the mitochondrial metallochaperone sco1 is …cell reports article the mitochondrial...
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Article
The Mitochondrial Metallo
chaperone SCO1 IsRequired to Sustain Expression of the High-AffinityCopper Transporter CTR1 and Preserve CopperHomeostasisGraphical Abstract
Highlights
d Liver-specific deletion of the mitochondrial
metallochaperone Sco1 is lethal
d Sco1-null livers are COX and Cu deficient, like affected
tissues of SCO1 patients
d Loss of SCO1 causes a Cu deficiency by reducing CTR1
abundance
d Sco1 deletion results in enhanced proteasomal degradation
of CTR1
Hlynialuk et al., 2015, Cell Reports 10, 933–943February 17, 2015 ª2015 The Authorshttp://dx.doi.org/10.1016/j.celrep.2015.01.019
Authors
Christopher J. Hlynialuk, Binbing Ling, ...,
Michael J. Petris, Scot C. Leary
In Brief
Hlynialuk et al. demonstrate that SCO1 is
integral to a mitochondrial signaling
pathway that regulates copper
homeostasis by controlling the
abundance and plasma membrane
localization of CTR1, the high-affinity
permease responsible for importing
copper into the cell.
Accession Numbers
GSE58997
Cell Reports
Article
The Mitochondrial Metallochaperone SCO1 Is Requiredto Sustain Expression of the High-Affinity CopperTransporter CTR1 and Preserve Copper HomeostasisChristopher J. Hlynialuk,1,7 Binbing Ling,1,7 Zakery N. Baker,1,7 Paul A. Cobine,2 Lisa D. Yu,1 Aren Boulet,1 Timothy Wai,3
Amzad Hossain,1 Amr M. El Zawily,1,6 Pamela J. McFie,1 Scot J. Stone,1 Francisca Diaz,4 Carlos T. Moraes,4
Deepa Viswanathan,5 Michael J. Petris,5 and Scot C. Leary1,*1Department of Biochemistry, University of Saskatchewan, Saskatoon, SK S7N 5E5, Canada2Department of Biological Sciences, Auburn University, Auburn, AL 36849, USA3Institute for Genetics, University of Cologne, 50931 Cologne, Germany4Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA5Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA6Faculty of Science, Damanhour University, Damanhour 22516, Egypt7Co-first author
*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.celrep.2015.01.019
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
SUMMARY
Human SCO1 fulfills essential roles in cytochrome coxidase (COX) assembly and the regulation of copper(Cu) homeostasis, yet it remains unclear why patho-genic mutations in this gene cause such clinicallyheterogeneous forms of disease. Here, we establisha Sco1 mouse model of human disease and showthat ablation of Sco1 expression in the liver is lethalowing to severe COX and Cu deficiencies. We furtherdemonstrate that the Cu deficiency is explained by afunctional connection between SCO1 and CTR1, thehigh-affinity transporter that imports Cu into the cell.CTR1 is rapidly degraded in the absence of SCO1protein, and we show that its levels are restored inSco1�/� mouse embryonic fibroblasts upon inhibi-tion of the proteasome. These data suggest thatmitochondrial signaling through SCO1 provides apost-translational mechanism to regulate CTR1-dependent Cu import into the cell, and they furtherunderpin the importance of mitochondria in cellularCu homeostasis.
INTRODUCTION
Copper (Cu) is an essential trace element that is acquired via the
diet and is required as a co-factor by several mammalian en-
zymes that collectively catalyze biochemical reactions critical
to early development and general physiology (Desai and Kaler,
2008). Because Cu is toxic in its unbound form, conserved
machinery has evolved to tightly regulate cellular Cu uptake
and efflux. Dedicated chaperones further ensure that Cu is safely
trafficked throughout the cell to specific cupro-proteins for their
metallation, or to labile pools for its storage (Kim et al., 2008;
Ce
Festa and Thiele, 2011). Genetic variants or environmental in-
sults that impair these Cu-handling systems negatively affect
innate immunity, impact the progression and treatment of
several types of cancer, and cause severe forms of human dis-
ease (Desai and Kaler, 2008; Turski and Thiele, 2009; Festa
and Thiele, 2012). Thus, there is considerable interest in
advancing our mechanistic understanding of how Cu is handled
at the cellular through to the systemic levels of organization.
Cellular Cu uptake is mediated by the permease copper trans-
porter 1 (CTR1) (Lee et al., 2002; Puig et al., 2002; Eisses and
Kaplan, 2005), the only high-affinity Cu importer that has been
identified to date. CTR1 cycles between the plasma membrane
(PM) and cytosolic vesicles in many cell types, and regulated
changes in its abundance or localization allow for the rate
of Cu uptake to be rapidly adjusted in response to altered cellular
Cu levels (Petris et al., 2003; Guo et al., 2004). Following import,
Cu is transferred from CTR1 to soluble chaperones for its deliv-
ery to various subcellular compartments. Copper chaperone for
superoxide dismutase 1 (CCS1) delivers and inserts Cu into
SOD1 within the cytosol (Lamb et al., 2001). Cu destined for
the trans-Golgi network and the secretory pathway is delivered
via ATOX1 to ATP7A or ATP7B (Hamza et al., 1999; Wernimont
et al., 2000), homologous P-type ATPases with analogous func-
tions but with limited overlap in their expression patterns across
adult tissues (Lutsenko et al., 2007). ATP7A and ATP7B translo-
cate Cu into the lumen of the trans-Golgi network for its incorpo-
ration into secreted cuproenzymes; however, both proteins also
traffic to the cell periphery to efflux Cu in response to increased
Cu concentrations (Hasan and Lutsenko, 2012). Loss-of-func-
tion mutations in ATP7A cause Menkes disease, a neurodegen-
erative disorder in which defective Cu transport from enterocytes
into the general circulation produces a severe Cu deficiency in
several tissues, particularly the brain (Mercer et al., 1993; Vulpe
et al., 1993). Mutations in ATP7B result in Wilson disease, a con-
dition in which patients suffer from hepatic Cu overload due to an
inability to export Cu into the bile, the main pathway for its
ll Reports 10, 933–943, February 17, 2015 ª2015 The Authors 933
excretion from the body (Bull et al., 1993; Yamaguchi et al.,
1993). How Cu is delivered to mitochondria, where it is used
to metallate cytochrome c oxidase (COX) and a small fraction
of the total cellular SOD1 pool, is unclear, as known Cu chaper-
ones do not appear to function in this capacity (Cobine et al.,
2006).
Historically, studies have investigated the various pathways
that handle Cu independently of each other; however, evidence
from a number of model systems has elegantly demonstrated
that, under limiting conditions, Cu utilization is prioritized at the
cellular, tissue, and systemic levels of organization (Mendelsohn
et al., 2006; Merchant et al., 2006; Nose et al., 2006; Kim et al.,
2008; White et al., 2009). These findings argue that mechanisms
exist to monitor global Cu status and reallocate labile Cu stores.
How discrete Cu-handling pathways are organized into a func-
tional framework that can be dynamically regulated remains an
open question, and one that is of fundamental importance to
our understanding of both basic biology and human disease.
An active role for mitochondria in this process is now appreci-
ated, and it is clear that Cu loading of COX within the mitochon-
drial intermembrane space is tied to the regulation of cellular Cu
efflux (Leary et al., 2007, 2013b). Cu delivery to COX occurs as
the holoenzyme is assembled and is facilitated by as many as
11 accessory proteins (Leary, 2010). The first of these to be iden-
tified was yeast Cox17 (Glerum et al., 1996a), and SCO1 and
SCO2 subsequently were identified as high copy suppressors
of a COX17 mutant strain (Glerum et al., 1996b). Although this
suggested that both Sco1 and Sco2 act downstream of Cox17
to deliver Cu to COX, only the deletion of yeast SCO1 resulted
in a respiratory phenotype (Glerum et al., 1996b). In contrast,
both SCO1 and SCO2 are essential in humans, with mutations
in either gene resulting in a severe, isolated COX deficiency
and an early onset, fatal clinical outcome. SCO2 mutations are
associated primarily with neonatal encephalocardiomyopathy
(Papadopoulou et al., 1999; Jaksch et al., 2000), and virtually
all of the �30 reported pedigrees carry at least one E140K allele
(DiMauro et al., 2012). Patients with SCO1 mutations are rare,
and only three pedigrees thus far have been described (Valnot
et al., 2000; Stiburek et al., 2009; Leary et al., 2013a). Curiously,
each SCO1 pedigree harbors a unique pathogenic allele whose
expression causes amarkedly different clinical course of disease
that primarily affects heart, liver, or brain function.
Affected tissues in SCO1 and SCO2 patients are both COX
and Cu deficient (Leary et al., 2007; Stiburek et al., 2009). While
the combined deficiency is likely a critical facet of disease pro-
gression, the basis for the tissue-specific manifestations in
SCO patients remains enigmatic, and its elucidation requires
the investigation of robust animal models of the associated dis-
eases. Two Sco2 transgenic mouse models have been charac-
terized thus far, and used in innovative translational research
(Viscomi et al., 2011; Cerutti et al., 2014); however, tissues
from these mice do not exhibit a global Cu deficiency, and the
reduction in COX activity is relativelymild compared toSCO2 pa-
tients (Yang et al., 2010). Here, we establish a conditional Sco1
mouse model and use the Cre-lox system to specifically delete
Sco1 in the liver. We show that this mousemodel exhibits a lethal
phenotype associated with profound hepatic COX and Cu defi-
ciencies, much like the patient from the original SCO1 pedigree
934 Cell Reports 10, 933–943, February 17, 2015 ª2015 The Authors
who succumbed to acute liver failure (Valnot et al., 2000). We
further demonstrate that the Cu deficiency in the Sco1-null liver
is explained by a loss of CTR1, via a mechanism that involves
increased proteasomal turnover of the protein. These findings
reveal that a SCO1-dependent mitochondrial signal regulates
Cu homeostasis by modulating cellular Cu import.
RESULTS
Generation of a Liver-Specific Sco1 Knockout MouseModelBecause the patient from the original SCO1 pedigree presented
with a fatal hepatopathy and the most severe clinical course of
disease of allSCO1 patients (Leary et al., 2013a), we constructed
a liver-specific mousemodel of the disease (Figure S1A; Supple-
mental Experimental Procedures). To do this, we first produced
a Sco1 conditional mouse on a C57BL/6N background
(Sco1loxP/loxP), and crossed it to mice expressing Cre recombi-
nase under the control of the liver-specific albumin promoter
(Alb-Cre, Jackson Laboratory) (Postic et al., 1999). We then
backcrossed Alb-Cretg/wt, Sco1loxP/wt animals to Sco1loxP/loxP
mice to generate liver-specificSco1 knockoutmice (Alb-Cretg/wt,
Sco1loxP/loxP), which we obtained at the expected Mendelian
ratios (c2 analysis, p = 0.72 [n = 150]). Molecular analyses
demonstrated that Cre-mediated deletion ofSco1was restricted
to the liver, and that SCO1 protein levels were unaffected in other
somatic tissues (Figures S1B and S1C). Liver-specific Sco1
knockout mice exhibited normal weight gain for roughly the first
28 days of life, but failed to thrive thereafter, with subsequent
weight loss producing a runted phenotype (Figures 1A and 1B).
Liver-specific Sco1 knockout mice had amedian life expectancy
of 70 days (c2 analysis, p < 0.0001) (Figure 1C), which was asso-
ciated with the loss of immunologically detectable SCO1 in the
liver (Figure 1D).
Lethality in Liver-Specific Sco1 Knockout Mice IsAssociated with a Severe Hepatic COX and CuDeficiencyTo further investigate the underlying cause of morbidity andmor-
tality in liver-specific Sco1 knockout mice, we quantified body
and tissue weights and examined gross liver morphology.
Liver-specific Sco1 knockout mice exhibited profound splenic
atrophy, with an initial loss of white pulp and reduced levels of
white blood cells in the circulation, in the absence of any detect-
able changes in the total number or properties of the red blood
cell population (Figures S1D–S1F). While the brain and liver
weights of liver-specific Sco1 knockout mice were unchanged
relative to wild-type littermates, despite the considerable loss
of total body weight (Figure S1D), Sco1-null livers differed mark-
edly with respect to the quality and consistency of their color
(Figure 2A) from roughly postnatal day 37 (P37) onward. Trans-
mission electron microscopy (TEM) analyses revealed that the
spotty nature of Sco1-null livers was attributable to an increase
in lipid droplet accumulation (Figure 2B, asterisks), and thin-layer
chromatography confirmed that there was an age-dependent in-
crease in hepatic cholesterol ester and triacylglycerol content
(Figures 2C and 2D). These findings were further corroborated
by Ingenuity pathway analysis of microarray data, which
Figure 1. Liver-Specific Sco1 Deletion Is
Lethal
(A) Change in body weight (g) over time in liver-
specific Sco1 knockout mice (Sco1liv/liv, n = 38)
and wild-type littermates (Controls, n = 184).
(B) Runted phenotype of a liver-specific Sco1
knockout mouse at P57.
(C) Kaplan-Meier survival curve (Sco1liv/liv, n = 17;
Controls, n = 96).
(D) SCO1, COX IV, and CCS1 abundance in wild-
type (flox) and Sco1-null (liv) livers at P47. Tubulin
served as an internal loading control (see also
Figure S1).
identified genetic reprogramming in Sco1-null livers consistent
with hepatic steatosis and impaired mitosis (Figure S2). TEM
also indicated that marked mitochondrial proliferation had
occurred in Sco1-null livers (Figure 2B, arrows). Consistent
with this observation, the activity of citrate synthase (CS), a
biochemical marker of mitochondrial content, increased signifi-
cantly with time in Sco1-null livers, as did the abundance of
Complexes I, II, and III of oxidative phosphorylation (OXPHOS),
and the steady-state levels of TOM40 and the a subunit of Com-
plex V (Figures 2E and 2F). In contrast, no changes in CS activity
(data not shown) or OXPHOS complex content (Figure 2F) were
observed in the heart, a representative unaffected tissue from
liver-specific Sco1 knockout mice.
In humans, mutations in SCO1 or SCO2 cause tissue-specific
diseases that likely reflect a combined COX and Cu deficiency
in affected tissues (Leary et al., 2007, 2013a). To determine
whether Sco1-null livers recapitulate these clinical hallmarks
of the human diseases, we serially measured the activity of
COX in Sco1-null and wild-type livers at P18, P27, and P57.
Sco1-null livers exhibited a progressive loss of COX activity
and holoenzyme content (Figures 2E and 2F). The total cellular
activity of SOD1, another Cu-dependent enzyme, was also
significantly lower in Sco1-null livers (Figure 2E, inset), and
was accompanied by a concordant increase in the activity (Fig-
ure 2E, inset) and content (Figure 2F) of the matrix-localized,
manganese-dependent enzyme SOD2. Consistent with the
reduced activity of these two Cu-dependent enzymes, Sco1-
null livers were Cu deficient as early as P18, with the severity
of the Cu deficiency worsening with time (Figures 3A and 3B).
The hepatic Cu deficiency preceded changes in tissue zinc
(Zn) and iron (Fe) content (Figure 3B), and H&E and Perls stain-
ing revealed Fe accumulation in the absence of necrosis in the
Cell Reports 10, 933–943,
Kupffer cells of the liver (Figures 3C and
3D) and the reticuloendothelial cells of
the spleen (Figure S1E) in liver-specific
Sco1 knockout mice at P57. Surprisingly,
the severe global Cu deficiency in Sco1-
null livers did not significantly affect the
total hepatic mitochondrial Cu pool (Fig-
ures S3A and S3B) or the Cu levels of
other peripheral organs (Figures 3A and
S3C), apart from a modest reduction in
renal Cu content. These data collectively
demonstrate that SCO1 fulfills essential
functions in COX assembly and the regulation of Cu homeosta-
sis in the liver.
SCO1 Depletion Causes a Severe Hepatic Cu Deficiencyby Attenuating CTR1 ExpressionSco1 deletion in the liver resulted in a marked, age-dependent
reduction in SCO1 abundance (Figure 4A, arrow), with the
upper band of the immunoreactive doublet being the mature
form of the protein, which was barely detectable in crude mito-
chondrial extracts at P18 (Figure S4). While the temporal loss
of SCO1 in Sco1-null livers accounts for the progressive
decrease in COX activity and content (Figures 2E and 2F), the
resultant hepatic Cu deficiency could be explained by enhanced
Cu export or impaired Cu uptake. To begin to distinguish be-
tween these two possibilities, we analyzed the abundance of
the Cu exporters ATP7A and ATP7B in wild-type and Sco1-null
livers at P18, P27, P37, and P57. The steady-state levels of
ATP7B were unchanged in Sco1-null livers over time (Figure 4A),
and biliary Cu content was comparable to that of wild-type litter-
mates (flox, 1.63 ng/ml Cu [pooled sample, n = 7]; liv, 0.92 ±
0.14 ng/ml Cu [n = 5, mean ± SEM]); however, the volume of
bile that was produced by Sco1-null livers increased markedly
starting at P37 (data not shown). Increases in ATP7A abundance
were only evident from P47 onward (Figure S5A), and coincided
with elevated serum content of Cu and Fe (Figure S5B). Consis-
tent with these observations, circulating levels of the Fe carrier
protein ferritin and the enzymatically active form of the Cu-
dependent ferroxidase ceruloplasmin, which both depend on
the secretory pathway for their maturation, also increased (Fig-
ures S5C and S5D). Although these data collectively suggest
that Cu transport into the secretory pathway and biliary Cu
excretion became more active with time in the Sco1-null liver,
February 17, 2015 ª2015 The Authors 935
Figure 2. Liver-Specific Sco1Deletion Is Associatedwith an Isolated COXDeficiency, Loss of SOD1Activity, Mitochondrial Proliferation, and
Accumulation of Intracellular Lipid Droplets
(A and B) (A) Gross morphology and (B) TEM images of a wild-type (flox) and Sco1-null (liv) liver at P57. For TEM images, arrows indicate representative
mitochondria, while the asterisks in the Sco1-null liver denote lipid droplets (3,3403 magnification; scale bar, 2 mm).
(C) Chromatographic analysis of cholesterol ester (CE), triglyceride (TG), fatty acid, and cholesterol content of representative wild-type (flox) and Sco1-null (liv)
livers at P27, P37, and P47.
(D) Quantitation of CE and TG content in Sco1-null livers (liv) and age-matched wild-type (flox) littermates (n = 6 [mean ± SEM]).
(E) COX, CS, and total cellular SOD activities in Sco1-null livers (liv) at P18, P27, and P57 expressed as a percentage of age-matched, wild-type littermates
(liv, n = 4–5; Controls, n = 5–7). (Inset) Percentage contribution of SOD1 and SOD2 to the total SOD activity (mean ± SEM) in Sco1-null (liv, white bars) and wild-
type livers (Controls, black bars) at P57 (liv, n = 4; Controls, n = 5).
(F) SDS-PAGE analysis of the steady-state levels of SOD2, ATPase a, and TOM40, and BN-PAGE analysis of the abundance of OXPHOS Complexes I–III and
COX in livers at P18 and in livers and hearts at P57 (liv, liver-specific Sco1 knockout mice; flox, wild-type littermates) (see also Table S1 and Figure S2).
these changes do not explain the onset of the Cu deficiency,
which occurred well before P37 (Figure 3B).
To evaluate whether the Cu deficiency in Sco1-null livers is
caused by impaired Cu uptake, we measured the abundance
of CTR1 in wild-type and Sco1-null livers at P18, P27, P37,
and P57. Unlike ATP7A and ATP7B, we observed a striking,
936 Cell Reports 10, 933–943, February 17, 2015 ª2015 The Authors
age-dependent reduction in the steady-state levels of the
mature, glycosylated form of CTR1 (Figure 4A, arrow). Indeed,
CTR1 abundance in Sco1-null livers was less than half that of
wild-type littermates at P27 (Figures S6A and S6B). Genome-
wide RNA expression profiling (Table S1) and semiquantitative
RT-PCR (Figure S6C) demonstrated that ablation of Sco1
Figure 3. Liver-Specific Sco1 Deletion
Causes a Severe Hepatic Cu Deficiency
without Markedly Perturbing Cu Homeosta-
sis in Peripheral Tissues
(A) Cu, Fe, and Zn content of tissues from liver-
specificSco1 knockoutmice (liv) at P47 expressed
as a percentage of wild-type littermates (Controls).
Li, liver; K, kidney; Sp, spleen; H, heart; and Br,
brain (liv, n = 5; Controls, n = 8 [mean ± SEM]).
(B) Cu, Fe, and Zn content of Sco1-null livers from
P18 to P57 expressed as a percentage of
age-matched, wild-type littermates (Controls) (liv,
n = 4–5; Controls, n = 4–11 [mean ± SEM]).
(C and D) (C) H&E and (D) Perls staining in wild-
type (flox) and Sco1-null (liv) livers at P57 (scale
bar, 50 mm) (see also Figures S3 and S5).
expression did not grossly affect Ctr1 mRNA levels, suggesting
that SCO1 may be required to regulate the abundance of
CTR1 post-transcriptionally. To determine whether this effect
is specific to SCO1, or a general phenomenon associated with
an isolated COX deficiency, we analyzed null livers from two
additional mouse models that exhibit a COX defect due to dele-
tion of either the heme A:farnesyltransferase gene Cox10 or
Lrpprc, which encodes a factor required for the stability and
handling of mature mitochondrial mRNAs (Sasarman et al.,
2010). The steady-state levels of SCO1weremarkedly increased
in both Lrpprc- and Cox10-null livers (Figures 4B, 4C, and S4C,
arrow); however, the abundance of the mature, glycosylated
form of CTR1 was only affected in Cox10-null livers, where it
was virtually undetectable (Figure 4C, arrow). In contrast, the
abundance of Cu exporter ATP7A was significantly higher in
Lrpprc-null livers compared with age-matched, wild-type litter-
mates (Figure 4B). Since Cox10- and Lrpprc-null livers exhibited
an equivalent COX deficiency (Figure 4D), we conclude that the
observed changes in CTR1 abundance in liver-specific Sco1
knockout mice cannot be explained by a general decrease in
COX activity, but may be a consequence of perturbed SCO1
function. Consistent with this idea, CTR1 levels were reduced
upon stable knockdown of SCO1 to immunologically undetect-
able levels in SCO1 patient fibroblasts (Figure 4E), and its
expression was attenuated but still supported in rho0 cells, which
harbor SCO1 but lack mitochondrial DNA and, therefore, cannot
assemble the COX holoenzyme (Figure 4F). These data collec-
tively suggest that mitochondrial signaling, through a subset of
COX assembly factors that includes SCO1, provides a post-
translational mechanism for the regulation of CTR1 abundance.
Lack of SCO1 Protein Reduces the Abundance of CTR1at the PM and Promotes Its Turnover by the ProteasomePrevious studies have demonstrated that CTR1 abundance is
inversely proportional to cellular Cu status, and that increases
Cell Reports 10, 933–943,
in cellular Cu levels promote the endocy-
tosis of CTR1 from the PM and facilitate
its subsequent degradation (Petris et al.,
2003; Guo et al., 2004). While the cellular
origin and nature of these regulatory in-
puts have yet to be fully defined, our
data implicate mitochondria in this signaling process. To further
investigate how SCO1 contributes to the regulation of CTR1
function, we harvested individual wild-type and Sco1-null livers
at P22, P27, P37, and P47, and used each one for multiple ana-
lyses to ensure the internal consistency of the data. Similar to our
previous findings, Sco1-null livers exhibited an age-dependent
decrease in the abundance of the mature, glycosylated form of
CTR1 (Figure 5A, arrow). While reductions in CTR1 abundance
were accompanied by a concomitant decrease in total liver Cu
content (Figure 5B), the small but significant Cu deficiency we
observed at P18 (Figure 3B) and P22 (Figure 5B) occurred in
the absence of obvious changes in CTR1 levels (Figures 4A
and 5A). We therefore conducted immunofluorescence analyses
to test the hypothesis that the onset of the Cu deficiency inSco1-
null livers is caused by reduced localization of the total CTR1
pool at the PM. We found that, although CTR1 was localized to
the cytosol and PM in both wild-type and Sco1-null livers at
P22 (Figures 5C and S7), there was indeed a modest reduction
in the extent of its coincident localization with Na+/K+ ATPase,
a PM marker protein (overlay correlation coefficients [OCC] =
89.2% ± 4.7% of wild-type, Figure 5B). The intensity of CTR1
staining at the PM in the Sco1-null liver had further declined by
P27, and the overall immunoreactivity was barely detectable at
P37 and P47 (Figure 5C), consistent with the marked reduction
in total CTR1 abundance (Figure 5A). Taken together, these
data suggest that mitochondrial signaling through SCO1 sup-
ports CTR1 localization at the PM and, therefore, Cu import
into the cell.
To further explore the relationship between SCO1 and CTR1,
we interrogated CTR1 in Sco1 knockout mouse embryonic fibro-
blasts (MEFs) (Figures 6A and 6B). Four independent clonal
Sco1�/� MEF lines (KO-2 through KO-5) were all COX and Cu
deficient, and had reduced steady-state levels of CTR1 relative
to control (flox) MEFs (Figures 6C and 6D). Stable overexpres-
sion of a Sco1 cDNA in Sco1�/� MEFs rescued CTR1 levels
February 17, 2015 ª2015 The Authors 937
Figure 4. Lack of SCO1 Protein Leads to a
Diminution in CTR1 Abundance
(A) ATP7A, ATP7B, CTR1, SCO1, and CCS1 abun-
dance in age-matched, wild-type (flox) and Sco1-null
(liv) livers from P18 to P57. Tubulin served as an in-
ternal loading control.
(B and C) ATP7A, CTR1, SCO1, and SCO2 abun-
dance in representative Lrpprc-null (P70); Cox10-null
(P47–P67); and age-matched, wild-type (flox) livers.
Extracts from P47 Sco1-null (liv) and wild-type (flox)
livers served as internal controls, while calnexin or
GAPDH served as loading controls.
(D) Cu content, COX, and SOD1 enzyme activities
(mean ± SEM) in Sco1-, Cox10- (n = 8), and Lrpprc-
(n = 3) null livers expressed as a percentage of that in
age-matched, wild-type littermates (Controls, Cox10
[n = 6]; Lrpprc [n = 3]). Data for Sco1-null livers are
from Figures 2E and 3B, and are shown here for
comparative purposes.
(E) SCO1 and CTR1 in control (C1) and SCO1-2
patient fibroblasts (Leary et al., 2013a) alone (�) or
stably overexpressing an shRNA to abolish SCO1
expression (Leary et al., 2007). Tubulin served as a
loading control.
(F) CTR1, SCO1, COX II, and porin abundance in a
rho+ (143B) and rho0 human osteosarcoma cell line.
Tubulin served as an internal loading control. For
(A–C), the left-hand-side arrow denotes the mature,
mitochondrial form of the SCO1 protein, and the
double asterisk highlights a second immunoreactive
band. For all except (D), the arrow and asterisk on the
right-hand side indicate the mature, glycosylated
CTR1 and the cleaved form of the protein, respec-
tively (see also Figures S4 and S6).
938 Cell Reports 10, 933–943, February 17, 2015 ª2015 The Authors
Figure 5. SCO1 Is Required to Maintain
CTR1 Abundance and Its Localization at
the PM in the Liver
(A) ATP7A, CTR1, and SCO1 abundance in age-
matched, wild-type (flox), and Sco1-null (liv) livers
from P22 to P47. The left-hand-side arrow denotes
the mature, mitochondrial form of the SCO1 pro-
tein, and the double asterisk highlights a second
immunoreactive band. The right-hand-side arrow
denotes mature, glycosylated CTR1, while the
asterisk indicates the cleaved form of the protein.
All samples were immunoblotted on the same
membrane, and tubulin served as an internal
loading control.
(B) Cu content and correlation coefficient (mean ±
SEM) of CTR1 and Na+/K+ ATPase costaining
(OCC, n = 7 for each time point) in Sco1-null livers
expressed as a percentage of age-matched, wild-
type livers (Controls).
(C) Individual and overlay images of the localization
of CTR1 and Na+/K+ATPase in wild-type (Controls)
and Sco1-null (Sco1liv/liv) livers (scale bar, 10 mm)
(see also Figure S7).
and the combined COX and Cu deficiency (Figures 6D and 6E),
demonstrating a SCO1-specific effect. CTR1 levels were also
increased by stable overexpression of a Ctr1 cDNA in
Sco1+/+MEFs; however, no change in its abundance was
observed in Sco1�/� MEFs, suggesting that CTR1 is actively
degraded by the proteasome in the absence of SCO1 expression
(Figure 6E). Consistent with this idea, incubation of Sco1�/�, butnot Sco1+/+, MEFs with the proteasomal inhibitors MG132 (Fig-
ure 7) or lactacystin (data not shown) resulted in a significant
increase in CTR1 abundance. These data further argue that dele-
tion of Sco1 affects a mitochondrial signaling pathway that reg-
ulates high-affinity Cu uptake into the cell by CTR1.
DISCUSSION
Several lines of evidence presented herein directly implicate
SCO1 in the post-translational regulation of the high-affinity Cu
importer CTR1. First, liver-specific deletion of Sco1 caused a
severe Cu deficiency associated with a lack of themature, glyco-
sylated form of CTR1. Second, reductions in SCO1 abundance
attenuated the PM localization of CTR1, and, ultimately, led to
a profound reduction in CTR1 protein levels, without affecting
its steady-state mRNA levels. Third, CTR1 protein levels were
Cell Reports 10, 933–943,
restored in Sco1�/� MEFs by inhibiting
the proteasome or overexpressing a
Sco1 cDNA. Finally, rho0 cells could not
assemble COX yet still expressed SCO1
and CTR1, while SCO1 patient fibroblasts
that lacked immunologically detectable
SCO1 but harbored residual COX activity
(�12% of control; Leary et al., 2013a)
had reduced levels of CTR1. These data
further argue that the observed effect on
CTR1 is largely attributable to SCO1 and
independent of COX activity, and are
consistent with our ability to dissociate the Cu and COX defi-
ciency phenotypes in patient cells (Leary et al., 2007). When
combined with our previous work, which supports the idea that
SCO1 function also impinges on the trafficking and Cu efflux ac-
tivity of ATP7A (Leary et al., 2007, 2013b), these investigations
emphasize that a SCO1-mediated mitochondrial signal is at
the center of a coordinated cellular response to limit Cu
availability.
Like affected tissues in SCO1 and SCO2 patients (Leary et al.,
2007; Stiburek et al., 2009), Sco1-null mouse livers exhibit a
combined COX and Cu deficiency. Ultrastructural, biochemical,
and molecular analyses all strongly suggest that Sco1-null livers
attempt to compensate for the severe, isolated COX deficiency
by increasing mitochondrial content, a classic response to
impaired organelle function observed in many mitochondrial
disorders (DiMauro et al., 2012). Lipid homeostasis is also signif-
icantly perturbed in Sco1-null livers, and there is an age-depen-
dent increase in lipid droplet accumulation. It is, therefore,
tempting to speculate that hepatic steatosis is responsible for
the death of liver-specific Sco1 knockout mice. However, liver-
specific Cox10 deletion also results in profound steatosis,
yet hepatic function eventually normalizes and these mice
remain viable (Diaz et al., 2008). Mice similarly recover following
February 17, 2015 ª2015 The Authors 939
Figure 6. Sco1�/� MEFs Are COX and Cu Deficient Owing to an
Inability to Express Adequate Levels of CTR1
(A and B) (A) PCR genotyping and (B) abundance of SCO1 and COX IV in Sco1-
floxedMEFs cocultured in the presence or absence of Cre recombinase. SD70
served as an internal loading control.
(C) SCO1, CTR1, and ATP7A abundance in parental (flox) and Sco1�/� MEF
clones (KO-2 through KO-4). SD70 served as an internal loading control.
(D) COX activity and total Cu content of 4 distinct Sco1�/� MEF clones alone
(�) or overexpressing a wild-type Sco1 or Ctr1 cDNA, expressed as a per-
centage of the parental MEF lines (flox) (mean ± SEM). ND, not determined.
(E) SCO1, COX I, COX IV, CTR1, and ATP7A abundance in parental (flox) and
Sco1�/�MEF clones (KO-5) alone (�) or overexpressing SCO1orCTR1. Tubulin
served as an internal loading control. For (C and E), the arrow denotes mature,
glycosylated CTR1, while the asterisk indicates the cleaved form of the protein.
liver-specific deletion of Ctr1 (Kim et al., 2009), and there is no
evidence of death in liver-specific Lrpprc knockout mice up to
3 months of age (E. Shoubridge, personal communication).
This suggests that the high frequency of mortality in liver-specific
Sco1 knockout mice also reflects the fact that Sco1-null livers
exhibit the most severe COX and Cu deficiencies of all of these
mouse models.
Perhaps milder models of liver failure are able to repopulate
the failing liver by engaging the proliferation of a subpopulation
of hepatocytes with low or no Cre recombinase activity. Sco1-
null livers appear to be particularly deficient in this regard,
because residual levels of the mature form of the SCO1 protein
are barely detectable at P18, and pathway analysis of our
microarray data indicates that Sco1 deletion significantly im-
pairs the mitotic potential of hepatocytes. It is also possible
that impaired function of other organ systems contributes to
940 Cell Reports 10, 933–943, February 17, 2015 ª2015 The Authors
the mortality of liver-specific Sco1 knockout mice. Fe levels
are ultimately elevated in the kidney and the spleen of liver-
specific Sco1 knockout mice, and the spleen is severely atro-
phied. Hematological and histological analyses indicate that
there is an initial and selective loss of white pulp; however, sub-
sequent Fe accumulation in the reticuloendothelial cells of the
spleen and in the Kupffer cells of the Sco1-null liver are both
consistent with the eventual loss of red pulp function, with
the latter being an established compensatory response aimed
at removing senescent red blood cells from the circulation.
While there are intriguing parallels between our mouse model
and patients with nonalcoholic fatty liver disease, who also pre-
sent with hepatic Cu deficiencies (Aigner et al., 2010), these
patients typically exhibit splenomegaly and not hyposplenism.
Additional studies will be required to tease apart how hepatic
ablation of Sco1 expression impairs the ability of the liver to
communicate with the spleen, thereby impinging upon its
function.
Our data strongly suggest that the Cu deficiency in Sco1-null
livers is driven by changes in the localization and, ultimately,
abundance of CTR1. The mild but significant Cu deficiency in
Sco1-null livers at P18 and P22 occurs in the absence of a
detectable change in CTR1 abundance, but is accompanied
by a modest attenuation in the PM localization of the protein.
The Cu deficiency in Sco1-null livers is markedly worse at P27,
and coincides with a clear reduction in the PM localization and
abundance of CTR1, which is further exacerbated with age. Re-
sidual COX activity is less than 10% of wild-type and barely de-
clines thereafter, arguing that Sco1-null liver mitochondria
already lack SCO1 protein at P27. The progressive reduction in
CTR1 abundance in Sco1-null livers likely reflects its enhanced
proteasomal turnover, because hepatic Ctr1 mRNA levels are
normal and treatment of Sco1�/� MEFs with the proteasomal in-
hibitor MG132 restored CTR1 abundance to wild-type levels.
These findings collectively suggest that SCO1 contributes to
the generation of a mitochondrial signal that is required to pro-
mote the PM localization of CTR1 and/or inhibit its turnover by
the proteasome. Because endocytosis of CTR1 from the PM
and its subsequent degradation are appropriate responses to
cellular Cu overload and reflect an attempt to normalize Cu levels
(Petris et al., 2003), or at least limit further Cu uptake, we favor
the idea that the lack of SCO1mis-signals a state of Cu overload
in the Sco1-null liver. Consistent with this idea, the abundance of
the known biomarker of Cu deficiency CCS1 is lower inSco1-null
livers than it is in wild-type livers, even though it is well known
that CCS1 levels are increased in a Cu-deficient state (Car-
uano-Yzermans et al., 2006). To the best of our knowledge,
this represents the first example of a stimulus other than direct
manipulation of Cu status that regulates CTR1 function. Whether
the SCO1-dependent mitochondrial redox signal that regulates
the trafficking and Cu efflux activity of ATP7A in human fibro-
blasts (Leary et al., 2007, 2013b) also functions to modulate
the activity of CTR1 is not yet known. However, it seems unlikely
that CTR2, which physically interacts with the mature, glycosy-
lated form of CTR1 and facilitates its proteolytic processing (Ohr-
vik et al., 2013), plays a role in this pathway in the liver because
the cleaved CTR1 product does not accumulate in Ctr2- (Ohrvik
et al., 2013), Cox10-, and Sco1-null livers.
Figure 7. Proteasome Inhibition Restores CTR1 Levels in Sco1�/�
MEFs
(A) CTR1 and ATP7A abundance in parental (flox) and Sco1�/� MEF clones
(KO-5) grown in basal media or in media containing MG132. Tubulin served as
an internal loading control. The arrow denotes mature, glycosylated CTR1,
while the asterisk indicates the cleaved form of the protein.
(B) Individual and overlay images of the localization of CTR1 and wheat germ
agglutinin (WGA) in parental (flox) and Sco1�/� MEF clones (KO-5) grown
in basal media or in media containing the MG132 (scale bar, 20 mm). For
(A and B), cells were grown for 6 hr in media containing vehicle (i.e., DMSO) or
40 mM MG132.
Even though there was a clear connection between SCO1 and
CTR1 that, when perturbed, drove the onset and early progres-
sion of the Cu deficiency in Sco1-null livers, hepatic Cu levels
never dropped below �20% of wild-type. Biochemical analyses
indicated that very little of this residual Cu was contained within
COX or SOD1. Yet, like affected cell types from SCO patients
(Dodani et al., 2011), Sco1-null livers preserved the total size of
their mitochondrial Cu pool even in the face of a severe cellular
Cu deficiency. SCO2 abundance is significantly increased in
Sco1-, Cox10-, and Lrpprc-null livers, while livers of transgenic
mice expressing a SCO2 variant equivalent to the pathogenic
E140K allele in SCO2 patients exhibit a mild reduction in mito-
chondrial Cu content in the absence of a change in cellular Cu
Ce
status (Yang et al., 2010). Thus, an attractive postulate is that
SCO2 acts alone or in concert with SCO1 tomodulate the activity
of a transporter that is essential for Cu import (Vest et al., 2013) or
export across the inner mitochondrial membrane. Our data sug-
gest that, although the total mitochondrial Cu pool remains intact
in Sco1-null livers, Cu is preferentially routed to the secretory
pathway once CTR1 levels are barely detectable and the Cu
deficiency has stabilized. Serum levels of Cu and of the secreted
Cu-loaded ferroxidase ceruloplasmin increased considerably
beginning at P37, and the latter observation is unlikely to be
explained by reduced protein turnover because the half-life of
holo-ceruloplasmin in mouse serum is estimated to be roughly
1 day (Gitlin and Janeway, 1960). The increased rate of holo-
ceruloplasmin maturation also was accompanied by a marked
increase in biliary volume in Sco1-null livers relative to wild-
type littermates (�10–203), but we did not observe a concordant
increase in the Cu content of feces (data not shown). At present,
it is unclear if lower affinity Cu uptake systems allow the Sco1-
null liver to maintain a fixed residual Cu content in the absence
of immunologically detectable CTR1.
In summary, we have developed the first animal model of hu-
man disease associated with mutations in SCO1, and used it to
uncover a novel connection between SCO1 function and the
post-translational regulation of the high-affinity Cu importer
CTR1. Our findings expand upon the known role of the mito-
chondrion in the regulation of cellular Cu homeostasis (Leary
et al., 2007, 2013b), and suggest that SCO1 is an essential
component of a mitochondrial signaling pathway that modulates
the activity of both the cellular Cu uptake and efflux machinery.
Why the abundance of this machinery is influenced by the dele-
tion of other COX assembly factors, like Cox10 and Lrpprc, and
whether this involves significant changes in the redox state and
therefore functionality of SCO1 warrant further attention. Future
studies will further explore the functional coupling of SCO1 and
CTR1, and investigate the significance of this coupling to the
regulation of Cu homeostasis in other tissues.
EXPERIMENTAL PROCEDURES
Transgenic Animal Generation and Husbandry
A detailed description of how Sco1 transgenic mice were generated, main-
tained, and manipulated is provided in the Supplemental Experimental Proce-
dures. All animal-related experiments were approved by the Animal Ethics
Review Board at the University of Saskatchewan (AUP #20100091).
Immunoblot Analyses
Mitochondria were isolated frommouse tissues as previously described (Leary
et al., 2013b). For denaturing SDS-PAGE,mitochondrial, whole-tissue, and cell
extracts were prepared on ice in extraction buffer (Potting et al., 2013) supple-
mentedwith a complete protease inhibitor cocktail (Roche) and 0.5mMPMSF,
clarified by centrifugation at 16,600 3 g for 10 min at 4�C, and equal amounts
of protein (10–25 mg/lane) were electrophoresed on 12, 15, or 4%–20% Tris-
HCl gels. For native PAGE, tissue extracts were prepared and equal amounts
of protein (10 mg/lane) were electrophoresed by blue native PAGE on a 5%–
13% gradient gel, as described elsewhere (McKenzie et al., 2007). Equal vol-
umes of serum (2 ml/lane) were electrophoresed at 4�C on 4%–20% Tris-HCl
gels under native conditions to detect the Cu-loaded and apo forms of cerulo-
plasmin (Sato andGitlin, 1991), and under denaturing conditions to detect total
ceruloplasmin content. All gels were transferred to nitrocellulose under semi-
dry conditions and blotted with the appropriate antibodies (see Supplemental
Experimental Procedures). Following incubation with secondary antibody,
ll Reports 10, 933–943, February 17, 2015 ª2015 The Authors 941
immunoreactive proteins were detected by luminol-enhanced chemilumines-
cence (Pierce). The abundance of the mature, glycosylated form of CTR1
was quantified densitometrically for multiple exposures within the linear range
of the film using ImageQuant software (Leary et al., 2009).
Imaging Analyses
Tissue preparation for various applications is described in detail in the Supple-
mental Experimental Procedures. For immunofluorescence analyses, six
micrometer fixed liver sections mounted on slides were incubated in Tris-buff-
ered saline (TBS [pH 7.4], 25 mM Tris-HCl, 137 mM NaCl, and 2.7 mM KCl)
containing 1% BSA, 0.2% fat free milk, 5% v/v goat serum, and 0.2% Triton
X-100. Slides were washed with PBS, and incubated overnight at 4�C in block-
ing buffer containing polyclonal CTR1 (1:100; Jack Kaplan, University of
Chicago) and monoclonal Na+/K+ ATPase (1:500; Millipore) antibodies. Slides
were washed with PBS, and incubated in blocking buffer containing Alexa
Fluor 488 goat anti-rabbit (1:1,000, Life Technologies) and Alexa Fluor 568
goat anti-mouse (1:500, Life Technologies) secondary antibodies. Slides
were once again washed with PBS, mounted with Prolong Gold Antifade
Mountant containing DAPI (Life Technologies), and dried for 10–15 min.
Stained slides were visualized by confocal laser-scanning microscopy (LSM
700, AxioObserver, Carl Zeiss). Green, red, and DAPI fluorescence was de-
tected using a laser Diodes tuned to 488, 555, and 405 nm, respectively. Fluo-
rescence images were collected simultaneously and OCC were calculated
using a PC running ZEN 2012 software (version 8.0), while Fiji (Schindelin
et al., 2012) or ImageJ software was used to split the channels.
Miscellaneous
Protein concentration and the enzymatic activities of COX, CS, SOD1, and
SOD2 were measured as previously described (Leary et al., 2002, 2004). Sta-
tistical analyses were conducted using Prism (GraphPad), and are detailed
wherever appropriate in the text. Methods related to cell culture experiments
and elemental, lipid, serum, microarray, RT-PCR, and genotyping analyses
are detailed in the Supplemental Experimental Procedures.
ACCESSION NUMBERS
The GEO accession number for the microarray data reported here is
GSE58997.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
http://dx.doi.org/10.1016/j.celrep.2015.01.019.
AUTHOR CONTRIBUTIONS
S.C.L. generated the floxed Sco1mouse. C.J.H., B.L., A.H., and Z.N.B. devel-
oped the liver-specific Sco1 knockout mouse model. S.C.L., P.A.C., T.W., and
M.J.P. designed the experiments. F.D. and C.T.M. provided Cox10 floxed
and knockout livers. P.J.M. and S.J.S. conducted the lipid analyses. D.V.
and M.J.P. did the initial IF experiments. C.J.H., B.L., Z.N.B., P.A.C., L.D.Y.,
A.B., T.W., A.H., A.M.E., and S.C.L. executed all other experiments. C.J.H.,
B.L., Z.N.B., and S.C.L. analyzed the data. S.C.L. drafted the manuscript
and P.A.C., T.W., A.M.E., S.J.S., F.D., C.T.M., and M.J.P. provided editorial
feedback.
ACKNOWLEDGMENTS
We wish to acknowledge Drs. Joe Prohaska (University of Minnesota Medical
School), Dennis Winge (University of Utah), Dennis Thiele (Duke University),
Jonathan Gitlin (Marine Biological Laboratory), and Jaekwon Lee (University
of Nebraska-Lincoln) for helpful discussions; Drs. Dennis Thiele, Joe Pro-
haska, and Jack Kaplan (University of Chicago) for CTR1 or CCS1 antiserum;
and Dr. Eric Shoubridge (McGill University) for Lrrprc liver samples. Grants-in-
aid of research to S.C.L. from the Saskatchewan Health Research Foundation,
942 Cell Reports 10, 933–943, February 17, 2015 ª2015 The Authors
the Canadian Institutes for Health Research (CIHR), and the Canadian Gene
Cure Foundation, and to P.A.C. from the National Science Foundation funded
this research. S.C.L. is a New Investigator of the CIHR, and T.W. is a long-term
fellow of the Human Frontiers Science Program.
Received: July 28, 2014
Revised: December 15, 2014
Accepted: January 8, 2015
Published: February 12, 2015
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