to reconstitute any apo-Fet3p in vitro. 30 µg of Triton X-100 solubilized membrane proteins were run on an SDS-polyacrylamide electrophoresis gel without prior reduction or heat denaturation. The gels were equilibrated in oxidase buffer containing glycerol to renature the Fet3p protein, and then the gels were soaked in oxidase buffer containing p-phenylenediamine dihydrochoride substrate. Treated gels were incubated overnight in a dark, humidified box to develop bands of oxidized substrate corresponding to Fet3p oxidase activity in the gels.

Polyclonal Antibody against ATP7B-- A rabbit polyclonal antiserum against the C-terminal 10-kDa fragment of ATP7B was prepared as described (17). Purification of specific antibody from the antiserum was done by affinity chromatography. Seven mg of the purified histidine-tagged fusion protein, used as the antigen for antiserum production, was coupled to a 5-ml gel volume of N-hydroxysuccinimide-activated Sepharose column (Hi-trap, Pharmacia) according to the manufacturer's protocol, except that the coupling buffer contained 100 mM guanidine-HCl to maintain solubility of the fusion protein. One milliliter of rabbit antiserum was diluted to 5 ml in TBS and then applied to the affinity column. After 3 h incubation at room temperature, the column was washed extensively with TBS and the bound antibody eluted with 6-column volumes of Gentle Ab/Ag Elution buffer (Pierce Chemical Co.). The eluted antibody was dialyzed against TBS and then concentrated using an Amicon ultrafiltration device with a 30-kDa molecular mass cut-off. The concentrated antibody was stored in TBS buffer containing 0.1% bovine serum albumin, 0.1% Thimersol (as preservative), and 50% glycerol. The purified antibody was designated "anti-ATP7B.C10."

Protein Preparations and Western Blotting-- Solubilized yeast membrane proteins (10 µg) prepared for the Fet3p oxidase assay were mixed with Laemmli loading buffer (26) containing dithiothreitol. The samples were not heated prior to electrophoresis to reduce ATP7B aggregation. SDS-polyacrylamide gel electrophoresis was performed using 7.5% polyacrylamide gels. Protein was transferred electrophoretically to polyvinylidene difluoride membrane for 450 Volt hours in Towbin buffer (27) containing 15% methanol and 0.01% SDS. Western blotting was performed using anti-ATP7B.C10 as primary antibody at a 1/7,500 dilution. Secondary antibody was horseradish peroxidase-conjugated goat anti-rabbit antibody at a dilution of 1/10,000 (Pierce Chemical Co.). Bound antibody was detected by ECL using Supersignal substrate (Pierce).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The copper-binding domains of the mutant ATP7B expression constructs analyzed for function in this study are shown in Fig. 1. All constructs were expressed in ccc2 mutant yeast from the single copy integrating vector unless otherwise noted.


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Fig. 1.   ATP7B expression constructs. Normal copper-binding motifs (CXXC) are indicated by open squares. Crossed squares denote copper-binding motifs in which the cysteine residues were mutated to serine (SXXS). Deleted regions of ATP7B are indicated by open bars. The upright black bar represents the first transmembrane segment. The schematics are oriented N- to C-terminal (L-R) and are not to scale. Only the copper-binding domains of the full-length constructs are shown.

Series 1 constructs were those in which both cysteine residues in each of the copper-binding motifs were mutated to serine sequentially from the N-terminal to C-terminal end of the copper-binding domain (Fig. 1). These were analyzed for function based on their ability to complement the high affinity iron uptake deficiency phenotype of ccc2 mutant yeast. When expressed in yeast, all proteins but Cu1-6C/S were able to complement the ccc2 yeast mutant allowing the cells to grow on iron-limited medium (Fig. 2A). We have shown previously that mutant proteins unable to fully complement ccc2 mutant yeast at single copy expression levels could often complement when overexpressed from a multicopy vector (17). Overexpression from the multicopy vector produced approximately 30-fold more ATP7B protein than from the single copy vector, and saturated the yeast cell membranes with ATP7B protein (data not shown). A mutant protein which failed to complement when expressed in multicopy was considered completely non-functional. Cu1-6C/S also failed to complement when expressed from the multicopy vector indicating that the mutant protein was nonfunctional (data not shown).


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Fig. 2.   Complementation of ccc2 mutant yeast by ATP7B copper-binding domain mutation series 1 constructs (Fig. 1). A, plating assays were performed as described under "Experimental Procedures." B, Fet3p oxidase assays were performed as described under "Experimental Procedures." Holo-Fet3p activity, Fet3p copper loaded in vivo, was detected by homogenizing yeast in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent artifactual copper loading of apo-Fet3p during processing. Total-Fet3p activity, holo-Fet3p plus apo-Fet3p activity, was detected by homogenizing yeast in the presence of copper to reconstitute apo-Fet3p in vitro. Western blots were done, using anti-ATP7B.C10 antibody, on 10 µg of solubilized membrane protein prepared for the oxidase assay.

Fet3p oxidase assays were employed to measure the function of Series 1 ATP7B copper-binding domain mutants. Fet3p receives its copper from Ccc2p within a vesicular compartment and therefore serves as a marker protein for copper transport across the vesicular membrane (19). In wild-type yeast and in yeast expressing normal ATP7B, there is little difference between holo- and total-Fet3p activity indicating little or no excess apo-Fet3p production. If high affinity iron uptake is reduced or absent, Fet3p expression is induced in an attempt to compensate (28), therefore a high ratio of total-Fet3p to holo-Fet3p activity indicates an ATP7B mutant protein with absent or reduced function (17). The Fet3p assay results for the first series of ATP7B mutant proteins showed, with the exception of Cu1-6C/S, little or no difference between holo- and total-Fet3p activity indicating that the first series of copper-binding domain mutant ATP7B proteins analyzed have activity comparable to normal ATP7B (Fig. 2B). Only the sixth motif was necessary for ATP7B function. Cu1-6C/S protein was nonfunctional judged by its inability to generate detectable holo-Fet3p activity, and the high level of total-Fet3p activity.

We have shown previously, by measuring growth curves, that ccc2 mutant yeast expressing ATP7B grows at a rate equal to the wild-type strain in iron-limited medium (17). We were able to quantitate differences in the ability of mutant ATP7B proteins to complement ccc2 mutant yeast, which was useful as a relative measure of ATP7B function. Growth rates, in iron-limited medium, of yeast expressing ATP7B and Cu1-5C/S were calculated from the linear exponential phase of growth curves done in triplicate (Table I). The ccc2 yeast strain expressing the Cu1-5C/S grew at a rate identical to normal ATP7B. These results confirm that only the sixth copper-binding motif was required for normal transport activity of ATP7B.

                              
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Table I
Growth rates of ccc2 mutant yeast expressing ATP7B copper-binding domain mutant proteins
Growth curves in iron-limited medium were generated over 24-h period. Optical density at 600 nm of triplicate cultures was measured at times 0, 3, 6, 12, and 24 h. Growth rates were calculated from the linear exponential growth phase of the cultures using the 3-, 6-, and 12-h time points.

To determine if copper-binding motifs at the N-terminal end of the copper-binding domain were sufficient for function, Series 2 copper-binding domain mutants were made (Fig. 1). Cu4-6C/S and Cu3-6C/S were made in which the cysteines of copper-binding motifs four to six and three to six were mutated to serine, respectively. These proteins, when expressed, did not restore the ability of ccc2 mutant yeast to grow on iron-limited medium, and generated no detectable holo-Fet3p activity, but had a high total-Fet3p activity indicating they were non-functional (Fig. 3). Additionally, expression of these constructs from the multicopy vector failed to complement ccc2 mutant yeast (data not shown). However, Cu3-5C/S was able to complement ccc2 mutant yeast and deliver copper to Fet3p as well as normal ATP7B, as indicated by similar levels of holo- and total-Fet3p oxidase activity (Fig. 3). These data demonstrate that two or three N-terminal motifs alone are not sufficient for ATP7B function, and reinforce the functional importance of the sixth copper-binding motif.


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Fig. 3.   Complementation of ccc2 mutant yeast by ATP7B copper-binding domain mutation series 2 constructs (Fig. 1). A, plating assays were performed as described under "Experimental Procedures." B, Fet3p oxidase assays were performed as described under "Experimental Procedures." Holo-Fet3p activity, Fet3p copper loaded in vivo, was detected by homogenizing yeast in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent adventitious copper loading of apo-Fet3p during processing. Total-Fet3p activity, holo-Fet3p plus apo-Fet3p activity, was detected by homogenizing yeast in the presence of copper to reconstitute apo-Fet3p in vitro. Western blots were done, using anti-ATP7B.C10 antibody, on 10 µg of solubilized membrane protein prepared for the oxidase assay.

To determine if the sixth copper-binding motif was essential for ATP7B function, a construct was made (Cu6C/S) in which the cysteine residues within the sixth motif only were mutated to serine (Fig. 1). This protein was able to complement ccc2 mutant yeast, and deliver copper to Fet3p as well as normal ATP7B judged by the similar levels of holo- and total-Fet3p oxidase activity (Fig. 3). These results indicate that the sixth copper-binding motif is sufficient but not essential for ATP7B function.

The rodent orthologues of ATP7B all are lacking the fourth copper-binding motif (29, 30). However, the spacing and sequence properties between the third and fifth motif are conserved leading to the conclusion that the copper binding property of the fourth motif is not essential for function. As expected, Cu4C/S complemented ccc2 mutant yeast cells, allowing normal growth on iron-limited medium, and delivered copper to Fet3p as well as normal ATP7B (ratio of holo- and total-Fet3p oxidase activity similar to ATP7B; Fig. 3).

A series of copper-binding domain deletion constructs were analyzed for function. Cu4-6del and Cu3-6del were made so that the third or second copper-binding motifs, respectively, were in the same position relative to the beginning of the first membrane spanning segment as was the sixth copper-binding motif. Neither of these constructs could restore the ability of ccc2 mutant yeast to grow on iron-limited medium (Fig. 4A). Expression of Cu4-6del or Cu3-6del proteins generated no detectable holo-Fet3p activity, but had a high level of total-Fet3p activity indicating they were nonfunctional (Fig. 4B). These data indicate that the N-terminal motifs of the copper-binding domain are not equivalent to, and cannot replace, the C-terminal motifs. Deletion of the third to fifth copper-binding motifs (Cu3-5del) resulted in a protein unable to complement ccc2 mutant yeast (Fig. 4A). Cu3-5del protein was nonfunctional, generating no detectable holo-Fet3p activity, and a high level of total-Fet3p activity (Fig. 4B). None of these deletion constructs could complement ccc2 mutant yeast when overexpressed from the multicopy vector, further supporting that they were nonfunctional (data not shown).


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Fig. 4.   Complementation of ccc2 mutant yeast by ATP7B copper-binding domain deletion series constructs (Fig. 1). A, plating assays were performed as described under "Experimental Procedures." B, Fet3p oxidase assays were performed as described under "Experimental Procedures." Holo-Fet3p activity, Fet3p copper loaded in vivo, was detected by homogenizing yeast in buffer containing the copper chelator bathocuproine disulfonate and reducing agent ascorbate to prevent artifactual copper loading of apo-Fet3p during processing. Total-Fet3p activity, holo-Fet3p plus apo-Fet3p activity, was detected by homogenizing yeast in the presence of copper to reconstitute apo-Fet3p in vitro. Western blots were done, using anti-ATP7B.C10 antibody, on 10 µg of solubilized membrane protein prepared for the oxidase assay.

Cu1-5del was able to complement ccc2 mutant yeast (Fig. 4). The mutant protein delivered copper to Fet3p, and restored the ability of the mutant yeast strain to grow on iron-limited medium. Growth curve analysis revealed that Cu1-5del protein allowed ccc2 mutant yeast to grow at a rate 95% of normal ATP7B expressing ccc2 mutant yeast (Table I). Deletion of the entire copper-binding domain (Cudel) resulted in a protein unable to complement ccc2 mutant yeast (Fig. 4) even when overexpressed from a multicopy vector (data not shown). Expression of Cudel generated no detectable holo-Fet3p activity, but had a high level of total-Fet3p activity indicating the protein was nonfunctional.

Judged by Western blot analysis, several mutant proteins appeared to have a higher steady-state protein level with respect to normal ATP7B (Figs. 2B, 3B, and 4B). This result was reproduced in multiple protein preparations (membrane and total cell extracts) and Western blotting experiments. All of these mutant constructs were confirmed to be correctly integrated in single copy by Southern blot analysis of genomic DNA isolated from these strains probed with ATP7B cDNA (data not shown). In most cases, the mutants exhibiting higher protein levels do not complement ccc2 mutant yeast. Constructs Cu1-3C/S, Cu1-4C/S, and Cu3-5C/S do fully complement ccc2 mutant yeast and the proteins appeared to be more abundant relative to normal ATP7B. However, since Cu1-5C/S, which was detected at a level equal to normal ATP7B was able to fully complement ccc2 mutant yeast, we believe that the relatively high steady-state protein level of these constructs was not significant to our results. We are currently investigating the cause for the increased protein levels observed.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have used the yeast complementation assay developed in our laboratory to study the functional consequences of mutation and deletions in the copper-binding domain of ATP7B. Fet3p serves as a marker enzyme for the putative copper-transport function of Ccc2p across an as yet unidentified post-Golgi membrane (19). ATP7B is able to replace Ccc2p in yeast demonstrating that it too is a putative copper transporter. ATP-dependent copper uptake has been demonstrated in basolateral membranes of rat and human liver (31, 32), and in Golgi membranes from rat hepatocytes (33). However, these activities have not been specifically assigned to Atp7b. Recent reports have shown that expression of ATP7B in Menkes patient fibroblast cell lines was able to reduce copper accumulation in these cells (34, 35). In light of these observations, together with the impaired hepatic copper efflux found in patients with Wilson disease in which ATP7B is mutated (36), ATP7B appears to be copper transporter. However, copper transport by ATP7B remains to be directly demonstrated as has been done for ATP7A (37).

The copper-binding domain of ATP7B was required absolutely for its copper transport function. Mutation or deletion of all six copper-binding motifs resulted in a protein unable to complement ccc2 mutant yeast or deliver copper to Fet3p (Figs. 2 and 4). These data indicate that binding of copper to this domain is a requirement for transport across membranes by ATP7B. Copper transiently bound to the copper-binding domain, perhaps delivered by a chaperone protein, is likely the source of copper transferred to the transmembrane domain for subsequent transport.

Our data indicate that the copper-binding motifs of the copper-binding domain nearer to the transmembrane domain of ATP7B were more important for the copper transporting activity of ATP7B than were the more N-terminal ones. Only the sixth motif was necessary, but not essential, for normal function of the protein (Figs. 2 and 4 and Table I). Mutation or deletion of the first five motifs resulted in a protein with normal or near normal function. However, the presence of the first two or three N-terminal motifs alone was not sufficient for function (Fig. 3). This result is consistent with the fact that most prokaryotic and lower eukaryotic CPx-type ATPases have only one to three heavy-metal binding motifs, suggesting that motifs close to the transmembrane domain are required for copper delivery to the channel prior to transport through the membrane (1). However, our results are considerably different from results reported by Payne and Gitlin (21). That paper presented a series of copper-binding domain mutations in ATP7A, which corresponded to our mutation Series 1 constructs, analyzed by complementation of ccc2 mutant yeast. The authors found that mutation (cysteine to serine) of any more that the first two copper-binding motifs of ATP7A resulted in complete loss of ATP7A function. These results suggested that the N-terminal motifs of the ATP7A copper-binding domain are more critical for function than the C-terminal motifs. When compared with our results, this suggests a structural or functional difference between the copper-binding domains of ATP7A and ATP7B. Sequence alignments between the copper-binding domains of ATP7A and ATP7B reveal that while the copper-binding motifs are highly conserved, the spacing between them is not (9). There are several, multiple amino acid insertions and deletions in the ATP7A domain compared with ATP7B. Most notably there is a large 78-amino acid insertion between copper-binding motifs one and two, and an 18-amino acid deletion between copper-binding motifs four and five. Overall, motifs two to six of ATP7A are more closely spaced than ATP7B, with the first motif of ATP7A being further N-terminal in primary structure. Additionally, the sixth copper-binding motif of ATP7B is closer in primary structure to its first predicted transmembrane segment, in relation to the sixth motif of ATP7A to its first predicted transmembrane segment (5, 9). This difference in motif spacing between ATP7A and ATP7B may represent a different domain structure between the two proteins. As a result, the N-terminal motifs of the ATP7A copper-binding domain may play a more critical role in copper transport than in ATP7B.

Lutsenko et al. (2) reported that during purification of a fusion protein between the maltose-binding protein and the N-terminal copper-binding domain of ATP7B, a major proteolytic fragment was co-purified. This fragment was recognized by antibodies against the copper-binding domain, and was able to bind to metal-chelate chromatography resin charged with copper. Based on these observations and molecular weight estimates, the degradation product was judged to contain two or three copper-binding motifs and was proposed to represent a proteolytically sensitive subdomain of the copper-binding domain (2). We also observed consistent degradation of full-length ATP7B protein when isolating 100,000 × g membrane pellets from yeast (data not shown). Based on molecular weight estimates, this degradation fragment would contain two or three copper-binding motifs. To test the hypothesis that the copper-binding domain may be divided into two subdomains, and to determine if these domains were functionally interchangeable, ATP7B deletion constructs were used (Fig. 1). Constructs in which either the second or third copper-binding motif were placed in the same position as the sixth (Cu3-6del and Cu4-6del, respectively), were unable to complement ccc2 mutant yeast or deliver copper to Fet3p indicating that they were nonfunctional. These data suggest that the N-terminal copper-binding motifs of the ATP7B copper-binding domain are not functionally identical to, and cannot replace, the C-terminal motifs even when placed in the same sequence position. The copper-binding motifs from the N-terminal end of the copper-binding domain may fold, with respect to each other, such that the orientation of the copper-binding cysteine residues was not correct to deliver copper to the transmembrane domain of ATP7B for transport. This hypothesis is supported by the result from Cu3-5del. This ATP7B deletion mutant was unable to complement ccc2 mutant yeast even when overexpressed from a multicopy vector suggesting that is was unable to transport copper. Since Cu1-5del, with the sixth motif alone intact was able to complement ccc2 mutant yeast, addition of copper-binding motifs one and two to the sixth motif may change the overall fold of the mutant copper-binding domain so that even the sixth motif was no longer in the correct position to deliver copper to the transmembrane channel. These results support the hypothesis that the N-terminal two or three copper-binding motifs may represent an independently, differently folded second subdomain of the ATP7B copper-binding domain. These data also suggest that, while the folded structure of individual copper-binding motifs is likely conserved (4), it is the fold of multiple motifs with respect to each other and the transmembrane channel that is essential for the overall function of the copper-binding domain.

Iida et al. (22) used deletion analysis and a yeast complementation assay similar to ours to study the copper-binding domain of ATP7B. The authors sequentially deleted copper-binding motifs from the C to N terminus of the copper-binding domain. They found that none of these constructs, including one in which the sixth motif alone was deleted, were able to complement ccc2 mutant yeast. A construct containing only the sixth motif (one to five deleted) was functional. From these data it was concluded that the sixth motif was essential for ATP7B copper transport function. This conclusion does not agree with our data since we have shown that mutation of the sixth repeat only does not affect ATP7B function, indicating that the motif is important but not essential. Since Iida et al. analyzed only deletion constructs, probably the spacing and folding orientation with respect to the transmembrane channel provided by the sixth motif is essential for ATP7B function, rather than its copper binding capacity.

Our data has shown that mutant ATP7B protein with only one copper-binding motif is capable of fully complementing the iron-uptake deficiency of the ccc2 mutant yeast. Biochemical evidence suggests that copper binding to the copper-binding domain of ATP7B may be cooperative (3). If the observed cooperative binding was required for copper transport, one motif alone would not be predicted to be sufficient for normal function. We propose that in light of our results, cooperative copper binding is not critical for copper transport function of ATP7B. Instead, cooperative copper binding to the N-terminal domain may induce conformation changes in the protein, which acts as a signal to initiate copper-induced trafficking from the trans-Golgi network to the membrane vesicles or plasma membrane. Preliminary results support this hypothesis. DiDonato et al.1 have observed, using circular dichroism spectroscopy, that the apo-form of purified ATP7B copper-binding domain protein had secondary structure that consisted of mostly beta -type structures (beta -sheets, beta -turns, etc.). Upon addition of a 2-fold molar excess of copper to the apo-protein, secondary structure switched to predominantly alpha -helix. Further addition of copper had little effect on secondary structure, however, the tertiary structure changed. These data were supported by quenching of tryptophan fluorescence upon addition of copper to the apo-protein. Therefore, conformation changes in the copper-binding domain may be acting cooperatively as a copper sensor to trigger movement of ATP7B out of the trans-Golgi network in response to excess cellular copper. Although cooperative copper binding to the N-terminal domain of ATP7A has not been demonstrated, cooperativity may explain the apparent functional difference between ATP7A and ATP7B copper-binding domains that we have observed. ATP7A may rely on cooperative copper binding for normal transport activity, perhaps explaining the observed complete loss of function after mutation of only the first three copper-binding motifs (22).

Our data may also explain in part why ATP7B has six copper-binding motifs while most other CPx-type ATPase have only one to three. For example, Ccc2p has only two copper-binding motifs and does not alter its subcellular localization in response to copper (19). Based on our data, we propose that the copper-binding motifs closest to the transmembrane channel of ATP7B are directly involved in copper transport, transferring copper to residues within the channel, for subsequent translocation across the membrane. The remaining N-terminal motifs may not be directly involved in copper transport. Instead they may act cooperatively to induce conformational changes in the domain, sensing cytosolic copper concentrations, thereby inducing redistribution of ATP7B within the cell.

    ACKNOWLEDGEMENTS

We thank Michael DiDonato and Bibudhendra Sarkar for sharing unpublished data to include in this manuscript. We also thank Steven Moore for critical review of this manuscript.

    FOOTNOTES

* This work was supported in part by grants from the National Science and Research Council, Canada, and the Canadian Genetic Diseases Network.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.

Dagger Supported by a graduate scholarship from the Medical Research Council, Canada.

§ Supported by a graduate scholarship from National Science and Research Council, Canada.

To whom correspondence should be addressed: Dept. of Medical Genetics, 8-39, Medical Sciences Bldg., University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-7501; Fax: 780-492-1998; E-mail: diane.cox{at}ualberta.ca.

1 M. DiDonato and B. Sarkar, personal communication.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

  1. Solioz, M. (1998) in Advances in Molecular and Cell Biology (Bittar, E. E., and Anderson, J. P., eds), pp. 167-203, JAI Press, London
  2. Lutsenko, S., Petrukhin, K., Cooper, M. J., Gilliam, C. T., and Kaplan, J. H. (1997) J. Biol. Chem. 272, 18939-18944[Abstract/Free Full Text]
  3. DiDonato, M., Narindrasorasak, S., Forbes, J. R., Cox, D. W., and Sarkar, B. (1997) J. Biol. Chem. 272, 33279-33282[Abstract/Free Full Text]
  4. Gitschier, J., Moffat, B., Reilly, D., Wood, W. I., and Fairbrother, W. J. (1998) Nat. Struct. Biol. 5, 47-54[Medline] [Order article via Infotrieve]
  5. Vulpe, C., Levinson, B., Whitney, S., Packman, S., and Gitschier, J. (1993) Nat. Genet. 3, 7-13[Medline] [Order article via Infotrieve]
  6. Mercer, J. F. B., Livingstone, J., Hall, B., Paynter, J. A., Begy, C., Chandrasekharappa, S., Lockhart, P., Grimes, A., Bhave, M., Siemieniak, D., and Glover, T. W. (1993) Nat. Genet. 3, 20-25[Medline] [Order article via Infotrieve]
  7. Chelly, J., Tumer, Z., Tonnesen, T., Petterson, A., Ishikawa-Brush, Y., Tommerup, N., Horn, N., and Monaco, A. P. (1993) Nat. Genet. 3, 14-19[Medline] [Order article via Infotrieve]
  8. Tanzi, R. E., Petrukhin, K. E., Chernov, I., Pellequer, J. L., Wasco, W., Ross, B., Romano, D. M., Parano, E., Pavone, L., Brzustowicz, L. M., Devoto, M., Peppercorn, J., Bush, A. I., Sternlieb, I., Pirastu, M., Gusella, J. F., Evgrafov, O., Penchaszadeh, G. K., Honig, B., Edelman, I. S., Soares, M. B., Scheinberg, I. H., and Gilliam, T. C. (1993) Nat. Genet. 5, 344-350[Medline] [Order article via Infotrieve]
  9. Bull, P. C., Thomas, G. R., Rommens, J. M., Forbes, J. R., and Cox, D. W. (1993) Nat. Genet. 5, 327-337[Medline] [Order article via Infotrieve]
  10. Yuan, D. S., Stearman, R., Dancis, A., Dunn, T., Beeler, T., and Klausner, R. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2632-2636[Abstract]
  11. Bull, P. C., and Cox, D. W. (1994) Trends Genet. 10, 246-252[CrossRef][Medline] [Order article via Infotrieve]
  12. Pufahl, R. A., Singer, C. P., Peariso, K. L., Lin, S. J., Schmidt, P. J., Fahrni, C. J., Culotta, V. C., Penner-Hahn, J. E., and O'Halloran, T. V. (1997) Science 278, 853-856[Abstract/Free Full Text]
  13. Lin, S.-J., Pufahl, R. A., Dancis, A., O'Halloran, T. V., and Culotta, V. C. (1997) J. Biol. Chem. 272, 9215-9220[Abstract/Free Full Text]
  14. Klomp, L. W. J., Lin, S.-J., Yuan, D. S., Klausner, R. D., Culotta, V. C., and Gitlin, J. D. (1997) J. Biol. Chem. 272, 9221-9226[Abstract/Free Full Text]
  15. Petris, M. J., Mercer, J. F. B., Culvenor, J. G., Lockhart, P., Gleeson, P. A., and Camakaris, J. (1996) EMBO J. 15, 6084-6095[Abstract]
  16. Hung, I. H., Suzuki, M., Yamaguchi, Y., Yuan, D. S., Klausner, R. D., and Gitlin, J. D. (1997) J. Biol. Chem. 272, 21461-21466[Abstract/Free Full Text]
  17. Forbes, J. R., and Cox, D. W. (1998) Am. J. Hum. Genet. 63, 1663-1674[CrossRef][Medline] [Order article via Infotrieve]
  18. Dancis, A., Yuan, D. S., Haile, D., Askwith, C., Eide, D., Moehle, C., Kaplan, J., and Klausner, R. D. (1994) Cell. 76, 393-402[Medline] [Order article via Infotrieve]
  19. Yuan, D. S., Dancis, A., and Klausner, R. D. (1997) J. Biol. Chem. 272, 25787-25793[Abstract/Free Full Text]
  20. Stearman, R., Yuan, D. S., Yamaguchi-Iwai, Y., Klausner, R. D., and Dancis, A. (1996) Science 271, 1552-1557[Abstract]
  21. Payne, A. S., and Gitlin, J. D. (1998) J. Biol. Chem. 273, 3765-3770[Abstract/Free Full Text]
  22. Iida, M., Terada, K., Sambongi, Y., Wakabayashi, T., Miuna, N., Koyama, K., Futai, M., and Sugiyama, T. (1998) FEBS Lett. 428, 281-285[CrossRef][Medline] [Order article via Infotrieve]
  23. Zubenko, G. S., Mitchell, A. P., and Jones, E. W. (1980) Genetics 96, 137-146[Abstract/Free Full Text]
  24. Elble, R. (1992) BioTechniques 13, 18-20[Medline] [Order article via Infotrieve]
  25. Schena, M., Picard, D., and Yamamoto, K. R. (1991) Methods Enzmol. 194, 389-398
  26. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
  27. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract]
  28. Askwith, C., Eide, D., Van Ho, A., Bernard, P. S., Li, L., Davis-Kaplan, S., Sipe, D. M., and Kaplan, J. (1994) Cell. 76, 403-410[Medline] [Order article via Infotrieve]
  29. Wu, J., Forbes, J. R., Shiene Chen, H., and Cox, D. W. (1994) Nat. Genet. 7, 541-545[CrossRef][Medline] [Order article via Infotrieve]
  30. Theophilos, M. B., Cox, D. W., and Mercer, J. F. (1996) Am. J. Hum. Genet. 5, 1619-1624
  31. Dijkstra, M., In t'Veld, G., van den Berg, G. J., Muller, M., Kuipers, F., and Vonk, R. J. (1995) J. Clin. Invest. 95, 412-416[Medline] [Order article via Infotrieve]
  32. Dijkstra, M., van den Berg, G. S., Wolters, H., In't Veld, G., Sloof, M. J., Heymans, H. S., Kuipers, F., and Vonk, R. J. (1996) J. Hepatol. 25, 37-42[CrossRef][Medline] [Order article via Infotrieve]
  33. Bingham, M. J., Ong, T. J., Ingledew, W. J., and McArdle, H. J. (1996) Am. J. Physiol. 271, G741-G746[Abstract/Free Full Text]
  34. La Fontaine, S., Firth, S. D., Camakaris, J., Englezou, A., Theophilos, M. B., Petris, M. J., Howie, M., Lockhart, P. J., Greenough, M., Brooks, H., Reddel, R. R., and Mercer, J. F. B. (1998) J. Biol. Chem. 273, 31375-31380[Abstract/Free Full Text]
  35. Payne, A. S., Kelly, E. J., and Gitlin, J. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 10854-10859[Abstract/Free Full Text]
  36. Danks, D. M. (1995) in The Metabolic and Molecular Basis of Inherited Disease (Scriver, C. R., Beaudet, A. L., Sly, W. S., and Valle, D., eds), pp. 2211-2235, McGraw-Hill, New York
  37. Voskoboinik, I., Brooks, H., Smith, S., Shen, P., and Camakaris, J. (1998) FEBS Lett. 435, 178-182[CrossRef][Medline] [Order article via Infotrieve]


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