From the Edward Mallinckrodt Department of Pediatrics, Washington
University School of Medicine, St. Louis, Missouri 63110
Menkes disease is a fatal neurodegenerative
disorder of childhood caused by the absence or dysfunction of a
putative P-type ATPase encoded on the X chromosome. To elucidate the
function of the Menkes disease protein, a plasmid containing the open
reading frame of the human Menkes disease gene was constructed and used to transform a strain of Saccharomyces cerevisiae deficient
in CCC2, the yeast Menkes/Wilson disease gene homologue.
ccc2
yeast are deficient in copper transport into the
secretory pathway, and expression of a wild type human Menkes cDNA
complemented this defect, as evidenced by the restoration of copper
incorporation into the multicopper oxidase Fet3p. Site-directed
mutagenesis demonstrated the essential role of four specific amino
acids in this process, including a conserved histidine, which is the
site of the most common disease mutation in the homologous Wilson
disease protein. The expression of Menkes cDNAs with successive
mutations of the conserved cysteine residues in the six amino-terminal
MXCXXC metal binding domains confirmed the
essential role of these cysteine residues in copper transport but
revealed that each of these domains is not functionally equivalent.
These data demonstrate that the Menkes disease protein functions to
deliver copper into the secretory pathway of the cell and that this
process involves biochemical mechanisms common to previously
characterized members of this P-type ATPase family.
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INTRODUCTION |
Copper is a trace element that is essential to human physiology
and the development of the central nervous system. This vital role of
copper is revealed in patients with Menkes disease, a disorder of
copper metabolism characterized by progressive neurodegeneration and
death in early childhood (1, 2). Although the pathogenesis of this
disorder is poorly understood, the Menkes disease gene has been cloned
(3-5) and identified as a member of a unique family of
metal-transporting P-type ATPases (6, 7). The Menkes protein is a
single-chain 178-kDa polypeptide that is localized to the
trans-Golgi network and undergoes a
copper-dependent relocalization in human and rodent cell
lines (8-10). Recent studies have revealed similar findings for the
homologous Wilson disease ATPase (11, 12), suggesting a common
mechanism for copper transport by these proteins into the secretory
pathway of the cell.
Despite these immunocytochemical studies, little data are currently
available regarding the function of the Menkes disease protein.
Copper-resistant rodent cell lines have been shown to overexpress the
Menkes protein, supporting the concept that this protein functions to
maintain cellular copper homeostasis (13). Saccharomyces
cerevisiae deficient in CCC2, the yeast Menkes/Wilson disease gene homologue (14), are defective in high affinity iron
transport due to the lack of copper incorporation into Fet3p, a
multicopper oxidase homologous to human ceruloplasmin (15, 16).
ccc2
yeast have been utilized to evaluate the function of
the Caenorhabditis elegans P-type ATPase (17) as well as to
demonstrate copper transport by the Wilson disease protein and to
define specific amino acid residues of the protein involved in the
delivery of copper into the yeast secretory compartment (11). As the
direct connection between holoFet3p biosynthesis and the phenotype of
yeast defective in copper transport into the secretory pathway provides
an opportunity to assay the function of putative copper-transporting
ATPases, the current study employed this approach to define the
structure and function relationship of the Menkes disease protein.
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EXPERIMENTAL PROCEDURES |
Menkes Antibody Production--
Oligonucleotide primers
synthesized with EcoRI linkers were used to amplify the
portion of the Menkes cDNA corresponding to amino acids 1046-1356
in the polymerase chain reaction
(PCR)1 using Klentaq
polymerase (CLONTECH) according to manufacturer's recommendations. The PCR product was subsequently ligated into pGEX4T-3, confirmed by automated sequencing (Perkin-Elmer), and transformed into Escherichia coli BL21 for protein
expression and purification as described (18). Briefly, cultures were
grown to an optical density at 600 nm (A600) of
0.6 absorbance units and induced with 0.1 mM isopropyl
1-thio-
-D-galactopyranoside for 3 h at 37 °C.
Harvested bacteria were treated with lysozyme and sonicated at 4 °C,
and soluble and insoluble proteins were separated by centrifugation at
16,000 × g for 20 min at 4 °C. The pellet was
redissolved in phosphate-buffered saline (PBS) containing 6 M urea and again centrifuged at 16,000 × g
for 20 min at 4 °C. The supernatant was diluted to 1 M
urea with PBS, pooled with the initial soluble fraction, and applied to
glutathione-agarose beads. Bound fusion protein was eluted with 5 mM reduced glutathione in 50 mM Tris, pH 8, and
dialyzed extensively against PBS, pH 7.4. The purified glutathione
S-transferase fusion protein was used to produce rabbit
polyclonal antisera (Animal Pharm Services), which was characterized as
described previously (8).
Cell Culture, Immunoblotting, and Immunofluorescence--
HeLa
and HepG2 cells were obtained from the American Type Culture Collection
and were maintained in Dulbecco's modified Eagle's medium containing
10% fetal bovine serum and supplemented with glutamine and
penicillin/streptomycin. Cells were lysed in 1% Nonidet P-40, 20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM
EDTA, and 10% glycerol supplemented with protease inhibitors for 20 min at 4 °C followed by centrifugation for 20 min at 16,000 × g at 4 °C. Proteins (100 µg) from the supernatant were
separated by reducing SDS-PAGE and transferred to nitrocellulose by
semi-dry transfer (Novablot). Membranes were blocked with 5% milk in
PBS and then incubated with a 1:2,000 dilution of antiserum for 1 h at room temperature, followed by several washes with PBS, 0.1% Tween-20. For blocking experiments, antisera was preincubated with an
excess of fusion protein for 2 h at 4 °C. Membranes were then
incubated with horseradish peroxidase-coupled secondary antibody, washed, and developed using enhanced chemiluminescence reagent (Amersham Life Science, Corp.) according to the manufacturer's protocol. For indirect immunofluorescence, cells were grown on coverslips, fixed in ice-cold acetone, and analyzed as described previously (8, 11).
Construction and Mutagenesis of the Menkes cDNA--
The
coding region of the human Menkes disease gene was obtained by PCR of a
human HeLa cDNA library (CLONTECH) using
oligonucleotides corresponding to known Menkes gene sequence. cDNAs
were amplified with Klentaq polymerase (CLONTECH).
The gene was isolated in three portions, and each of the gene fragments
was subcloned by T overhang into pCRII (Invitrogen): 1) initiator
ATG-BamHI (amino acids 1-591), 2)
BamHI-KpnI (amino acids 592-1107), and 3)
KpnI-stop codon TAA (amino acids 1108-1500). An
XbaI site and consensus Kozak sequence (GCCACC) were
engineered into the cDNA immediately preceding the initiator ATG,
and an XbaI site was introduced after the stop codon. A
silent mutation (T to C) was introduced at nucleotide 6 to abolish the
BamHI site at the start of the gene to allow for three-part
ligation of the fragments into the XbaI site of yeast vector
pVT103U (19). Site-directed mutagenesis was performed by PCR on the
appropriate gene fragment with Klentaq polymerase and oligonucleotide
primer pairs corresponding to the P1001A, H1086Q, A629P, G1019D, and
CXXC to SXXS mutations along with flanking 5
sense and 3
antisense oligonucleotides (20). Ligated vectors were used
to transform Top10F
(Invitrogen) and screened by restriction digest.
The presence of specific mutations and the fidelity of the entire
cDNA sequence were verified by automated fluorescent sequencing
(Perkin-Elmer) according to the manufacturer's recommendations.
Yeast Strains and Protocols--
S. cerevisiae
strains used in this study were as follows: fet3
: MAT
,
ura3-52, lys2-801, ade2-101, his3-200, leu2-
1, trp1-
1, FET3
::TRP1 (16); IHY4 (wild type): MAT
,
his3-
1, trp1-289, ura3-52, leu2, GAL; and IHY5
(ccc2
): MAT
, his3 (his3-200 or his3-
1), trp1 (trp1-
1 or
trp1-289), ura3-52, leu2, ccc2
::LEU2, GAL (11). All strains were grown at 30 °C in complete synthetic media (Bio101) or the appropriate dropout medium supplemented with 2%
glucose. IHY5 yeast cells were transformed by the lithium acetate
method (21) and selected on uracil-deficient plates. Total yeast
lysates were prepared as described previously, without boiling samples
before electrophoresis (22).
Evaluation of growth in copper- and iron-deficient media was based upon
a ferri reductase assay previously described (15). The copper- and
iron-deficient medium was previously reported (23). Yeast were grown to
saturation in the appropriate synthetic media, transferred to copper-
and iron-deficient media, grown for 6 h at 30 °C, and then
diluted to an A600 of 0.01 into parallel cultures containing either copper- and iron-deficient media or the
appropriate synthetic media. Growth was evaluated after 36-64 h by
measuring the A600 of each culture. Growth rates
were normalized by dividing the A600 in copper-
and iron-deficient media by the A600 in
synthetic media and represented as a percentage of wild type growth.
The assay for 64Cu incorporation into Fet3p was performed
as described (23). Fet3p oxidase activity in membrane extracts was
assayed as described (16), and gels were developed from 4 h to
overnight in a humid chamber at room temperature and then
photographed.
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RESULTS |
The analysis of the Menkes protein presented in this study was
guided by the previously predicted polytopic membrane structure of the
protein based on sequence homology and hydropathy analyses (3-5). Fig.
1 presents a schematic topological model
of the Menkes protein, which highlights the conserved protein domains.
The Menkes protein contains four signature motifs of the P-type
ATPases: the phosphatase domain (TGEA), an invariant aspartate residue (DKTGT), a conserved cysteine and proline (CPC)
in the proposed cation transduction channel, and an ATP binding
sequence (GDGIND). The protein also contains six characteristic copper
binding domains at its amino terminus (MXCXXC) as
well as a conserved histidine and proline within an SEHPL
motif of unknown function, which is present in the homologous Wilson
protein and in which a histidine to glutamine alteration is the most
commonly identified mutation in patients with Wilson disease (24, 25).
In addition, point mutations in the DNA of patients with Menkes disease
have been described at positions corresponding to amino acid residues
alanine 629 and glycine 1,019 (26).

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Fig. 1.
Proposed topological model of the human
Menkes protein. A schematic membrane structure of the human Menkes
protein is shown, as previously derived from hydropathy analysis of the primary structure of the Menkes protein. Conserved amino acid motifs
are indicated in circles, and residues mutated in this study
are in boldface. The dotted line marks the region
of the protein used for antibody production.
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As the Menkes-specific antiserum previously reported (8) had limited
utility in exploring amino-terminal mutations of the Menkes protein, a
rabbit polyclonal antiserum was produced against the large cytoplasmic
loop of the Menkes protein (indicated by a dotted line in
Fig. 1) to detect the expression of the wild type and mutant Menkes
proteins within cells. This antiserum recognized a protein of
approximately 178 kDa in HeLa cell lysates but not in HepG2 cell
lysates (Fig. 2), and the protein signal
was not observed when the antiserum was preincubated with excess fusion protein (Fig. 2B). A lower nonspecific band at approximately
80 kDa was also detected. Indirect immunofluorescence with the
antiserum on HeLa cells resulted in perinuclear staining (Fig.
2C), consistent with previous reports on the
trans-Golgi localization of the Menkes protein (8-10).

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Fig. 2.
Analysis of Menkes antiserum by immunoblot
and immunofluorescence. A, 100 µg of protein from cell
lysates was separated by SDS-PAGE, transferred to nitrocellulose,
incubated with Menkes antiserum, and developed with
chemiluminescence. B, parallel blot as in A,
except that Menkes antiserum was preincubated with an excess of fusion
protein. C, HeLa cells were fixed with ice-cold acetone,
incubated with Menkes antisera followed by rhodamine-labeled secondary
reagent, and viewed by epifluorescence microscopy with a 60 × PlanApo oil immersion lens.
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For the structure and function analysis of the Menkes protein, a
cDNA encompassing the open reading frame of the Menkes gene was
constructed as described under "Experimental Procedures." A yeast
expression vector harboring the Menkes cDNA was used to transform a strain of S. cerevisiae deficient in
CCC2, the yeast Menkes/Wilson disease gene homologue.
Immunoblot analysis of lysates from ccc2
(IHY5) yeast
transformed with this Menkes cDNA indicated that the transformed
yeast abundantly expressed Menkes protein (Fig.
3A, lane 3).
Differential glycosylation or aberrant migration of the protein during
SDS-PAGE under the conditions used for the yeast lysate may have
accounted for the decrease in apparent molecular weight. To evaluate
the function of the expressed protein, wild type (IHY4) yeast and
ccc2
(IHY5) yeast transformed with either vector or the
Menkes cDNA were pulse-labeled with 64Cu, and copper
incorporation into the multicopper oxidase Fet3p was directly detected
by autoradiography of membrane extracts separated by nonreducing
SDS-PAGE. As demonstrated in Fig. 3B, expression of the
Menkes protein in ccc2
yeast resulted in copper incorporation into Fet3p (lane 3) similar to the level
observed in wild type yeast (lane 1). Since the oxidase
activity of Fet3p is copper-dependent, the enzymatic
function of Fet3p from the 64Cu-labeled membrane extracts
was assayed. Fig. 3C shows that Fet3p oxidase activity
paralleled copper incorporation into Fet3p, both in wild type yeast
(lane 1) and ccc2
yeast transformed with the Menkes cDNA (lane 3). Because the assay of Fet3p oxidase
activity accurately reflected the incorporation of copper into the
protein, the copper status of Fet3p was evaluated by this method
throughout the remainder of the study.

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Fig. 3.
Functional expression of the human Menkes
protein in S. cerevisiae. A, 100 µg of protein
from human cell lysates and 20 µg of protein from ccc2
yeast transformed with the human Menkes cDNA (IHY5.MNK)
were analyzed by immunoblot with Menkes antiserum. B, IHY4
(wild type (wt)) yeast and ccc2 yeast
transformed with vector or the Menkes cDNA (MNK) were
pulse-labeled with 64Cu, 200 µg of protein from membrane
fractions were separated by nonreducing SDS-PAGE, and copper
incorporation into Fet3p was detected by autoradiography. C,
samples in B were separated by nonreducing SDS-PAGE and
analyzed for Fet3p oxidase activity as described under "Experimental
Procedures."
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To examine the structure-function relationship of the Menkes
protein, point mutations corresponding to the amino acid substitutions P1001A, H1086Q, A629P, and G1019D were introduced into the Menkes cDNA sequence (indicated in boldface in Fig. 1). In
addition, the six copper binding domains at the amino terminus of the
Menkes protein were mutated from MXCXXC to
MXSXXS to create Menkes protein mutants with
five, four, three, two, one, or no functional copper binding sites
remaining (Fig. 1). Immunoblot analyses of lysates from
ccc2
yeast transformed with these cDNAs demonstrated
that each of the mutant proteins was expressed at a level comparable to
the wild type Menkes protein (Fig. 4,
lane 2 versus lanes 3-6 and lane 8 versus lanes
9-14).

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Fig. 4.
Immunoblot analysis of human Menkes proteins
in ccc2 transformants. 40 µg of protein from
total lysates of ccc2 yeast transformed with vector or
mutant Menkes cDNAs were separated by SDS-PAGE, transferred to
nitrocellulose, incubated with Menkes antiserum, and analyzed by
chemiluminescence. MNK, Menkes cDNA; wt, wild
type.
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To evaluate the effect of these structural mutations on Menkes
protein function, the growth rate of ccc2
yeast
transformants in copper-deficient media was examined. Fet3p activity
decreases when yeast are grown in copper-deficient media, and this
decrease has been correlated to a decrease in growth rate (15).
Consistent with these findings, the growth of fet3
yeast
in copper- and iron-deficient media was 3% that of the growth of wild
type (IHY4) yeast (Fig. 5A).
Since Fet3p activity in ccc2
yeast is restored upon
expression of functional Menkes protein (Fig. 3C), the
growth of ccc2
yeast transformed with the mutant Menkes
constructs in copper- and iron-deficient media was
evaluated. ccc2
yeast transformed with vector exhibited
2% of the growth of yeast transformed with the wild type Menkes
protein, and the P1001A, H1086Q, A629P, and G1019D mutations all
demonstrated reduced growth (5, 11, 47, and 11%, respectively) as
compared with yeast expressing the wild type Menkes protein (Fig.
5B). Evaluated through the same assay, the Menkes proteins
with mutations in one or two copper binding domains exhibited 83 and
79% that of wild type growth, whereas Menkes proteins with mutations
in the first three, four, five, or all six copper binding domains
demonstrated negligible growth in the copper- and iron-deficient
media (Fig. 5C).

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Fig. 5.
Growth of ccc2 transformants
in copper- and iron-deficient media. Growth rates of
fet3 and wild type (IHY4) yeast (A) or
ccc2 yeast transformed with vector, wild type Menkes
protein, and Menkes missense mutant proteins (B) or Menkes
copper binding mutants (C) were evaluated in copper- and
iron-deficient media as described under "Experimental
Procedures." MNK, Menkes cDNA; wt, wild
type.
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The examination of growth in copper-deficient media suggested that
copper-dependent Fet3p activity was compromised in the ccc2
yeast expressing the mutant Menkes proteins. To
assess copper transport into the secretory pathway by the mutant Menkes
proteins more directly, Fet3p oxidase activity in membrane extracts of the transformed ccc2
yeast was evaluated. The expression
of Menkes mutant proteins P1001A, H1086Q, and G1019D in
ccc2
yeast did not restore copper incorporation into
Fet3p, as evidenced by negligible Fet3p oxidase activity (upper
panel of Fig. 6, lanes 5, 6, and 8). In contrast, the A629P substitution in the
Menkes protein resulted in a decrease in Fet3p oxidase activity
(lane 7) as compared with the wild type Menkes transformant
(lane 4), indicating some Fet3p copper incorporation. These
results were not due to variations in the amount of Fet3p among
transformants, as evidenced by immunoblot analysis of Fet3p on
identical membrane fractions (Fig. 6, lower panel).

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Fig. 6.
Analysis of Fet3p activity in ccc2
transformants. 200 µg of protein from membrane fractions
of fet3 , wild type, and ccc2 yeast
transformed with Menkes missense mutant proteins were evaluated for
Fet3p oxidase activity by nonreducing SDS-PAGE (upper panel)
or for Fet3p expression levels by reducing SDS-PAGE followed by
immunoblot analysis with Fet3p antiserum (lower panel).
MNK, Menkes cDNA; wt, wild type.
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The Fet3p oxidase assay was subsequently used to analyze the role of
the copper binding domains in Menkes protein function. Successive
rounds of site-directed mutagenesis on the Menkes cDNA produced
Menkes mutant proteins defective in one to six copper binding domains
as diagrammed in Fig. 7A.
Evaluation of Fet3p copper incorporation indicated that expression of
the Menkes protein lacking one copper binding domain resulted in Fet3p
oxidase activity comparable to that observed with the wild type Menkes
protein (upper panel of Fig. 7B, compare
lanes 2 and 3), the mutant Menkes protein lacking
two copper binding domains resulted in slightly less Fet3p activity
(lane 4), and the mutants lacking three to six copper
binding domains were devoid of copper-dependent Fet3p oxidase activity (lanes 6-8), despite equal expression of
Fet3p between strains as indicated by immunoblot analysis of Fet3p on identical membrane fractions (Fig. 7B, lower panel).

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Fig. 7.
Mutational analysis of Menkes copper binding
domains. A, successive rounds of site-directed mutagenesis
were performed to construct Menkes cDNAs with mutations in one to
six amino-terminal copper binding domains as indicated. B,
200 µg of protein from membrane fractions of ccc2 yeast
transformed with vector, wild type Menkes, or Menkes copper binding
mutant cDNAs were evaluated for Fet3p oxidase activity (upper
panel) or for Fet3p expression levels (lower panel) as
described under "Experimental Procedures." MNK, Menkes
cDNA; wt, wild type.
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DISCUSSION |
The data presented in this study demonstrate that the Menkes
disease protein functions to deliver copper into the secretory pathway
of eukaryotic cells. The ability of the Menkes protein to deliver
copper to Fet3p is common to Ccc2p (16) and the Wilson disease protein
(11). These findings are consistent with the original observations in
Menkes patient fibroblasts of elevated intracellular copper (27, 28),
concomitant with reduced activity of the secreted
copper-dependent enzyme lysyl oxidase (29). The data are
also consistent with recent studies on the localization of the yeast
Menkes homologue Ccc2p to a late or post-Golgi compartment (30) as well
as immunocytochemical studies that have localized the human and rodent
Menkes protein to the trans-Golgi network (8-10) where this
ATPase would be positioned to transport copper for incorporation into
nascent cupro enzymes within the secretory pathway.
The discovery that the Menkes protein can deliver copper to a
ceruloplasmin homologue, a role normally attributed to the Wilson protein in humans and Ccc2p in yeast, indicates that the Menkes protein
can interact with upstream and downstream proteins within the Fet3p
pathway of copper transport and thus appears to be functionally synonymous with other copper-transporting ATPases. This concept is
further supported by the finding that Menkes proteins harboring the
P1001A and H1086Q mutations cannot restore copper incorporation into
Fet3p (Fig. 6), comparable to previous findings with the analogous
mutants of the Wilson protein (11). The inability of the P1001A mutant
to restore Fet3p oxidase activity supports the proposed role of the
conserved proline residue in the transduction of copper through the
membrane channel formed by the copper-transporting ATPases (7). The
H1086Q Menkes mutation parallels the H1070Q mutation in the Wilson
protein, the most common point mutation identified in patients with
Wilson disease (24). Although the H1086Q mutation is not a known Menkes
disease mutation, the similar abrogation of Fet3p activity observed to
result from these Wilson and Menkes mutant proteins, together with
the conservation of the histidine among all the reported
copper-transporting ATPases, suggests that the histidine plays a
fundamental structural role in the function of these proteins, perhaps
as a site for protein-protein interaction (25). Although the Menkes
gene locus demonstrates a propensity toward frameshifts, splice site
mutations, and nonsense mutations (26, 31), the data presented here
suggest that the spectrum of mutations uncovered by patient DNA
analysis may be influenced more by chromosomal dynamics of the gene
locus rather than the biology of the transporter, since the most
commonly occurring mutation in Wilson disease similarly compromises
Menkes protein function. The common function of these ATPases has been
questioned by a recent report that certain murine cell types express
both the Menkes and the Wilson disease proteins, leading to the
proposal of disparate functions for these ATPases (32). The current
data, together with recent findings of copper-dependent
relocalization for both the Wilson and Menkes proteins in cells,
suggest that the physiologically different roles that the Menkes and
Wilson proteins assume in human biology may be attributed to the
differences in their expression patterns and not differences in
cellular function.
The A629P and G1019D disease mutations in the Menkes protein are the
only reported missense mutations that occur in predicted soluble
domains of the protein (26). Although no function has yet been
attributed to glycine 1,019, alanine 629 resides in the "stalk"
region of the Menkes protein, which is prone to mutations in Menkes
disease and has been proposed to function in joining the copper binding
domains to the ATPase core (26). The expression of both the A629P and
G1019D mutants in ccc2
yeast results in reduced growth in
copper-deficient media (Fig. 5) and a decrease but not a deficiency in
Fet3p oxidase activity for the A629P mutation (Fig. 6), suggesting that
copper transport in patients with this mutation is not absent but only
impaired. This finding implies that disease may result when lowered
levels of copper transport by the Menkes protein cannot meet the
metabolic demands of the cell. The distinction between decreased and
deficient copper transport by the Menkes protein mutants may have
significant clinical relevance and may in part explain the variable
response of patients to copper therapy (33, 34). Alternatively, since
in mammalian cells the Menkes and Wilson proteins traffic within the
cell in response to the cellular copper concentration (9, 11), these
Menkes protein mutants may be defective in copper-dependent
trafficking, analogous to observed mutations in the cystic fibrosis
transmembrane regulator that affect transport indirectly through
mislocalization of the protein (35). Analysis of trafficking by the
Menkes protein and its mutants will require expression in mammalian
cells, as specific post-Golgi sorting compartments do not appear to be
morphologically conserved between yeast and mammalian cells.
Previous studies have demonstrated that a soluble form of the Menkes
amino-terminal domain containing the six MXCXXC
motifs binds six molar equivalents of copper, suggesting one copper
atom/metal binding motif (36). The current data (Fig. 7) suggest that
the Menkes protein may not bind six copper atoms in vivo or
that the bound copper atoms are not directly transported through the
protein channel. The sudden interruption of copper transport upon
mutation of the third copper binding motif, as opposed to a linear
decrease in Fet3p activity, suggests either the loss of a critical
interaction site or steric constraints in transferring copper from the
remaining copper binding motifs to the ATPase core. Steric hindrance is unlikely since Ccc2p and the C. elegans ATPase contain only
two and three copper binding motifs, respectively, within a
commensurately shorter amino terminus. The potential loss of an
interaction site has supporting evidence in recent data demonstrating
an interaction between the copper binding motifs of Ccc2p and Atx1p,
the copper chaperone to Ccc2p in the yeast Fet3p copper transport
pathway (37). A recent study also indicates that mutation of the two cysteine residues in the copper binding domain of the human Atx1p homologue, HAH1 (23), abrogates copper binding by the motif as well as
copper delivery to Fet3p (38). The current data may thus suggest that
the third copper binding domain plays a critical role in the transfer
of copper from HAH1 to the Menkes protein. The finding that the copper
binding domains within the Menkes amino terminus are not functionally
equivalent despite the conservation of the copper binding amino acids
between domains indicates that additional residues must dictate
specificity in the transfer of copper between proteins. These residues
may reside within or near the copper binding sites or alternatively,
may occur distantly within the protein where they would function as a
site for protein-protein interaction.
The experiments presented in this study identify the Menkes protein as
a member of a mechanistically conserved family of copper-transporting P-type ATPases, which function to transport copper into the secretory compartment of eukaryotic cells. These studies lend insight into pathophysiologic mechanisms in Menkes disease, in which defective placental copper transport during a critical window period in central
nervous system development may result in the irreversible neurological
deficits observed in Menkes patients. The intracellular transport and
export of copper through this family of proteins are likely to occur
through an ordered mechanism of copper transfer from the point of
cellular entry to exit, guided by specific interactions between
proximal proteins within the pathway. Disorders of copper transport
caused by mutations within the Menkes protein may thus be classified
into categories of functional disruptions affecting chaperone
interaction, copper binding, copper transport, or protein trafficking.
Further molecular analysis of Menkes protein function based upon this
framework may thus provide novel therapeutic approaches to prevent or
ameliorate the consequences of disordered copper metabolism in affected
patients.