(Received for publication, October 25, 1996, and in revised form, January 17, 1997)
From the § Division of Toxicological Sciences,
Department of Environmental Health Sciences, The Johns Hopkins
University School of Hygiene and Public Health, Baltimore, Maryland
21205, ¶ Cell Biology and Metabolism Branch, NICHHD, National
Institutes of Health, Bethesda, Maryland 20892, and
Edward Mallinckrodt Department of Pediatrics, Washington
University School of Medicine, St. Louis, Missouri 63110
To search for a mammalian homologue of ATX1, a human liver cDNA library was screened and a cDNA clone was isolated, which encodes a protein with 47% amino acid identity to Atx1p including conservation of the MTCXGC copper-binding domain. RNA blot analysis using this cDNA identified an abundant 0.5-kilobase mRNA in all human tissues and cell lines examined. Southern blot analysis using this same clone indicated that the corresponding gene exists as a single copy in the haploid genome, and chromosomal localization by fluorescence in situ hybridization detected this locus at the interface between bands 5q32 and 5q33. Yeast strains lacking copper/zinc superoxide dismutase (SOD1) are sensitive to redox cycling agents and dioxygen and are auxotrophic for lysine when grown in air, and expression of this human ATX1 homologue (HAH1) in these strains restored growth on lysine-deficient media. Yeast strains lacking ATX1 are deficient in high affinity iron uptake and expression of HAH1 in these strains permits growth on iron-depleted media and results in restoration of copper incorporation into newly synthesized Fet3p. These results identify HAH1 as a novel ubiquitously expressed protein, which may play an essential role in antioxidant defense and copper homeostasis in humans.
The biological activation of dioxygen by copper is essential for the survival of all living organisms (1). In humans, copper functions in oxidative catalysis by permitting facile electron transfer reactions in a number of enzymes, which play a critical role in the biochemistry of iron homeostasis, cellular respiration, antioxidant defense, neurotransmitter biosynthesis, connective tissue formation, pigment production, and endocrine organ regulation (2). Despite the essential requirement for copper in these cellular processes, excess copper is highly toxic, and therefore cells have evolved highly specialized systems for the transport and compartmentalization of this transition metal (3).
Insight into the mechanisms of cellular copper homeostasis in humans has come from the molecular characterization of two inherited disorders of copper metabolism, Wilson disease and Menkes disease. Although patients affected with these diseases have markedly different clinical phenotypes due to either copper overload or deficiency, each disorder is due to the absence or dysfunction of a homologous member of the cation transport P-type ATPase family (4-9). These proteins reside in the trans-Golgi network of cells and transfer copper into the secretory pathway for subsequent export or incorporation into newly synthesized cuproproteins (10). Most recently, a homologous ATPase, Ccc2p, was identified in Saccharomyces cerevisiae and shown to play a central role along with the multicopper oxidase Fet3p in copper and iron metabolism in yeast (11-13). The subsequent elucidation of the essential role of the Fet3p homologue ceruloplasmin in human iron homeostasis (14, 15) has revealed a remarkable evolutionary conservation of the mechanisms of cellular copper and iron metabolism.
Little is currently known about the pathways and mechanisms of copper
trafficking in mammalian cells. ATX1 encodes a cytosolic copper-binding protein in S. cerevisiae, which is a
multicopy suppressor of sod1 mutants functioning to
protect cells from toxicity in a copper-dependent manner
(16). Yeast strains deficient in ATX1 are impaired in high
affinity iron uptake, and this defect is presumably due to a failure of
copper delivery to Ccc2p for subsequent transport into the secretory
pathway and incorporation into Fet3p (17). As the structure and
function of Ccc2p and Fet3p are highly conserved between yeast and
humans, this current study was undertaken to search for a homologue of
Atx1p and to elucidate any functional role for such a protein in
antioxidant defense and copper homeostasis.
General chemicals and reagents were purchased
from Sigma. DNA restriction and modifying enzymes were
purchased from Promega Corp. and used according to manufacturer's
specifications. [32P]CTP and [32P]dCTP were
purchased from ICN Radiochemicals. Hybridization membranes were
obtained from Amersham Corp. 64Cu (600 Ci/mmol) was
obtained by fast neutron bombardment of a natural zinc target as
described previously (18). Yeast strains used in this study were as
follows: KS107, MAT leu2, 3-112 his3
1 trp-289a ura3-52
GAL+sod1
::TRP1 (19); SL215,
MATa ura3-52 lys2-801 ade2-101 trp1-
1 his3-
200 leu2-
1
atx1
::LEU2 (17); strain 7 (wt), MAT
his 3-200 leu 2 trp1-101 ura3-52 ade5 (13); and strain 8 (ccc2
), MAT
his 3-200 leu2 trp1-101 ura3-52 ade5
ccc2
::LEU2 (13).
A GenBank search for
sequences homologous to yeast ATX1 identified a partial
cDNA sequence (accession no. F14674[GenBank]) from a porcine small intestine
cDNA library. To identify a human homologue of ATX1,
250,000 recombinant clones from a human liver gt11 cDNA library
were transferred to nitrocellulose membranes and hybridized utilizing a
32P-labeled cDNA probe derived from this porcine clone.
Hybridized filters were washed as described previously, and positive
clones were purified by subsequent rounds of screening (7). Phage DNA
was isolated from positive clones by liquid lysis, and inserts were
analyzed by agarose gel electrophoresis following restriction digestion
with EcoRI (20). The largest size inserts were subcloned and
the nucleotide sequence determined by dideoxy chain termination (21).
To isolate genomic clones encoding HAH1, a 32P-labeled
full-length HAH1 cDNA was used to screen 800,000 recombinants of a human genomic library in
FixII (Stratagene).
Isolated phage DNA was characterized by restriction enzyme digestion
and nucleotide sequence determination as described (22).
RNA blot analysis was performed using nitrocellulose
membranes containing poly(A)+ RNA from different human
tissues (Clontech). RNA was isolated by dissolution of human tissues
and cell lines in guanidinium isothiocyanate followed by CsCl gradient
centrifugation (23). RNA samples were subjected to agarose-formaldehyde
gel electrophoresis, transferred to nylon membranes, and analyzed using
a 32P-labeled HAH1 cRNA as described previously (20). Human
genomic DNA was isolated from peripheral blood leukocytes, digested
with restriction enzymes overnight, electrophoresed on 0.8% agarose gels, transferred to nylon membranes, and analyzed using a
32P-labeled HAH1 cDNA probe (24). A
bacteriophage clone encompassing the HAH1 gene was labeled
with digoxygenin dUTP by nick translation (Genome Systems, St. Louis,
MO). Labeled probe was hybridized to normal metaphase chromosomes from
phytohemagglutinin-stimulated peripheral blood lymphocytes in 50%
formamide, 10% dextran sulfate, and 2 × SSC. Specific
hybridization signals were detected by incubation with
fluorescein-conjugated anti-digoxygenin antibodies followed by
counterstaining of the slides with 4,6-diamidino-2-phenylindole. A
total of 57 specifically labeled metaphase cells were analyzed, and
localization was confirmed using a probe known to localize to 5q21.
To express HAH1 in
S. cerevisiae, the coding region encompassing nucleotides
114-318 (see Fig. 1A) was amplified by polymerase chain
reaction and directionally subcloned into the EcoRI and BamHI sites of pSM703. The resulting construct pHAH703 was
verified by nucleotide sequencing and transformed into KS107
(sod1) and SL215 (atx1
) strains as
described (17). The empty CEN pRS413 (25) and 2µ pSM703 vectors, and
the CEN p413-A1 and 2µ pRS-A1 plasmids containing a functional copy
of the ATX1 gene were transformed into these same strains
(16, 17). To test for reversal of lysine auxotrophy, cells were grown
in air on SD1 plates with or without lysine
for 3 days at 30 °C (26). Iron-dependent growth was
determined on SD complete media buffered with 50 mM Na-MES,
pH 6.1, and 3 µM ferrozine with or without 350 µM ferrous ammonium sulfate for 5 days at 30 °C
(26).
HoloFet3p Biosynthesis
To examine the biosynthesis of holoFet3p (copper incorporation into Fet3p), saturated cultures were used to inoculate 100 ml of yeast nitrogen base culture medium without copper, iron, and dextrose, supplemented with amino acids, 2% glucose, 50 mM Na-MES, pH 6.1, and 100 µM ferrozine. All stock solutions were treated with Chelex-100. After growth to A600 of 0.4, cells were labeled for 2 h with 2.5 µCi/ml 64Cu. Cells were washed extensively in 150 mM NaCl, 25 mM Tris-HCl pH 7.5, and an aliquot of cells was used to determine cell-associated radioactivity. Cells were lysed by vigorous shaking with glass beads for 1 h, and protein extracts of crude membrane fractions were prepared as described (13). Samples (100 µg) were analyzed on 7.5% nonreducing SDS-PAGE without heat denaturation followed by direct autoradiography of the gels as previous studies revealed that copper is retained in multicopper oxidases under these conditions (27). For Western blot analysis of Fet3p, crude membrane extracts were separated by SDS-PAGE and transferred to nitrocellulose by semidry transfer. Following blocking in 5% nonfat dry milk, 0.1% Tween 20, 0.02% Nonidet P-40 in phosphate-buffered saline, membranes were incubated for 1 h with a 1:1000 dilution of an anti-peptide antiserum specific for Fet3p.2 Membranes were subsequently washed in phosphate-buffered saline containing 0.1% Tween 20 and incubated for 1 h with horseradish peroxidase-conjugated secondary antibody, and the antibody-antigen complex was detected using enhanced chemiluminescence (Amersham).
To search for a mammalian Atx1p homologue, an ATX1
genomic DNA fragment was used to screen a human liver cDNA library
by colony hybridization at low stringency and by polymerase chain
reaction using degenerate oligonucleotides corresponding to the
conserved amino- and carboxyl-terminal domains of ATX1 (Fig.
1B). This approach did not result in
isolation of full-length clones, and thus a partial porcine cDNA
with homology to the 3 region of ATX1 was used to rescreen
this human liver cDNA library. Seven clones were isolated by this
method, and the largest size insert was characterized by nucleotide
sequencing. This clone, termed HAH1 (human ATX
homologue 1), encompassed 113 bp of 5
untranslated region, a 204-bp
open reading frame, and 185 bp of 3
untranslated sequence (GenBank accession no. U70660[GenBank]) (Fig. 1A). A polyadenylation consensus sequence was identified 18 bp from the poly(A) tail. The open reading
frame of HAH1 encoded a protein of 68 amino acids with 47%
identity and 58% similarity to Atx1p including conservation of both
the copper-binding domain (MTCXGC) and the lysine-rich carboxyl terminus (Fig. 1B). A GenBank search identified
homologous open reading frames with these consensus regions in other
eukaryotic species including plants and worms, supporting the concept
that Atx1p has been phylogenetically conserved (Fig.
1B).
To determine the tissue-specific expression of HAH1, RNA
blot analysis was performed using a HAH1 cDNA probe.
This analysis revealed a single transcript of 0.5 kilobases in
agreement with the size of the HAH1 cDNA isolated by
library screening. HAH1 mRNA was abundant in all tissues examined
both in the peripheral tissues and in the central nervous system (Fig.
2, A and B). Similar analysis
detected a HAH1 transcript in RNA isolated from more than 20 additional
human tissues including fetal lung, liver, and kidney (data not shown).
These experiments suggested that the HAH1 gene is widely
expressed under normal conditions, and, consistent with this concept, a
HAH1-specific transcript was also detected in RNA isolated from a
variety of human tissue-derived cell lines (Fig. 2C).
Although the abundance of HAH1 mRNA varied slightly in
these different cell lines, all samples contained the HAH1
gene transcript, and no change in abundance was observed with
alteration of the intracellular copper content of these cells (data not
shown).
To characterize the gene encoding HAH1, the full-length cDNA clone
was used as a probe to analyze human genomic DNA by Southern blot
analysis. As shown in Fig. 3, hybridization of
restriction endonuclease-digested genomic DNA revealed a simple pattern
consistent with the presence of single copy gene in the haploid genome.
Three independent clones encompassing the HAH1 gene were
then isolated from a genomic library and characterized using Southern
blot analysis and sequencing. The largest of these clones was used to
determine the chromosomal localization of the HAH1 gene.
Fig. 4A shows fluorescent in situ
hybridization analysis of normal metaphase chromosomes derived from
peripheral blood lymphocytes utilizing this 15-kilobase genomic
fragment as a probe. Consistent with the Southern blot experiments,
this analysis revealed specific labeling of a single locus on the
distal long arm of chromosome 5 in a total of 57 metaphase cells.
Cohybridization of the HAH1 probe with a probe that precisely mapped to
5q21 resulted in double-labeling of the long arm of chromosome 5. Southern blot analysis of DNA from human hamster cell hybrids confirmed
this localization to chromosome 5 (data not shown). Measurements of 10 specifically labeled chromosome 5 preparations demonstrated that the
HAH1 gene is located at a position that is 75% of the
distance from the centromere to the telomere at an area that
corresponds to the interface between bands 5q32 and 5q33 (Fig.
4B).
The ATX1 gene was isolated by virtue of its ability to
reverse the aerobic lysine auxotrophy observed in sod1)
strains. To ascertain the function of HAH1, the coding region of the
HAH1 cDNA was placed under control of the S. cerevisiae phosphoglycerate kinase promoter and introduced into
the sod1
null mutant. This strain was subsequently
analyzed for its ability to grow aerobically on plates without lysine.
As can be seen in Fig. 5A, yeast strains transformed with either ATX1 or HAH1 were able to
suppress the lysine auxotrophy of a sod1
mutant strain.
Since complementation in these experiments is accomplished by over
expression of these proteins, the slight difference in growth rate
between the ATX1 and the HAH1 transformants (Fig.
5A, b versus c) may reflect a difference in the
amount of protein expressed under these circumstances.
As revealed by studies in an atx1 null strain, Atx1p is
essential for efficient high affinity iron uptake in S. cerevisiae (17). As shown in Fig. 5B, this
atx1
strain is dependent upon high concentrations of
exogenous iron for growth, but will grow on iron-depleted media after
reintroduction of the ATX1 gene. Transformation of the
atx1
strain with HAH1 also suppressed this iron-dependent phenotype, as evidenced by support of growth
on low iron medium (Fig. 5B, c). Cells
transformed with vector alone failed to grow under these conditions
(Fig. 5B, b and d), indicating that
the observed effect is specific for HAH1. Further studies with an HAH1 antibody will be important to ascertain the amount of HAH1
produced under these circumstances.
The role of copper in high affinity iron uptake in S. cerevisiae has been shown to be due to the plasma membrane
multicopper oxidase Fet3p (11, 12), and the iron deficiency in the
atx1 strain therefore presumably results from a failure
of copper incorporation into Fet3p. To directly test this hypothesis
and to obtain functional data on the role of HAH1 in copper metabolism,
yeast strains were cultured in copper-free medium, pulse-labeled with
64Cu, and then newly synthesized holoFet3p was detected in
membrane fractions. Determination of radioactivity in aliquots of the
cell suspensions indicated that the amount of 64Cu uptake
by each strain was identical (data not shown). When membrane fractions
were analyzed in this fashion, a protein with the appropriate molecular
mass of Fet3p was detected in wild type (Fig.
6A, lane 1) but not in a
ccc2
mutant strain (Fig. 6A, lane 2) previously shown to have Fet3p oxidase activity (13).
Consistent with the partial iron deficiency observed in
atx1
null mutants (17), a marked reduction, but not
complete impairment of copper incorporation into Fet3p was observed in
the atx1
strains transformed with either the pRS413 or
pSM703 vectors alone (Fig. 6A, lanes 3 and
5). However, radiolabeled Fet3p equivalent in amount to that
observed in the wild type strain was detected in the atx1
mutant following introduction of ATX1 on an episomal plasmid
(Fig. 6, lane 4). Furthermore, introduction of
HAH1 also resulted in restoration of holoFet3p biosynthesis
to amounts observed in the wild-type strain, indicating that
HAH1 can function to facilitate copper incorporation in an
analogous fashion to Atx1p (Fig. 6A, lane 6).
Consistent with this finding, HAH1 expression restored Fet3p
oxidase activity in the atx1
null mutants (data not
shown). These same membrane samples were examined for immunoreactive
Fet3p by Western blotting using a polyclonal antiserum to Fet3p. As can
be seen in Fig. 6B, the amount of Fet3p detected by this
technique was equivalent in each of the atx1
strains,
thus confirming that the changes in holoFet3p biosynthesis observed in
the copper-labeling experiment were not due to an effect of Atx1p on
the amount of Fet3p present in these strains. As has been observed for
other multicopper oxidases, the mobility of Fet3p under these
circumstances was lower than that observed for holoFet3p analyzed in
nonreducing SDS-PAGE gels (Fig. 6, A versus B).
The sequence data reported in this study suggest that HAH1 is a
human homologue of the S. cerevisiae copper-binding protein Atx1p. The considerable amino acid identity between these two proteins,
including conservation of the MTCXGC copper-binding motif,
as well as the occurrence of homologous sequences as open reading
frames in the DNA of other diverse eukaryotic organisms, supports this
concept and suggests a remarkable evolutionary conservation of the
structure of this protein moiety similar to what has been observed for
other proteins involved in copper homeostasis (2). Perhaps most
importantly, the expression of HAH1 in sod1
and atx1
null yeast strains reconstitutes the defects in
antioxidant defense and iron homeostasis in these mutants, indicating
that the HAH1 open reading frame encodes a functional
protein.
RNA blot analysis of HAH1 expression identified a single 0.5-kilobase transcript in all tissues and cell lines examined, with no evidence of qualitative differences in gene expression among tissues. These data are consistent with the size of the isolated full-length cDNA (Fig. 1A) and the structure of the HAH1 gene identified from sequencing of genomic clones (data not shown). This analysis also revealed that HAH1 is abundantly expressed in each of these tissues in amounts equivalent to that observed for copper/zinc superoxide dismutase when this transcript was similarly analyzed (data not shown). Although no HAH1 protein expression data are yet available, the observation that HAH1 is ubiquitously and abundantly expressed supports the concept that this protein may play an important and previously unappreciated role in cellular copper homeostasis. Consistent with this concept, recent experiments in transgenic mice reveal that neither metallothionein I and II nor copper/zinc superoxide dismutase is essential for cellular copper metabolism under normal circumstances, suggesting that additional proteins must be present to fulfill this function (28-30).
Based upon the complementation of atx1 null strains by
HAH1 and the recently defined role of Atx1p in iron metabolism in S. cerevisiae (17) it is reasonable to assume that HAH1
functions in an analogous fashion in mammalian cells. In this context,
HAH1 would be responsible for the delivery of cytosolic copper to the Menkes and Wilson disease proteins in the trans-Golgi
network prior to transport of this metal by these ATPases into the
secretory pathway. This model is consistent with the known
intracellular localization of these copper transport ATPases in
mammalian cells (10), as well as the clearly defined role of the Wilson
disease ATPase in providing copper for the biosynthesis of the Fet3p
multicopper oxidase homologue ceruloplasmin.2 Although such
a model is consistent with the data on Fet3p biosynthesis (Fig. 6),
further understanding and confirmation of this pathway must await the
development of HAH1 antibodies to define the expression and
localization of this protein within the cell. The overall concept of
specific proteins essential for copper trafficking within cells is also
supported by recent studies in S. cerevisiae, which indicate
the existence of a distinct set of intracellular proteins necessary for
copper delivery to mitochondrial cytochrome c oxidase (31,
32).
In the model of cytosolic copper delivery described above inherited abnormalities in HAH1 might be sufficient to interrupt the normal traffic of copper to the secretory pathway. As such, HAH1 serves as a likely gene candidate for the ecogenetic disorders of hepatic copper metabolism known as Indian childhood cirrhosis or idiopathic copper toxicosis, where an increase in dietary copper appears to result in cytosolic copper accumulation in genetically susceptible individuals (33-35). Although the genetic loci for such disorders have not yet been mapped, the identification of the chromosomal location of HAH1 may facilitate genetic analysis for inherited abnormalities in HAH1.
In addition to, and independent of, its role in iron homeostasis, Atx1p
functions as an antioxidant protecting yeast from the toxic effects of
superoxide and hydrogen peroxide (16). As Atx1p itself does not appear
to have antioxidant properties,3 the most
likely explanation for these observations is that Atx1p and HAH1
deliver copper to an as yet uncharacterized protein, which functions in
cellular antioxidant defense. This hypothesis would be consistent with
the observed role for Atx1p and HAH1 in the production of newly
synthesized copper proteins in addition to Fet3p that are detected in
wild type but not atx1 null strains following metabolic
labeling with 64Cu (data not shown). These findings suggest
a potential role for HAH1 or additional copper proteins dependent upon
HAH1 in the antioxidant defense of mammalian cells. Given recent
observations on the role of gain-of-function mutations in copper/zinc
superoxide dismutase in patients with familial amyotrophic lateral
sclerosis and data that implicate hydrogen peroxide in the pathogenesis of the neuronal degeneration resulting from these mutant enzymes, HAH1 may be a likely gene candidate for loci in other
affected families as well as a potential therapeutic target for the
responses to such injury (36-38).
Collectively, the data presented here suggest that HAH1 is a novel human copper-binding protein, which is abundantly and ubiquitously expressed and which functions in copper homeostasis and antioxidant defense. Although the pathways and proteins involved in copper trafficking in mammalian cells remain largely unknown, the results of this study suggest that the marked evolutionary conservation of these pathways will permit utilization of the techniques of yeast genetics to identify and characterize such proteins. Given the increasing recognition of the role of both copper and iron in inherited and acquired neurodegenerative diseases, as well as the known importance of host antioxidant defenses in human disease, such an approach is likely to continue to contribute new information to this area of biology (39, 40).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U70660[GenBank].
We thank A. Winteroe for the porcine F14674 cDNA clone, S. Michaelis for the pSM703 plasmid, Irene Hung for advice and assistance with the copper experiments, and Aimee Payne for critical review of the manuscript.