ARTICLE
Received 30 Jan 2013 | Accepted 14 Jun 2013 | Published 16 Jul 2013

DOI: 10.1038/ncomms3147

A stabilizing factor for mitochondrial respiratory
supercomplex assembly regulates energy
metabolism in muscle
Kazuhiro Ikeda1, Sachiko Shiba1, Kuniko Horie-Inoue1, Kunitoshi Shimokata1,2 & Satoshi Inoue1,2

The mitochondrial respiratory chain is essential for oxidative phosphorylation and comprises
multiple complexes, including cytochrome c oxidase, assembled in macromolecular supercomplexes. Little is known about factors that contribute to supercomplex organization. Here
we identify COX7RP as a factor that promotes supercomplex assembly. Cox7rp-knockout mice
exhibit decreased muscular activity and heat production failure in the cold due to reduced
COX activity. In contrast, COX7RP-transgenic mice exhibit increased exercise performancewith increased cytochrome c oxidase activity. Two-dimensional blue native electrophoresis
reveals that COX7RP is a key molecule that promotes assembly of the III2/IVn supercomplex
with complex I. Our study identiﬁed COX7RP as a protein that functions in I/III2/IVnsupercomplex assembly and is required for full activity of mitochondrial respiration.

1 Division of Gene Regulation and Signal Transduction, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka-shi,

Saitama 350 1241, Japan. 2 Department of Anti-Aging Medicine, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, 7-3-1 Hongo, Tokyo 113
8865, Japan. Correspondence and requests for materials should be addressed to S.I. (email: sinoue07@saitama-med.ac.jp).
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3147

he respiratory chain supercomplexes in the inner membrane of mitochondria form ‘respirasome’ structures,
which mainly comprise complexes I and III, and theterminal enzyme COX (complex IV) in mammalian cells1,2.
Complexes II and V are usually not associated with any of the othercomplexes3. Complex IV is comprised of three subunits encoded by
mitochondrial DNA and 10 subunits encoded by nuclear DNA4. In
addition to mutations in mitochondrial DNA-encoded COX
genes5,6, mutations in nuclear DNA-encoded COX component
genes and COX accessory factors cause COX deﬁciency6–8. For
example, SURF1 is responsible for Leigh syndrome9,10, SCO1 forneonatal-onset hepatic failure and encephalopathy11, SCO2 for fatal
infantile cardioencephalomyopathy12, and COX15 for fatal
hypertrophic cardiomyopathy13. SCO1 and SCO2 are copper
metallochaperones that are necessary for copper insertion into thecatalytic core of COX assembly14. The latter has a particular role in
the regulation of cellular copper homoeostasis as the overexpressionof SCO2 can suppress cellular copper deﬁciency in ﬁbroblasts from
SCO2, SCO1 and COX15 mutation patients15.
Supercomplex assembly has been shown to enhance respiratory
chain activity through spatial restriction of electron carrier
diffusion3, although the factors involved in the formation andmaintenance of supercomplex assembly remain to be elucidated.
As a phospholipid that maintains the integrity of themitochondrial membrane, cardiolipin is required for the speciﬁc
association between complexes and a decrease in its content
might deeply affect inter-complex assembly16. A mutation of the
cardiolipin-remodeling enzyme TAZ forms a labile supercomplex
in Barth syndrome patients’ cells, causing complex IV todissociate readily from the supercomplex17. In various types ofheart failure, excessive sympathetic stimulation may also
dysregulate mitochondrial function by affecting either
individual complexes or their supramolecular assemblies18.
Recently, respiratory supercomplex factor 1 (Rcf1) and its
homologue Rcf2 have been identiﬁed in yeast as factors required
for the normal assembly and complete enzymatic activity ofcomplex IV19–21. Rcf1 and Rcf2 are both required for complete
complex IV assembly and enzyme activity. Rcf1 is required forcomplex assembly of subunits Cox13 (mammalian Cox6a) and
Rcf2, as well as for the oligomerization of a subclass of cytochromeoxidase complexes into respirasomes. COX6a has been shown to be
involved in the stabilization of the dimeric state of COX at a laterstage of complex IV assembly, together with COX7a or COX7b
subunits, and may contribute to the formation of an interaction site
for cytochrome c (ref. 4). In the absence of Rcf1 and Rcf2, theassembly of Cox13 and Cox12 (mammalian Cox6b) are adversely
affected, but the core of complex IV can associate with complex III(ref. 19). Thus, the Rcf1 and Rcf2 proteins are rather assumed
to be complex IV late-stage assembly factors. Yet, factors
responsible for supercomplex assembly per se have not been wellcharacterized.
Here we focus our study on a COX-related molecule, cytochromec oxidase (COX) subunit 7a-related protein (COX7RP), which
was originally identiﬁed as an oestrogen-responsive gene in MCF7
cells22. COX7RP encodes a 114-amino-acid protein, which issimilar to the mitochondrial respiratory enzyme COX subunit 7a(ref. 23). In the present study, we assess whether COX7RP is a
novel modulator of mitochondrial metabolism in vivo. We generate
Cox7rp-knockout mice (Cox7rpKO) and show that homozygous
KO mice have muscle weakness. Biochemically, these animals
exhibit decreased mitochondrial respiratory activities and impaired
formation of mitochondrial supercomplexes in muscles. Cox7rpKO
mice also exhibit heat production failure in the cold. In contrast,
COX7RP-transgenic mice (COX7RP-Tg) exhibit increased exercise
performance with increased COX activity. Thus, COX7RP isshown to be a factor that functions in supercomplex assembly,
2

which is necessary for the full activity of mitochondrial
respiration.
Results
COX7RP promotes oxidative phosphorylation and ATP
synthesis. COX7a-related protein (COX7RP) is an oestrogenresponsive gene identiﬁed by genomic-binding site cloning22.
Compared with the COX7a in complex IV, the amino-acidresidues of the COX7RP protein are highly conserved23, suggesting
its role in mitochondrial oxidative phosphorylation. We generated
Cox7rpKO mice from the library of embryonic stem cells
harbouring mutations by random retroviral gene trapping24.
Genomic sequencing showed that the retroviral vector (7.4 kb)
was inserted into the ﬁrst intron of mouse Cox7rp, (Fig. 1a,b). The
resulting integration caused the disruption of full-length transcriptsof Cox7rp gene and the production of 2 fusion transcripts, a
50 portion with neo and a 30 portion with BTK. Cox7rp protein was
abolished in Cox7rpKO mouse embryonic ﬁbroblasts (MEFs)(Fig. 1c). In general, Cox7rpKO mice were obtained with the
expected Mendelian frequency and without sexual bias.
Both male and female Cox7rpKO mice were fertile and theirappearance was not overtly different from that of wild-type(WT) littermates; however, COX activity and mitochondrial
ATP production rate were reduced in Cox7rpKO
MEFs compared with WT MEFs (Fig. 1d,e). Adenovirusmediated introduction of COX7RP elevated COX activity and
ATP production. DNA amounts in WT MEF cultures increased
steadily over time, whereas those in Cox7rpKO MEFs only slightly
increased over time (Fig. 1f). Furthermore, cellular
ATP production was substantially reduced in Cox7rpKO MEFs
compared with controls (Fig. 1g). Oxygen consumption rates
dependent on complex I (pyruvate and malate as substrates),
complex II (succinate as a substrate) and complex IV (N,N,N0 ,N0 tetramethyl-p-phenylenediamine
dihydrochloride,ascorbate,
and carbonyl cyanide-p-triﬂuoromethoxyphenylhydrazone as
substrates) were signiﬁcantly impaired in Cox7rpKO MEFs
compared with controls (Fig. 1h). We also generated transgenic
mice that overexpress human COX7RP under the control of CAG
promoter (COX7RP-Tg, Supplementary Fig. S1a,b). COX7RP-Tg
mice were also obtained with the expected Mendelian frequency
and without sexual bias. Both male and female COX7RP-Tg mice
were fertile and their appearance was not overtly different fromthat of WT littermates. COX7RP-Tg:Cox7rpKO (TgKO) mice weregenerated by intercross mating between COX7RP-Tg andCox7rpKO mice. Both male and female COX7RP-Tg mice werealso apparently normal. COX7RP mRNA was increased in skeletal
muscle and brown adipose tissue (BAT) of COX7RP-Tg mice
(Supplementary Fig. S1c,d). COX activity and cell proliferation
ability were enhanced in COX7RP-Tg MEFs compared with WT
MEFs (Supplementary Fig. S1e,f).
Cox7rp null mice have reduced running endurance. In
4-month-old Cox7rpKO mice, the weights of quadriceps femorismuscles were reduced compared with WT littermates, although
the body weights were not signiﬁcantly different (Supplementary
Fig. S2a,b). Histochemical staining revealed a reduction of COX
activity in quadriceps femoris muscles of 4-month-old Cox7rpKO
mice, whereas COX activity was restored in a cross-bred strain of
COX7RP-Tg:Cox7rpKO (TgKO) (Fig. 2a). COX activities inquadriceps femoris, gastrocnemius and cardiac muscles of KO
mice were 15–30% reduced compared with WT littermates(Supplementary Fig. S3). In the forced treadmill test, Cox7rpKO
mice showed 70% of the running capacity of their age- andweight-matched WT counterparts (Fig. 2b, Supplementary Fig. S4and Supplementary Movies 1–4). COX7RP-Tg mice exhibited

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3147

Termination
codons
LTR

SA

NEO

pA

PGK

BTK

L

SD

1 kb

LTR
TGA

ATG
Chr 17F

R
Cox7rp:Cox7rp:
WT

(bp)

+/–

KO

500

WT

300

Mut

**
Mitochondrial ATP(nmol min–1 per mg protein)

COX activity(OD 450 nm mg–1 per protein)

0.6
0.4
0.2

0
Cox7rp:

WT KO

WT KO

AdDsRedAdCOX7RP

COX7RP

β-Actin

0.3

2

**

**

0.4

**

KO

38

1
0.8

+/–

Relative DNA amount

**

WT

(kDa)
19

**

0.2

0.1

0
Cox7rp:WT KO WT KO
AdDsRedAdCOX7RP

*
* **
1

0.5

0
Days

1 2 341 2 34

Cox7rp:

WT

KO

160

40

70

1.5

90

WT
60

**

70
60
50
40
30
20

**

KO

120

30

50

*

40

**

20

80

10

40

30
20
10

10
0
Cox7rp:

Oxygen consumption(nmol min–1 per mg)

Cellular ATP(nmol mg–1 protein)

80

0

0
WT

KO

Pyruvate+
Malate0
Succinate

TMPD+
Ascorbate+
FCCP

Figure 1 | Reduction of mitochondrial respiratory activity in MEFs derived from Cox7rpKO mice. (a) Generation of Cox7rpKO mice. Schematic
representation of the gene trap vector insertion site in the mutant gene. BTK, bruton tyrosine kinase cassette; LTR, long terminal repeat; NEO, neomycinresistant gene cassette; pA, polyadenylation sequence; PGK, phosphoglycerate kinase-1 promoter; SA, splicing acceptor site; SD, splicing donor site.
(b) Genotyping of Cox7rpKO mice. Representative litters containing WT, Cox7rp þ /  ( þ /  ) and Cox7rpKO (KO) mice were shown. Sense primer-F and -L
within intron 1 and the vector, and common antisense primer-R within intron 1 (a) were used to amplify WT (489 bp) and mutant (Mut: 289 bp)
alleles, respectively. (c) Western blot analysis of Cox7rpKO MEFs. Cell extracts from MEFs were resolved on SDS–polyacrylamide gel and immunoblottedwith anti-COX7RP. (d) Decrease in COX activity in Cox7rpKO MEFs. COX activity was assessed using MEFs from WT and Cox7rpKO mice infected withrecombinant adenovirus expressing control DsRed (Ad-DsRed) or COX7RP (Ad-COX7RP). Data are presented as means±s.d. (n ¼ 3). *Po0.05 for
WT versus KO; Student’s t-test. (e) Decrease in mitochondrial ATP production in Cox7rpKO MEFs. ATP production was assessed using MEFs from WT and
Cox7rpKO mice infected with Ad-DsRed or Ad-COX7RP. Data are presented as means±s.d. (n ¼ 3). *Po0.05 for WT versus KO; Student’s t-test.
(f) Retardation of growth in Cox7rpKO MEFs. Data are presented as means±s.d. (n ¼ 3). *Po0.05, **Po0.01 for WT versus KO at each indicated time
point; Student’s t-test. (g) Decline of cellular ATP content in Cox7rpKO MEFs. Data are presented as means±s.d. (n ¼ 3). **Po0.01 for WT versus KO;
Student’s t-test. (h) Decreased oxygen consumption rates of Cox7rpKO MEFs. MEFs were permeabilized with digitonin, and subsequently substrates
and inhibitors were added. Rates of oxygen consumption are expressed as nanomoles of oxygen per minute per milligram of protein. Data are presented asmeans±s.d. (n ¼ 3) *Po0.05, **Po0.01 for WT versus KO under each condition; Student’s t-test.

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COX staining(Quadriceps femoris muscle)****

Running time (min)

100

KO

WT

*

80
60
40
20

0
Cox7rp: WT

0.13
0.12
0.11
0.10

** ** **************

0.11

KO

0.06

0.09

* *

0.08

KO
Tg

0.85

TgKO

0.80

*

0.07

WT

**
0.75
0.70

0.05
14 20 26 32 38(min)* * * **

0.90

0.10

0.06

0.07

** *

0.95

RQ

VO2 (mI min–1 per g)

0.14

VCO2 (mI min–1 per g)

0.12

8

TgKO

**

1.00

0.13
0.15

0.08

Tg

TgKO

Tg

0.09

**

8

14 20 26 32 38
(min)

8

14 20 26 32 38(min)

Figure 2 | Reduction of COX activity in muscles and compromised exercise performance in Cox7rpKO mice. (a) Reduced COX activity in Cox7rpKO mice.
COX staining of the quadriceps femoris of 4-month-old WT, Cox7rpKO (KO), COX7RP-Tg (Tg) and COX7RP-Tg:Cox7rpKO (TgKO) mice. Scale bar,
100 mm. (b) Compromised exercise performance in Cox7rpKO mice. Four-month-old KO, Tg and TgKO mice, as well as WT controls were subjected
to a forced treadmill exercise test. Data are presented as means±s.d. (n ¼ 7 or 8). *Po0.05, **Po0.01; Dunnett’s post hoc test. (c) Increase in the
respiration quotient in Cox7rpKO mice. CO2 production, O2 consumption and RQ were analysed during the above treadmill exercise test. Data are presented
as means±s.d. (n ¼ 7 or 8). *Po0.05, **Po0.01; Dunnett’s post hoc test.

130% of the running capacity of WT mice and TgKO micerecovered the running ability to the level of WT mice. Expression
microarray analysis revealed the dysregulation of pathways
involved in muscle physiology and diseases in Cox7rpKO muscles(Supplementary Tables S1 and S2). Thus, Cox7rp loss-of-function
leads to reduced mitochondrial oxidative capacity and impaired
muscle function in response to forced physical exertion. In the
study of indirect calorimetry during treadmill exercise, Cox7rpKO
had lower CO2 production (VCO2) and O2 consumption (VO2),
while the respiration quotient (RQ) was higher compared with
WT controls (Fig. 2c), indicating a decrease in the metabolic rate
and a metabolic shift to glucose consumption. Reduction of VO2
and VCO2 in Cox7rpKO mice was also shown in the steady state
before exercise (Supplementary Fig. S5). In contrast, COX7RP-Tg
mice exhibited an increase in VO2 and VCO2 even in the steadystate.
COX7RP regulates heat production in BAT. BAT weights of
WT and COX7RP-Tg mice were almost the same, whereas
Cox7rpKO BAT was substantially enlarged with white adipose tissue-like appearance, resulting from the accumulation of lipid droplets (Fig. 3a,b, and Supplementary Fig. S2c). The weights
of BAT in TgKO mice were intermediate. In the temperaturechallenge study, Cox7rpKO mice showed severe cold sensitivity 4 h
4after cold exposure at 4 °C, whereas WT mice could maintain theirbody temperature under the same condition (Fig. 3c). In the cold
exposure study in which the experimental end point was the bodytemperature r15 °C, Cox7rpKO mice exhibited the shortest dropout time among the four genotypes (Fig. 3d). Notably, the mRNAlevel of uncoupling protein Ucp1, which is known to have a criticalrole in the energy dissipation in BAT, was severely decreased in
Cox7rpKO BAT (Fig. 3e). Expression microarray analysis revealed
the dysregulation of pathways involved in mitochondria metabolism, oxidation and reduction reactions, fatty acid and lipid
metabolism, and fat cell differentiation in Cox7rpKO BAT(Supplementary Tables S3 and S4). Thus, disruption of Cox7rp
function leads to transition of BAT to white adipose tissue-likestructure and thermogenic defects in cold environments. In addition, by assessing expression levels of transcriptional regulatory
factors crucial for mitochondrial function, Ppara, Pparg, Pgc-1a andPgc-1b were revealed to be downregulated in BAT of Cox7rpKO
mice (Supplementary Fig. S6).
COX7RP is involved in respiratory supercomplex assembly.
We next investigated the contribution of COX7RP to supercomplex assembly. In two-dimensional blue native polyacrylamide
gel electrophoresis (PAGE) with subsequent immunoblotting for
digitonin-solubilized mitochondria in muscles, Cox7rp was

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KO

Tg

TgKO

d
35

WT
KO

30
25
20

**
**

15
10

Rate of dropout

Body temperature (°C)

1.0

P < 0.01 (KO versus WT)
P < 0.01 (TgKO versus WT)
P < 0.05 (KO versus TgKO)

0.8

WT
Tg
TgKO
KO

0.6
0.4
0.2
0

0

1

2

3
(h)

4

5

0

10

20
(h)

30

WT

KO

Tg

TgKO

e 1.0
mRNA expression/GAPDH

c

WT

40

WT
Tg
TgKO
KO

0.8
0.6
0.4

**
0.2

**

0
Ucp1

Ucp2

Ucp3

Fasn

Figure 3 | Increased mass and fat droplets of BAT and impaired resistance to cold exposure in Cox7rpKO mice. (a) Appearance of brown fat pads of
4-month-old WT, KO, Tg and TgKO mice. Scale bar, 5 mm. (b) Histological section and oil red O staining of brown fats of 4-month-old WT, KO, Tg and
TgKO mice. Scale bar, 100 mm. (c) Cox7rpKO mice are cold-sensitive. Body temperature of WT (blue box) and Cox7rpKO (red triangle) mice exposed
to 4 °C for 5 h was measured by using a thermofocus infrared thermometer. Data are the means±s.d. of ﬁve animals per group. **Po0.01; Student’s t-test.
(d) COX7RP-Tg mice demonstrate improved cold tolerance. Kaplan–Meier analysis of the body temperature was performed for mice exposed to 4 °C
for 5 h, in which the end point was deﬁned as r15 °C (n ¼ 7 or 8). Log-rank P-values after application of a Bonferroni correction were indicated.
(e) Decreased mRNA expression of uncoupling proteins and fatty acid synthase (Fasn) in Cox7rpKO BAT. qPCR analysis was performed using total RNA
from WT, KO, Tg and TgKO BAT. **Po0.01; Dunnett’s post hoc test.

detected predominantly in I/III2/IVn supercomplex in WT but notin Cox7rpKO mitochondria (Fig. 4a,b). Intensity of Cox4 signal insupercomplex I/III2/IVn was signiﬁcantly reduced by Cox7rp loss,
as B50 and 5% of Cox4 were involved in the supercomplex of
WT and KO mitochondria, respectively. We also noted that Cox4
signal in monomeric complex IV was increased, whereas the signal
in dimeric complex IV was decreased in Cox7rpKO mitochondria.
In terms of complex III assembly, the signal of Rieske iron-sulfur
protein (Risp), a late-stage subunit of supercomplex25, wasdecreased in I/III2/IVn in Cox7rpKO muscle compared with WT
muscle (Fig. 4c,d). An overexpression study of COX7RP in 293T
cells also revealed that COX7RP has a role in stabilizing complex
IV dimerization and supercomplex assembly (Supplementary
Fig. S7a,b). The RISP signal in I/III2/IVn supercomplex was
substantially intense in COX7RP-overexpressing cells comparedwith control cells (Supplementary Fig. S7c,d). Taken together, thedata reveal that COX7RP is a promoting factor for theorganization of I/III2/IVn supercomplex. The contribution of
COX7RP to the assembly of III2/IVn supercomplex is not clear so
far based on our results.
Discussion
Our ﬁndings provide a new insight into the normal andpathological relevance of mitochondrial supercomplex assemblyin mammalian systems. COX7RP is a nuclear DNA-encoded
mitochondrial gene that can facilitate supercomplex assembly ofthe mitochondrial respiratory chain and can modulate mitochondrial energy metabolism. The characteristics of Cox7rpKO mice
reveal that the gene has a critical role in muscle activity, as well as
adaptive thermogenesis in vivo. The present study demonstrates

the ﬁrst mammalian phenotype of the disorder of a supercomplex-promoting factor, which enables researchers to dissect
the function of COX7RP in normal and disease states of
mitochondria and to manipulate the structure of supercomplex
assembly in vivo.
As structurally COX7RP-related proteins, COX7A family
originally contains two members, COX7A1 and COX7A2(ref. 26). COX7A has been shown as one of the 13 subunits ofbovine heart complex IV by crystal structure analysis4. KO miceof heart-type Cox7a1 showed reduced COX activity, dilated
cardiomyopathy at 6 weeks of age27 and resulted in exercise
intolerance28. These ﬁndings indicate that COX7A family may
have important roles in COX activity and its regulation; however,the precise molecular mechanism has not been understood.
COX7A has been considered as a late-assembling subunit similar
to COX6A ref. 29, and in vitro translated COX7A was ﬁrst
incorporated into complex IV and supercomplex III þ IV basedon in vitro import and assembly assays30. Thus, it is likely that
COX7A has a role in the late steps of holo-complex IV formation,
as well as in the assembly of III and IV, and is located in the
periphery of complex IV. In regard to the stoichiometry of
COX7A in complex IV, either COX7A1 or COX7A2 would be
involved in complex IV as preferential expression of COX7A1
and COX7A2 are known in muscles and liver, respectively.
Compared with other COX7A family proteins, COX7RP protein
harbours a speciﬁc N-terminal domain through which COX7RP
may exert unique function in the regulation of supercomplex
assembly and mitochondrial respiration. Our data reveal that
COX7RP is a unique factor that promotes the assembly ofsupercomplex III þ IV with complex I, (as opposed to theassembly of III and IV). Considering possible differential

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(%)

68.8

31.2

38
(%)
18.2 4.5 28.4

26
48.9

WB:
Uqcrc2
Ndufa919

SDS-PAGE
Cox4

(kDa)
46

BN-PAGE
I/III2/IVn I/III2(%) III2/IVn IV2 IV

45.5

38
26

(%)
20.6 0.5 73.9

5.0
19

Cox4
Cox7rpCox7rpWT muscle

Cox7rpKO muscle
BN-PAGE

BN-PAGE
I/III2/IVn I/III2

III2/IVn

I/III2/IVn I/III2
WB:

(kDa)
46

56.9

43.1

(%)

Uqcrc2
Ndufa9

38
66.4

33.6

19
WT muscle

(%)

Risp
SDS-PAGE

26

WB:
Uqcrc2
Ndufa9

54.5

(kDa)
46

III2/IVn

31.8 68.2

WB:(%)

Uqcrc2
Ndufa9

(%)

Risp

38
26

10.4

89.6

19
Cox7rp KO muscle

SDS-PAGE(kDa)
46

III2/IVn IV2 IV

SDS-PAGE

BN-PAGE
I/III2/IVn I/III2

Figure 4 | COX7RP promotes assembly of respiratory chain supercomplexes. Mitochondrial proteins of skeletal muscle from WT (a,c) and Cox7rpKO
mice (b,d) were solubilized with digitonin in the concentration of 2 g g  1 protein (a,b) or 0.5 g g  1 protein (c,d), and subjected to blue nativepolyacrylamide gel electrophoresis (BN-PAGE) followed by second-dimensional SDS–PAGE. Western blot analysis was performed with antibodies for
NDUFA9 of complex I, UQCRC2 of complex III, RISP of complex III, COX4 of complex IV and COX7RP. Positions corresponding to mitochondrialsupercomplex species I/III2/IVn, I/III2, III2/IVn, dimerized complex IV (IV2) and monomerized complex IV (IV) are indicated. Percentages of UQCRC2, RISP
or COX4 signal intensities in each whole amount are indicated in the images.

functions of COX7RP and COX7A, COX7RP might exist in the
more peripheral region of complex IV after the incorporation of
COX7A into complex IV, and both COX7RP and a tissue-speciﬁc
COX7A may be incorporated into complex IV or supercomplex I/
III/IV. It is also speculated that once COX7RP binds tosupercomplex III þ IV, it would be quickly incorporatred into
supercomplex I/III/IV as COX7RP may function as a pilot. It may
be one reason why the role of COX7RP in the assembly of III andIV has not been clearly shown in our results. Questions are still
remained whether COX7A also has a role in the assembly ofsupercomplex III þ IV with I and whether COX7A can substitutefor COX7RP in this context. Taken together, COX7A and
COX7RP may have distinct roles in supercomplex assembly, as
COX7A supports COX assembly in the III2/IVn supercomplex,
while COX7RP supports COX’s ability to assemble with III into
the I/III2/IVn supercomplex.
We found that the brown adipocytes in Cox7rpKO mice
accumulate excess amounts of lipids, resulting in a cold-sensitive
phenotype of KO mice. BAT is an essential tissue that contains
abundant mitochondria and exerts energy dissipation and rapid
thermogenesis through mitochondrial uncoupling, utilizing
uncoupling proteins such as UCP1 (refs 31,32). Ucp1 and Fasn
mRNA expression is impaired in Cox7rpKO BAT. Thus, COX7RP
has a critical role in heat production or lipid metabolism in BAT.
It is also notable that RQ in Cox7rpKO mice is elevated during
exercise. The result implies that COX7RP may be preferentiallyinvolved in the utilization of fatty acids or amino acids rather
than glucose in a condition demanding high production ofenergy. Future study of Cox7rpKO and COX7RP-Tg mice fed
with high- or low-fat diet will further provide information inregard to the contribution of COX7RP to glucose and lipid
metabolism, obesity, insulin resistance and diabetes.
Interestingly, a couple of mice models deﬁcient in genes related
to energy metabolism also exhibit impaired thermogenesis, similar
to Cox7rpKO mice. Mice lacking oestrogen-related receptor alpha6(ERRa) are defective in adaptive thermogenesis due to the
reduction of mitochondrial mass and oxidative capacity in brown
adipocytes33, as this nuclear receptor is considered as an importanttranscription factor that regulates mitochondrial biogenesis andoxidative metabolism34,35. The ERRa coactivator peroxisome
proliferator-activated receptor-gamma coactivator 1a (PGC-1a) has
been also shown to regulate adaptive thermogenesis in BAT36–38.
Increased expression of PGC-1a delays the onset of mitochondrial
myopathy in mice with skeletal muscle-targeted ablation of the
COX assembly factor COX10 (ref. 39). Increased expression of
PGC-1b, as well as PGC-1a improved mitochondrial respiration incells with complex III or IV deﬁciency40. These studies may indicatethat COX7RP is one of the genes involved in pathways regulated by
ERRa and PGC-1a. Indeed, Cox7rp has been shown to be one ofthe downregulated metabolic genes in a heart failure mouse modelwith cardiac-speciﬁc disruption of Cdk7/cyclin H/ménage-à-trois 1,
which is critical for the transcriptional function of PGC-1a (ref. 41).
Impaired mitochondrial activity due to Cox7rp loss may also
lead to the altered expression of nuclear-encoded mitochondrial
genes, as we found that Pgc-1a and b mRNA expressions were
downregulated in Cox7rpKO BAT. It has been known thatmitochondrial dysfunction can activate retrograde signalling frommitochondria to the nucleus, which has a role in life span
expansion in yeasts, for example42. In mitochondrial dysfunction,
cellular NAD þ /NADH ratio, ATP/ADP ratio, cytosolic freeCa2 þ concentration, cellular oxidative stress may act as signalling
mediators that modulate the downstream transcription factors in
the nucleus43. In particular, PGC-1a has important roles
in mitochondrial retrograde signalling. For example, thetranscription of PGC-1a gene is activated by phosphorylated
CREB transcription coactivator triggered by increased cytosolicCa2 þ (ref. 44). Cellular stress-dependent ATP depletion activates
AMP-activated protein kinase, which also phosphorylates andactivates PGC-1a (ref. 45). In mammalian cells with an A3242G
mutation of mitochondrial DNA, mitochondrial dysfuction

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3147

altered expressions of several transcription factors including
RXRA, which led to the decrease in PGC-1a recruitment and therepression of oxidative phosphorylation genes46. The involvementof PGC-1b in the retrograde signalling has not been reported,
however, Pgc-1b may also have a role in the pathologicalcondition of Cox7rpKO mice similar to Pgc-1a.
In conclusion, COX7RP is a nuclear DNA-encoded mitochondrial gene that can promote the respiratory supercomplex
assembly of the III2/IVn supercomplex with complex I, rather
than the assembly of the III2/IVn supercomplex itself. COX7RP
has a critical role in muscle activity, as well as adaptive
thermogenesis in vivo. Our different mouse strains with altered
COX7RP expression will be useful tools for modulating the stateof supercomplex assembly and exploring energy metabolism innormal and disease states.
Methods
Anti-COX7RP polyclonal antibody. A polyclonal antibody against COX7RP wasraised in rabbits as described previously47. Brieﬂy, a peptide that comprised 14
amino-acid residues corresponding with the C-terminal region of human COX7RP
protein was used to immunize Japanese white rabbits. The serum was afﬁnity
puriﬁed using Afﬁgel 10 (Bio-Rad) according to the manufacturer’s instructions.
COX activity. The COX activity in cultured cells was assessed as described elsewhere48. Brieﬂy, cells permeabilized with 0.01% saponin were incubated for 15 min
in 4 nM 3,30 -diaminobenzidine tetra hydrocholoride (DAB) (Sigma), 100 mM
reduced cytochrome c (Sigma), 2 mg ml  1 of catalase (Sigma) in 0.1 M phosphatebuffer, pH 7.0. Absorbance was measured using a 450-nM ﬁlter. Muscle
homogenates were also applied to the COX activity-measuring method describedabove. For the histological study of COX activity, cross-sections (10 mm thick) offrozen muscles were stained as described above.
ATP synthesis. ATP synthesis in mitochondria was measured as described
previously49. Brieﬂy, cells were incubated in 150 mM KCl, 25 mM Tris-HCl, pH 7.4,
2 mM EDTA, 10 mM potassium phosphate, 0.1 mM MgCl2 and 0.1% bovine serum
albumin, supplemented with 50 mg ml  1 digitonin. Mitochondria were energizedusing 1 mM malate and 1 mM pyruvate as substrates in the presence of 1 mM ADP
and 0.15 mM adenylate kinase inhibitor P1,P5-di(adenosine-50 )pentaphosphate andincubated for 10 min at 37 °C. Parallel incubations were also carried out in thepresence of 2 mg ml  1 oligomycin to measure mitochondria-speciﬁc ATP synthesis.
ATP was measured by using the Enliten assay system (Promega).
Mice and MEF preparation. Cox7rpKO mice were generated by Lexicon Genetics
using random retroviral gene trapping in ES cells as previously described24.
Cox7rpKO mice were born at Mendelian ratios and were fertile. Two pairs of
Cox7rp heterozygous mice were purchased and backcrossed to the C57BL/6 inbred
strain through nine generations to generate Cox7rp-deﬁcient mice and WT
littermates used. The genotype of Cox7rpKO mice was determined by using PCR.
Three primers, named F, R and L, were designed based on the genomic sequencesurrounding the insertion site and the sequence of vector-speciﬁc oligonucleotides.
Primer sequences were as follows: Primer-F, 50 -TCTGGTTGAGGTGGCCTTTGT
GACTT-30 ; Primer-R, 50 -TTATCCAGCACCAACCATCTGCCAGA-30 ; andPrimer L, 50 -AAATGGCGTTACTTAAGCTAGCTTGC-30 . CAG-Flag-tagged
COX7RP transgene plasmid was constructed using pCXN2 vector that contains
CAG promoter, being a composite promoter consisting of a cytomegalovirus IE
enhancer, a chicken b-actin promoter, two functional loxP sites and a rabbit
b-globin polyadenylation signal50. CAG-Flag-tagged COX7RP plasmid waslinearized by restriction enzyme digestion. Transgenic mice were produced by
microinjection of the linearized plasmid into the pronuclei of fertilized eggs from
C57BL/6 mice as described previously51. Transgenic mice were identiﬁed by
PCR of genomic DNA extracted from the tail. Primers derived from the 50 - and30 -ﬂanking sequences of CAG promoter and polyA signal (50 -CCTACAGCTCCTG
GGCAACGTGCTGGTT-30 and 50 -AGAGGGAAAAAGATCTCAGTGGYAY-30 )were used to amplify a 600-bp product. Cox7rp-TgKO mice were generated byintercross mating between Cox7rpKO and COX7RP-Tg mice. MEFs were obtained
from 16-day-old embryos of WT, Cox7rpKO and COX7RP-Tg mice and used at
two to three generations. For assays of COX activity and mitochondrial ATP
synthesis, MEFs were infected with a recombinant adenovirus expressing COX7RP(Ad-COX7RP) or the ﬂuorescent protein DsRed (Ad-DsRed) at a multiplicity of
infection of 10 for 48 h. The adenoviral vectors were constructed using the
Adenovirus Expression Vector Kit (Takara, Tokyo, Japan) following the
manufacturer’s protocol. All mice used for the experiments were maintained in a
speciﬁc pathogen-free mouse facility. All studies involving animals were reviewed
and approved by the Institutional Animal Care and Use Committee of Siatama
Medical University.

Indirect calorimetry analysis in exercised and rested conditions. The
respiratory metabolic rate was measured using an individual open-circuit indirectcalorimetric system equipped with an airtight treadmill (TREADMILL FOR MICEMODEL MK-680AT/O2M, Muromachi, Japan). Before the exercise performance
test, mice were accustomed to the treadmill by subjecting them to a 3-min run at
10 m min  1 once per day for 2 days. The exercise test regimen was 15 m min  1
for the ﬁrst 10 min, followed by an incremental increase of 2 m min  1 at 10-min
intervals. Exhaustion was deﬁned as a state when the mice were unable to avoid
repetitive electrical shocks. The respiratory metabolic rate was also measured in the
non-exercise condition using an indirect calorimetric system equipped withan airtight metabolic chamber (Muromachi, Japan) for 1 h. During this time, thedata for each chamber were collected every 3 min with room air as reference tocalculate CO2 production, O2 consumption and RQ.
Oxygen consumption rate measurements. Oxygen consumption rates were
measured using a Strathkelvin 1302 oxygen electrode with an MT200 Mitocell
respiration chamber (Strathkelvin Instruments Ltd, Glasgow, UK) at 37 °C. Trypsinized cells were resuspended in KCl medium containing 80 mM KCl, 10 mM Tris-HCl(pH 7.4), 3 mM MgCl2, 1 mM EDTA and 5 mM potassium phosphate and permeabilized with digitonin (0.05 mg mg  1 protein). Substrates and inhibitors were addedin the following order and ﬁnal concentration: 10 mM pyruvate þ 2 mM malate þ 2
mM ADP as complex I substrates, 5 mM rotenone as complex I inhibitor, 10 mM
succinate as complex II substrate, 4 mM antimycin A as complex III inhibitorand 2 mM ascorbate þ 0.5 mM N, N, N0 , N0 -tetramethyl-p-phenylenediamine
dihydrochloride þ 0.5 mM carbonyl cyanide-p-triﬂuoromethoxyphenylhydrazone ascomplex IV substrates in uncoupled respiration. Rates of oxygen consumption are
expressed as nanomoles of oxygen per minute per milligrams of protein.
Oil red O staining. Interscapular BAT was ﬁxed in 4% PBS-bufferedparaformaldehyde solution and frozen in liquid nitrogen. Thirty-micrometer-thicksections were stained with oil red O and counter-stained with hematoxylin.
Thermogenic response to cold exposure. For acute cold exposure experiments,
4-month-old WT, Cox7rpKO and COX7RP-Tg littermates were transferred to 4 °C
and body temperature was measured every 60 min at the tail base using athermofocus infrared thermometer (Tecnimed, Varese, Italy) until any of themice showed hypothermia (tail base temperature r15 °C).
Blue native PAGE. Skeletal muscle or 293T cells transfected with COX7RP or
vector were homogenized with a glass-teﬂon homogenizer in buffer containing
10 mM HEPES-KOH (pH 7.4), 0.22 M mannitol, 0.07 M sucrose and 0.1 mM EDTA.
Cell extracts were centrifuged at 500g and the mitochondrial fraction was precipitatedby further centrifugation at 8,000g. Mitochondria (100 mg mitochondria protein) weresuspended in 10 ml of a buffer containing 50 mM Bis-Tris and 1 M 6-aminocaproic
acid. Digitonin (digitonin/protein ratio of 0.5–2 g g  1) was added to solubilize themitochondria. As RISP is known as a late-stage assembly subunit in mitochondrial
respiratory supercomplex25, a milder solubilization condition of 0.5 g digitonin pergram protein was employed to detect RISP. After 30-min incubation at 4 °C, thesolubilized proteins were obtained as supernatant fraction by centrifugation at
22,000g. The solubilized proteins were supplemented with 1 ml of sample buffer (5%Coomassie brilliant blue G-250 in 0.5 M 6-aminocaproic acid). A stacking gel (4%)
and separating gels with a stepwise 8, 9, 10 and 11% were cast and electrophoresedaccording to the method of Schägger and von Jagow52. The second-dimension SDS–
PAGE and immunoblotting were performed according to standard protocols, and theblot was probed with anti-NDUFA9 (Invitrogen) and anti-UQCRC2 antibodies(Abcam), together with anti-COX7RP anti-COX4 antibodies (Cell Signaling) or with
anti-RISP antibody (Abcam). Experiments were carried out four times andrepresentative results are shown in ﬁgures. full-length images of immunoblots areshown in Supplementary Fig. S8.
Quantitative real-time polymerase chain reaction. Total RNA was extracted
from the BAT of 4-month-old WT and Cox7rpKO male mice using the ISOGEN
reagent (Nippon Gene, Tokyo, Japan). Quantitative real-time RT–PCR (qPCR)
analysis was performed as described previously53. The sequences of the PCRprimers used were as follows: common primers for human and mouse Cox7rp,
50 -GTTCCACAGAAGCACCACCTATC-30 and 50 -GGTCCGGTAAAGCATTTGGTC-30 ; mouse speciﬁc Cox7rp, 50 -GCAGAAGTTGGCTGGAGCTT-30 and
50 -TATGCTGTCACACTGGAGGTCAG-30 ; Ucp1, 50 -CGTACCAAGCTGTGC
GATGT-30 and 50 -ACCCGAGTCGCAGAAAAGAA-30 ; Ucp2, 50 -AACTCTGCC
TTGGGCCAGTAC-30 and 50 -CGGAGAAAGGAAGGCATGAA-30 ; Ucp3,
50 -TTGGCCTCTACGACTCTGTCAA-30 and 50 -ATGGCTTGAAATCGGAC
CTTC-30 ; Fasn, 50 -GCTGTCCCCTGATGCCATT-30 and 50 -ACCTATCCCGGC
CTGAGAAC-30 ; Ppara, 50 -GATCGGCCTGGCCTTCTAA-30 and 50 -GGTGT
CATCTGGATGGTTGCT-30 ; Pparg, 50 -TTCCGAAGAACCATCCGATT-30 and
50 -TTTGTGGATCCGGCAGTTAAG-30 ; Ppard, 50 -ACACCATTCTGCGGGCTCTA-30 and 50 -GGGAAGAGGTACTGGCTGTCAG-30 ; Pgc-1a, 50 -GGCACGCA
GCCCTATTCAT-30 and 50 -CACGGAGAGTTAAAGGAAGAGCAA-30 ; Pgc-1b,
50 -CATCTGGGAAAAGCAAGTACGA-30 and 50 -CCTCGAAGGTTAAGGCT

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms3147

GATATCA-30 ; Erra, 50 -GGAGTACGTCCTGCTGAAAGCT-30 and 50 -CACAGCC
TCAGCATCTTCAATG-3’; Errb, 50 -CGATATCCCCGAGGGAGATATC-30 and
50 -CCAGTTGATGAGGAACACAAGCT-30 ; Errg, 50 -GACCCTACTGTCCCCG
ACAGT-30 and 50 -AACTCTCGGTCAGCCAAGTCA-30 ; Nrf1, 50 -CTTACAAGGTGGGGGACAGA-30 and 50 -CCGATATCCTGGTGGTCACT-30 ; Tfam, 50 -TGGA
AAGCATACAAAGAAGCTG-30 and 50 -GACAGATTTTTCCAAGCCTCA-30 ;and Gapdh, 50 -GCATGGCCTTCCGTGTTC-30 and 50 -TGTCATCATACTTGG
CAGGTTTCT-30 . Comparison of the amounts of PCR product was carried out by
employing the comparative cycle threshold (Ct) method, using Gapdh as control.
The experiments were independently performed in triplicate. Results were shownas means±s.d.
Microarray analysis. Gene expression in the muscle and BAT of 5-month-old WT
and Cox7rpKO male mice was measured with Affymetrix GeneChip (Mouse Gene
1.0 ST Array) according to the manufacturer’s protocol. The DAVID Functional
Annotation Clustering Tool (http://david.abcc.ncifcrf.gov/summary.jsp) was used
to perform a global analysis of gene expression and to sort differentially expressed
genes into pathways and clusters of functionally related genes.
Statistics. We determined the signiﬁcance of differences between the two groups
by using the unpaired Student’s t-test. Comparisons among several groups werecarried out with one-way analysis of variance followed by Dunnett’s post hoc test.
Survival analyses were carried out using Kaplan–Meier method. We considered avalue of Po0.05 to be statistically signiﬁcant. Bonferroni correction was used toadjust for multiple comparisons.

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Competing ﬁnancial interests: The authors declare no competing ﬁnancial interests.

Acknowledgements

Reprints and permission information is available online at http://npg.nature.com/
reprintsandpermissions/

We thank W. Sato, D. Tanaka, M. Fujitani and T. Hishinuma for their technicalassistance and Dr B. Blumberg for critical reading of the manuscript. This study was
supported by Cell Innovation Program and Support Project of Strategic Research Centerin Private Universities from the MEXT, grants from the Japan Society for the Promotion

Author contributions
K.I. designed and performed experiments, analysed and interpreted data, and wrote
manuscript. K.S. and S.S. performed experiments. K.H.-I. and S.I. designed andsupervised experiments, interpreted data and wrote manuscript.

Additional information
Supplementary Information accompanies this paper on http://www.nature.com/
naturecommunications

How to cite this article: Ikeda, K. et al. A stabilizing factor for mitochondrial respiratory
supercomplex assembly regulates energy metabolism in muscle. Nat. Commun. 4:2147
doi: 10.1038/ncomms3147 (2013).

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