From the Department of Medical Genetics, University of Alberta, Edmonton, T6G 2H7 Alberta, Canada
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ABSTRACT |
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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.
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.
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 ( 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).
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.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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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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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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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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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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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.
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DISCUSSION |
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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 -type structures (
-sheets,
-turns, etc.).
Upon addition of a 2-fold molar excess of copper to the apo-protein,
secondary structure switched to predominantly
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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.
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REFERENCES |
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