Tissue localization of the copper chaperone ATOX1 and
its potential role in disease
Steven D.P. Moore, Karmon E. Helmle, Lisa M. Prat, Diane W. Cox
University of Alberta, 8-39 Medical Sciences Building, Department of Medical Genetics, Edmonton, Alberta, Canada
Received: 7 February 2002 / Accepted: 17 June 2002
Abstract. ATOX1 is a cytoplasmic copper chaperone that in-
teracts with the copper-binding domain of the membrane
copper transporters ATP7A and ATP7B. ATOX1 has also
been suggested to have a potential anti-oxidant activity. This
study investigates the tissue-speci®c localization of the mouse
homolog, Atox1, in mouse liver and kidney. Immunohisto-
chemical studies in the liver localize the copper chaperone to
hepatocytes surrounding both hepatic and central veins. In the
kidney, Atox1 is localized to the cortex and the medulla.
Cortex immunostaining is speci®c to glomeruli in both the
juxtamedullary and cortical nephrons. Expression in the me-
dulla appears to be associated with the loops of Henle. These
data suggest that localized regions in the liver and kidney
express Atox1 and have a role in copper homeostasis and/or
anti-oxidant protection. Twenty-seven patients with Wilson
disease-like phenotypes and two patients with Menkes disease-
like phenotypes were screened for ATOX1 mutations with no
alterations detected. The human phenotype resulting from
mutations in ATOX1 remains unidenti®ed.
Copper is an essential cofactor for many enzymes involved in a
wide variety of cellular functions, such as cellular respiration,
iron transport, and free radical scavenging. Free copper within
the cell is potentially hazardous, given the oxidative potential
of the metal ion (Gutteridge 1984). Oxidative damage has been
implicated in numerous neurological disorders including Alz-
heimer disease (Markesbery and Carney 1999) and Parkinson
disease (Jenner and Olanow 1996; Jenner 1998). Normally,
copper is not free within the cell (Rae et al. 1999), but is as-
sociated with chaperones that transport copper to and from
donor and acceptor proteins. Atx1p, a yeast copper chaperone,
is necessary for the intracellular copper transport pathway and
is indirectly required for high-anity iron uptake (Lin et al.
1997). In higher organisms, ATOX1, the mammalian homolog
of Atx1p, is believed to be the only copper transporter that
delivers copper to the P-type ATPase copper transporters,
ATP7A and/or ATP7B, defective in Menkes and Wilson dis-
ease respectively. Menkes and Wilson disease result from a
disturbance of copper balance, resulting in either a de®ciency
(Menkes) or an accumulation of copper (Wilson) in the body.
The crystal structures of yeast Atx1p (Rosenzweig et al.
1999) and human ATOX1 (Wernimont et al. 2000) suggest a
molecular mechanism for protein recognition and metal ion
exchange between CXXC amino acid domains in ATP7A and
ATP7B (reviewed in Rosenzweig 2001). Yeast-two-hybrid
studies, ®rst using yeast Atx1p and Ccc2p (yeast ortholog to
human ATP7A/ATP7B) (Pufahl et al. 1997), and secondly
using the human homologs ATOX1 and ATP7B (Larin et al.
1999), demonstrate that the chaperone interacts directly with
the copper transporters. In addition to yeast studies, human
protein interactions were studied by mammalian two-hybrid
analysis using ATOX1 and ATP7B metal-binding domains
(Larin et al. 1999) as well as co-immunoprecipitation in HeLa
and HepG2 cells between ATOX1 and ATP7A and ATP7B
(Hamza et al. 1999).
Atx1 was initially isolated in yeast, on the basis of its ability
in multicopy suppression of oxidative toxicity in SOD1-de®-
cient yeast (Lin and Culotta 1995). This oxidative protection is
dependent both on copper and on conserved lysine residues in
the N-terminal domain, whereas copper transport is not de-
pendent on the lysine residues (Hung et al. 1998), suggesting
that this protein may have two distinct biological functions.
Recently, ATOX1 has been shown to have a protective role
against oxidative stress in neuronal cells (Kelner et al. 2000).
Expression studies in mouse brain indicate Atox1 expression in
several regions, with particularly strong presence in choroid
plexus, where Atp7a is also expressed (Nishihara et al. 1998;
Naeve et al. 1999). If ATOX1 has a critical role in neuronal
tissue, an alteration in function could lead to impaired brain
function, owing to a loss of cellular copper regulation, and/or
protective antioxidant activity. Neurological problems in the
mouse knockout of Atox1 (Hamza et al. 2001) may result from
a lack of copper-dependent enzymes such as dopamine-b-
hydroxylase, as well as a lack of antioxidant protection.
Both the liver and kidney are of particular interest because
of their role in copper excretion and homeostasis. When foods
containing copper are ingested, copper is distributed
throughout the body in two waves. In the ®rst wave, imme-
diately after intestinal absorption, copper enters the serum
associated with two high-molecular-weight, copper-binding
proteins, a high-molecular-weight complex (referred to as
transcuprein), and albumin (reviewed in Linder and Hazegh-
Azam 1996). Initial absorption does not involve any low-
molecular-weight substances. Once in the serum, radioactive
tracer copper is temporarily removed from the serum and
appears both in the liver and kidney. In the second phase,
copper re-enters the serum mainly bound to ceruloplasmin
secreted by the liver and possibly the kidney (Askwith et al.
1994), enabling copper to enter the serum and be taken up by
peripheral organs, including the heart and brain.
The copper transporter ATP7B is highly expressed in the
liver, with only a slight expression of ATP7A (Grimes et al.
1997). In contrast, the kidney expresses both ATP7A and
ATP7B. If Atox1 is the sole copper chaperone for the copper
transporters ATP7A and ATP7B, then the expression pattern
of the chaperone is expected to overlap with the known
expression pattern of these two transporters. Therefore, poly-
Mammalian Genome 13, 563±568 (2002).
Correspondence to: D.W. Cox; E-mail: firstname.lastname@example.org