Báo cáo khoa học: Frataxin deficiency causes upregulation of mitochondrial Lon and ClpP proteases and severe loss of mitochondrial Fe–S proteins pot

Frataxin deficiency causes upregulation of mitochondrial
Lon and ClpP proteases and severe loss of mitochondrial
Fe–S proteins
Blanche Guillon
1
, Anne-Laure Bulteau
2
, Marie Wattenhofer-Donze
´
3,4
, Ste
´
phane Schmucker
3,5
,
Bertrand Friguet
2
,He
´
le
`
ne Puccio
3,4,5,6,7
, Jean-Claude Drapier
1
and Ce
´
cile Bouton
1
1 Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France
2 Laboratoire de Biologie et Biochimie Cellulaire du Vieillissement, Universite

´
Paris 7, France
3 IGBMC (Institut de Ge
´
ne
´
tique et de Biologie Mole
´
culaire et Cellulaire), Illkirch, France
4 Colle
`
ge de France, Chaire de ge
´
ne
´
tique humaine, Illkirch, France
5 Universite
´
Louis Pasteur, Strasbourg, France
6 Inserm, U596, Illkirch, France
7 CNRS, UMR7104, Illkirch, France
Friedreich ataxia (FRDA) is an autosomal recessive
neurodegenerative and cardiodegenerative disease char-
acterized by progressive ataxia and cardiomyopathy
that is associated with deficit in Fe–S enzyme activities
and abnormal cellular iron metabolism [1]. FRDA
results from a greatly reduced level of the mitochon-
drial protein frataxin, due to a large GAA repeat
expansion in the gene, which inhibits the transcription
of the frataxin gene through heterochromatin silencing

of the locus [2]. Although the exact role of frataxin is
still controversial, pathophysiological studies from
patient autopsies demonstrated a specific loss of Fe–S
protein activities, with accumulation of iron being
thought to contribute to an increased level of oxidative
stress. FRDA mouse models with a tissue-targeted
frataxin deficiency have been developed to study the
pathophysiology of the disease and the function of
frataxin, and to test potential therapeutic agents [3].
Keywords
ClpP; frataxin; Friedreich ataxia; iron-sulfur
cluster; Lon protease
Correspondence
C. Bouton, ICSN-CNRS, Avenue de la
Terrasse, 91190 Gif-sur-Yvette, France
Fax: +33 1 69 07 72 47
Tel: +33 1 69 82 30 10
E-mail:
(Received 21 October 2008, revised 4
December 2008, accepted 9 December
2008)
doi:10.1111/j.1742-4658.2008.06847.x
Friedreich ataxia (FRDA) is a rare hereditary neurodegenerative disease
characterized by progressive ataxia and cardiomyopathy. The cause of the
disease is a defect in mitochondrial frataxin, an iron chaperone involved in
the maturation of Fe–S cluster proteins. Several human diseases, including
cardiomyopathies, have been found to result from deficiencies in the activ-
ity of specific proteases, which have important roles in protein turnover
and in the removal of damaged or unneeded protein. In this study, using
the muscle creatine kinase mouse heart model for FRDA, we show a clear

progressive increase in protein levels of two important mitochondrial ATP-
dependent proteases, Lon and ClpP, in the hearts of muscle creatine kinase
mutants. These proteases have been shown to degrade unfolded and dam-
aged proteins in the matrix of mitochondria. Their upregulation, which
was triggered at a mid-stage of the disease through separate pathways, was
accompanied by an increase in proteolytic activity. We also demonstrate a
simultaneous and significant progressive loss of mitochondrial Fe–S pro-
teins with no substantial change in their mRNA level. The correlative effect
of Lon and ClpP upregulation on loss of mitochondrial Fe–S proteins dur-
ing the progression of the disease may suggest that Fe–S proteins are
potential targets of Lon and ClpP proteases in FRDA.
Abbreviations
DNP, 2,4-dinitrophenylhydrazone; ER, endoplasmic reticulum; FRDA, Friedreich ataxia; MCK, muscle creatine kinase; SDHA, succinate
dehydrogenase complex subunit A; Yfh1p, yeast frataxin homolog.
1036 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS
The cardiac conditional model, in which frataxin has
been specifically deleted in striated muscles using a
recombinase expressed under control of the muscle
creatine kinase (MCK) promoter, and the animals
in which are hereafter referred to as MCK mutants,
reproduces important pathophysiological features and
biochemical aspects of the human disease [4,5]. These
animals show cardiodegeneration, deficiency of respira-
tory chain complexes I–III and aconitases, and mito-
chondrial iron accumulation, without the presence of
major oxidative stress. This model was used to demon-
strate that the deficit in mitochondrial Fe–S cluster
enzyme activities is an early event in FRDA disease,
followed by rapid cardiac dysfunction, whereas abnor-
mal iron accumulation within the mitochondria occurs

at a late stage, pointing to a role of frataxin in Fe–S
cluster biogenesis [5]. Since then, many studies have
reported the involvement of frataxin in the maturation
of the Fe–S cluster proteins in yeast [6–10], mammals
[11–14], Drosophila [15] and, recently, bacteria [16] and
plants [17]. Frataxin, which has been the focus of
extensive research in the yeast system, specifically
interacts with Fe–S scaffold Isu1 ⁄ 2 proteins [10,18–20],
and is thought to provide iron for the formation of the
cluster.
Evidence is accumulating that proteasome dysfunc-
tion might be associated with cardiomyopathies in
which accumulation of abnormal misfolded proteins
may lead to the formation of potentially toxic aggre-
gates [21]. In the mitochondrion, the main organelle
affected in FRDA, various proteases also have
important functions in protein quality control [22].
Among these, the ATP-stimulated Lon protease,
which forms homo-oligomeric complexes, degrades
misfolded and damaged proteins in the matrix space,
similarly to the proteasome function in the cytoplasm
[23]. By mediating complete proteolysis, Lon thereby
prevents aggregation and deleterious effects on mito-
chondrial functions. A second ATP-stimulated prote-
ase, named ClpP, has also been identified in the
matrix of mammalian cells [24] and associates with
the ATPase ClpX subunits in vitro to effect its ATP-
dependent proteolytic activity [25,26]. Although sub-
strate specificity has not been defined yet, several
studies have demonstrated that Fe–S cluster proteins

can be preferential substrates for Lon and ⁄ or ClpXP
proteases in different systems [27–30]. Indeed, it has
recently been demonstrated in yeast that Fe–S cluster
integrity in proteins is one of the major determinants
of susceptibility to degradation by Pim1, the yeast
homolog of human Lon protease [28]. By means of a
proteomic approach using wild-type and Pim-1
mutant strains, the authors identified five Pim-1
substrate proteins, including two Fe–S proteins (the
homoaconitase Lys4 and Yjl200c, a putative aconi-
tase isozyme). Using an in organello degradation
assay, they also demonstrated that improper assembly
of Fe–S clusters on Yjl200c and aconitase (aco1) led
to their increased susceptibility to degradation. Inter-
estingly, in mammals, mitochondrial aconitase has
been identified as a good proteolytic substrate for
Lon under mild oxidative conditions [29]. Finally,
one mutational study of ClpP performed in plants
showed that the Rieske Fe–S protein can be a sub-
strate for this protease [30].
In this study, we investigated whether mitochondrial
Lon and ClpP proteases are regulated in the heart of
conditional MCK mice during the progression of the
FRDA cardiac disease. We found a progressive
increase in mitochondrial Lon and ClpP protease
expression and activity in cardiac tissues of the MCK
mutant over the course of the disease. Moreover, the
proteases are upregulated through two distinct mecha-
nisms, as Lon upregulation is transcriptional, whereas
that of ClpP is post-transcriptional, acting either by

increasing its protein translation or by decreasing its
rate of turnover. We also addressed the fate of several
mitochondrial Fe–S proteins in cardiac tissue of
MCK mutants, and showed an overall clearance in
protein levels of key mitochondrial Fe–S cluster
enzymes that followed the elevated mitochondrial
ATP-stimulated proteolytic activity in this FRDA
model.
Results
Modulation of Lon and ClpP expression in cardiac
muscles of MCK mutants
Using the MCK mutants, we investigated the effect of
frataxin deficiency on the possible regulation of the
two mitochondrial matrix ATP-dependent proteases.
We first evaluated Lon and ClpP mRNA levels by
quantitative real-time PCR from total RNA heart
extracts of different age groups of control and MCK
mutants. A significant and progressive increase in Lon
mRNA levels was observed in mutant mice between 5
and 10 weeks of age, whereas ClpP mRNA levels were
not affected (Fig. 1A). Indeed, the Lon protease
mRNA level increased  2.5-fold at 5 weeks of age in
the hearts of MCK mutants as compared with control
mice, and continued to rise, increasing  4-fold at
10 weeks of age. We next investigated the possible
regulation of both proteases in MCK mutants at the
protein level. Immunoblot analysis using specific anti-
bodies against peptides clearly showed a progressive
B. Guillon et al. Upregulation of mitochondrial proteases in FRDA
FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1037

increase in Lon protein content, starting at the interme-
diate stage (5 weeks) of cardiomyopathy progression
[5] in the MCK mutants (Fig. 1B). Quantitative immu-
noblot analysis revealed  2.6-fold, 5.0-fold and 6.7-
fold increases in Lon protein expression at 5, 7 and
10 weeks of age, respectively, in MCK mutants as
compared with control mice. Interestingly, the ClpP
protein level was also progressively enhanced, with
 3-fold, 3.5-fold and 4.5-fold increases at 5, 7 and
10 weeks of age in MCK mutants, respectively, despite
no change in mRNA levels (Fig. 1A,B).
ATP-stimulated proteolytic activity of ClpP
and Lon proteases in heart mitochondria of
MCK mutants
To investigate whether the increase in Lon and ClpP
protein levels is accompanied by increased protein
functionality, ATP-stimulated proteolytic activity,
reflecting both ClpP and Lon activities [31], was
measured in heart mitochondria from frataxin-
deficient mice. The cytosolic contamination of mito-
chondrial fractions was checked by performing
A
B
Fig. 1. Regulation of mitochondrial Lon and ClpP protease expression in the hearts of control and MCK mutant mice. (A) Total RNA was
isolated from the hearts of control and MCK mutant mice at 3, 5 and 10 weeks of age and used to measure Lon protease and ClpP mRNA
levels by quantitative real-time PCR. The mRNA expression levels were expressed as fold change between MCK and control samples
(value 1) and normalized to 18S ribosomal RNA. The experiment was repeated at least three times with independent RNA samples, and the
average ± standard deviation of the three replicates is depicted in the bar graphs. Statistical analysis was performed using Student’s t-test:
***P < 0.0001. (B) Mitochondrial extracts from the hearts of control (C) and MCK (M) mice at 3, 5, 7 and 10 weeks of age were analyzed
by immunoblotting with antibodies against Lon and ClpP. Immunolabeled protein bands of interest were then quantified using a ChemiDoc

imaging system and
QUANTITY ONE software (BioRad, Marne-La-Coquette, France), and were normalized using antibodies against prohibitin
and ATP2 as mitochondrial loading controls. *P < 0.01.
Upregulation of mitochondrial proteases in FRDA B. Guillon et al.
1038 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS
immunoblotting using prohibitin as mitochondrial
marker and vinculin and proteasome 20S subunit
as cytosolic markers (Fig. 2A). As shown in Fig. 2A,
very little cytosolic contamination was observed in
the mitochondrial fraction. Values corresponding to
ATP-dependent proteolytic activities in control and
mutant mice (Table S1) were used to calculate
the fold changes in ClpP and Lon protease activities
found in MCK mutant versus control mice at 5, 7
and 8 weeks of age. We showed that ClpP ⁄ Lon
protease activity, which was low in the heart
mitochondria of 5-week-old MCK mutants, increased
 2-fold to  2.5-fold between 7 and 8 weeks of age
(Fig. 2B).
Level of carbonylated proteins in heart
mitochondria of MCK mutants at different ages
Stimulation of Lon proteolytic activity that may
depend on increases in carbonylated proteins has been
reported in an in vivo cardiac ischemia–reperfusion
model [32] and in yeast frataxin homolog (Yfh1p)-defi-
cient yeast cells [33]. In MCK mutants, Seznec et al.
[34] did not find any evidence of increased cellular oxi-
dative stress. Rather, a reduction in oxidized proteins
in the hearts of MCK mutants was detected from 7
to 10 weeks. In this previous report, carbonylated

proteins were measured in total extracts of frataxin-
deficient mice, leading to an underestimation of pos-
sible oxidative stress in mitochondria. We therefore
performed subcellular fractionation from cardiac tissue
of wild-type and MCK mutant mice in order to detect
carbonylated proteins in the mitochondrial protein
fractions. In mitochondria of control mice, appreciable
amounts of oxidized proteins were detected, probably
due to the oxidative metabolism of mitochondria
under normal conditions [35]. The total amount of oxi-
dized proteins did not increase in frataxin-deficient
mice as compared with control mice at any age tested
(Fig. 2C). Carbonylated proteins of mitochondrial frac-
tions were also quantified by ELISA using carbonylated
standards, and the results indicated that their levels were
similar in both control and MCK mutant mice at any
age and represented < 0.1 nmolÆmg
)1
of total proteins.
Therefore, in contrast to the Yfh1p-deficient yeast
model of FRDA, increase in Lon proteolytic activity in
Fig. 2. Lon ⁄ ClpP protease activity and level of oxidized proteins in
the heart mitochondria of MCK mutant mice. (A) Cytosolic contami-
nation of the mitochondrial fraction was checked by immunoblot-
ting with protein extracts (40 lg) from the first (500 g) and second
(10 000 g) pellets using antibodies against vinculin, proteasome
20S and prohibitin. A representative result of cell fractionation is
shown. C, control; M, MCK. (B) Mitochondrial fractions were pre-
pared from the hearts of control and MCK mutant mice at 5, 7 and
8 weeks of age and assayed for ATP-dependent proteolytic activity.

Two hearts of MCK mutant or control mice were pooled to mea-
sure proteolytic activity per dot. Each triangle, diamond or square
corresponds to the fold change in mitochondrial ATP-dependent
proteolytic activity of MCK versus control mice at the different
stages indicated. Detailed measures can be found in Table S1. (C)
Carbonylated proteins of mitochondrial fractions (10 lg) from
control (C) and MCK mutant (M) mice were detected after derivati-
zation of their carbonyl groups using a solution of dinitrophenylhydr-
azine, SDS ⁄ PAGE and immunoblotting using a primary antibody
against DNP as described in Experimental procedures. A digitized
image of Ponceau staining was used to check equal loading of each
lane (not shown). Experiments were performed at least three
times, and a representative result is shown.
A
B
C
B. Guillon et al. Upregulation of mitochondrial proteases in FRDA
FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1039
the MCK ⁄ FRDA mouse model is not linked to accumu-
lation of oxidized proteins.
Regulation of mitochondria-encoded genes in
MCK mutants at different ages
Beside its protease function, it has been reported that,
in vitro, Lon binds to specific regions of the light-
strand and heavy-strand promoters of mitochondrial
DNA [36,37], and this was recently confirmed in living
cells, pointing to another function of this protease in
the regulation of mitochondrial DNA replication and
gene expression [38]. We therefore examined whether
mitochondria-encoded gene expression was affected in

MCK mutant hearts. The mRNA expression profile of
12 mitochondria-encoded genes involved in com-
plexes I, III, IV and V of oxidative phosphorylation
was determined in the hearts of wild-type and MCK
mutant mice at different stages of the disease. Apart
from ND3 at 5 weeks, there was no significant decrease
or increase in gene expression of any of the mitochon-
dria-encoded genes tested at 3 and 5 weeks of age in
the hearts of MCK mutants as compared with control
littermate mice (Table 1). In contrast, at a late stage,
five genes of complex I, cytochrome b of complex III
and two genes of complex IV were slightly but signi-
ficantly downregulated in MCK mutants versus
controls.
Decrease in mitochondrial Fe–S protein levels in
MCK mutants
The effect of frataxin deficiency on the abundance of
mitochondrial Fe–S cluster-containing proteins was
also examined in the MCK mutants. Three Fe–S sub-
units of complex I (NDUFS3), complex II (SDHB)
and complex III (Rieske) of the respiratory chain were
selected for immunoblot analysis, as well as ferrochela-
tase, a [2Fe–2S] enzyme required for the last step in
heme biosynthesis [39], and the [4Fe–4S] aconitase of
the tricarboxylic acid cycle. A significant decrease in
the protein level of every mitochondrial Fe–S protein
tested was observed at 5 weeks in the heart of MCK
mutants, where frataxin is completely deleted, as com-
pared with control samples (Fig. 3A). Quantitative
immunoblot analysis revealed a similar pattern in the

time course of mitochondrial Fe–S cluster protein loss,
whereas expression of the mitochondrial Atp2 b-sub-
unit of the ATP synthase complex, which does not
contain an Fe–S cluster, was not significantly affected
in frataxin-deficient mice (Fig. 3B). At 3 weeks, the ini-
tial stage of the disease, in which the only phenotype
observed is the specific deficit in Fe–S enzyme activity,
very little change in Fe–S protein levels was observed,
suggesting a defect in Fe–S cluster assembly. The
decrease in mitochondrial Fe–S cluster proteins was
clearly apparent at 5 weeks ( 40–70% decrease) in
the hearts of MCK mutants, a stage corresponding to
the beginning of the cardiac dysfunction. Downregula-
tion further decreased at 7 weeks, residual Fe–S
protein levels reaching  30–20% in MCK mutants as
compared with controls, and stabilizing at a plateau of
 20% at 10 weeks. These results are in agreement
with the strong enzymatic deficiency of aconitase and
the respiratory chain previously observed. Protein
expression levels of other subunits of the respiratory
chain were also investigated (Fig. 4). The level of the
hydrophilic succinate dehydrogenase complex sub-
unit A (SDHA), the flavoprotein subunit of com-
plex II, which is not an Fe–S cluster protein but which
requires the Fe–S proteins to be properly folded into
the complex [40], was also visibly reduced at 5 weeks
by  40% as compared with control mice. The expres-
sion of the ND6, which is a partner subunit of Fe–S
complex I, was also significantly affected in frataxin-
deficient mice (Fig. 4A,B).

To determine whether the decrease in mitochondrial
Fe–S proteins and partners was due to a transcrip-
tional regulation, reverse transcription followed by
real-time qPCR was performed in control and MCK
mutants at 3, 5 and 10 weeks of age. As shown in
Fig. 5, no significant change in the mRNA expression
of mitochondrial aconitase and several subunits of the
respiratory chain (Rieske, ND6 and SDHA) was
observed in the hearts of either control or MCK
mutant mice, despite a marked reduction in their pro-
tein levels in the mutants. The ferrochelatase, Ndufs3
Table 1. mRNA expression of mitochondria-encoded genes in the
heart of control versus MCK mutant mice at 3, 5 and 10 weeks of
age. Cytb, cytochrome b; COX, cyclo-oxygenase.
Mitochondrial
subunits
mRNA fold change difference (WT ⁄ MCK)
3 weeks 5 weeks 10 weeks
ND1 0.83 ± 0.43 1.14 ± 0.09 1.22 ± 0.05
ND2 0.91 ± 0.47 1.08 ± 0.12 0.81 ± 0.03*
ND3 0.77 ± 0.41 1.23 ± 0.12* 0.78 ± 0.09*
ND4 0.79 ± 0.40 0.83 ± 0.06 0.74 ± 0.04*
ND4L 0.94 ± 0.16 0.89 ± 0.07 0.63 ± 0.07*
ND5 0.86 ± 0.27 0.81 ± 0.12 0.61 ± 0.06**
ND6 0.84 ± 0.28 0.83 ± 0.09 0.87 ± 0.09
COX1 1.24 ± 0.19 0.88 ± 0.05 0.81 ± 0.07*
COX2 0.93 ± 0.32 1.14 ± 0.22 1.08 ± 0.26
COX3 0.90 ± 0.31 0.99 ± 0.08 0.87 ± 0.08*
ATPase 6 0.80 ± 0.43 1.10 ± 0.09 1.05 ± 0.16
Cytb 0.91 ± 0.31 0.87 ± 0.11 0.59 ± 0.04*

*P < 0.05; **P < 0.001.
Upregulation of mitochondrial proteases in FRDA B. Guillon et al.
1040 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS
and Sdhb transcripts were slightly but significantly
decreased at a late stage in frataxin-deficient mice
(Fig. 5) [41], but this cannot explain the drastic change
seen earlier at the protein level in the hearts of MCK
mutants (Fig. 3).
Discussion
Disturbances of proteolytic systems have been associ-
ated with various human diseases [42]. A defect in these
enzymatic systems usually causes protein aggregation
and subsequent cellular damage [43]. In the matrix of
mitochondria, two ATP-stimulated serine proteases
have been identified, namely ClpP and Lon proteases,
which participate in the degradation of improperly
folded and damaged proteins [23,44]. Whereas Lon is a
homo-oligomeric complex, human ClpP forms a het-
erocomplex in vitro with ClpX, an ATP-dependent
AAA+ chaperone [45]. High expression levels of Lon
and ClpP have been reported in energy-hungry tissues
such as skeletal muscle and heart, suggesting an impor-
tant mitochondrial function of these proteases in these
tissues [46,47]. Little is known about Lon and ClpP in
mammals, and to date, regulation of these proteases
has never been studied in mitochondrial diseases. In the
present article, we report the first demonstration that
frataxin deficiency causes significant upregulation of
both mitochondrial Lon and ClpP proteases in the car-
diac mouse model for Friedreich ataxia. The increase in

protease levels started at the mid-stage of the disease,
and was rapidly followed by a boost of their proteolytic
activity. We also show that Lon upregulation and ClpP
upregulation in the hearts of MCK mutants operate
A
B
Fig. 3. Levels of mitochondrial Fe–S cluster-containing proteins in
cardiac muscle of control and MCK mutant mice. (A) Total protein
extracts (20 lg) from control (C) and MCK mutant (M) mice at 3, 5,
7 and 10 weeks of age were analyzed by SDS ⁄ PAGE and immuno-
blot with specific primary antibodies against frataxin, mitochondrial
aconitase, ferrochelatase, three Fe–S subunits of complex I
(NDUFS3), complex II (SDHB) and complex III (Rieske) and the
ATP2 b-subunit of mitochondrial ATP synthase. (B) Immunolabeled
protein bands of interest were quantified using a ChemiDoc imag-
ing system and
QUANTITY ONE software (BioRad), and were normal-
ized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
vinculin or b-tubulin as loading control. These experiments were
performed at least three times independently, and representative
data are shown. Statistical analysis was performed using Student’s
t-test: **P < 0.001; ***P < 0.0001.
A
B
Fig. 4. Levels of SDHA and ND6 respiratory chain subunits in the
hearts of control and MCK mutant mice. (A) Total protein extracts
(20 lg) from control (C) and MCK mutant (M) mice at 3, 5, 7 and
10 weeks of age were analyzed by immunoblot using specific pri-
mary antibodies against the ND6 subunit of complex I and the
flavoprotein (SDHA) of complex II. A representative result of three

independent experiments is shown. (B) Immunolabeled protein
bands of interest were quantified and normalized using vinculin as
loading control. Statistical analysis was performed using Student’s
t-test: **P < 0.001; ***P < 0.0001.
B. Guillon et al. Upregulation of mitochondrial proteases in FRDA
FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1041
through two distinct mechanisms. The increase in Lon
protein level was due to transcriptional regulation, as
the protein increase was mirrored at the mRNA level.
The same does not hold true for ClpP, which did not
exhibit a change in transcript level, suggesting transla-
tional or post-translational regulation. Regarding Lon,
although signals that trigger its upregulation in FRDA
are unknown, some proposals can be put forward. The
MCK mutants present signs of endoplasmic reticulum
(ER) stress (H. Puccio, unpublished data) simulta-
neously with the increase in Lon expression. As it has
been reported that cells subjected to hypoxia or ER
stress exhibit higher Lon mRNA levels [48], it is tempt-
ing to suggest that ER stress leads to Lon protease
upregulation in MCK mutants. Besides, depletion of
ATP, which has been shown in tissues of FRDA
patients [49], can lead to regulation of gene expression
in some stressful situations [50,51]. As Lon proteolytic
activity is stimulated up to nine-fold by ATP [44], it
can be hypothesized that lack of ATP may compensate
for low Lon activity by initiating upregulation of the
Lon gene and protein expression. Regarding the ClpP
protease, very little is known about its physiological
function and regulation in mammals. One study

reported ClpP gene upregulation after accumulation of
unfolded proteins within the mitochondrial matrix,
which appears to depend on CHOP and C ⁄ EBPb ele-
ments identified in its promoter [52]. However, as the
change in ClpP expression in MCK mutants occurred
at a post-transcriptional level, protein accumulation in
mitochondria, described by Zhao et al., is not the sig-
nal that triggers ClpP protein upregulation in the
MCK mutants.
Lon protease has been described as a multifunctional
enzyme, which behaves like an ATP-stimulated prote-
ase, a chaperone or a regulator of mitochondrial DNA
replication and gene expression [38,47,53]. We have
shown that the major activity displayed by Lon in
the heart of frataxin-deficient mice was its proteolytic
activity, which contrasts with the small change in mito-
chondria-encoded gene expression. In addition, the
prominent accumulation of mitochondria observed in
MCK mutants at 6–7 weeks, which becomes excessive
at the final stage of the disease [34], may be related to
high Lon expression. Indeed, two reports showed that
the expression and activity of Lon were increased in cells
with enhanced mitochondrial biogenesis [54] and that a
population of Lon-deficient cells exhibited fewer mito-
chondria [55]. Therefore, Lon could, at least in part, be
responsible for the prominent accumulation of mito-
chondria observed in the hearts of MCK mutants.
In the bacterial and yeast systems, it has recently
been shown that integrity of Fe–S clusters is a main
determinant of susceptibility to Lon and ⁄ or ClpP deg-

radation [27,28]. Interestingly, an important biochemi-
cal feature associated with frataxin deficiency in
Friedreich ataxia is the specific defect in Fe–S cluster
enzyme activities, a very early step in the disease pro-
cess [5,14,34]. This phenomenon was in part attributed
to imperfect Fe–S protein maturation, as frataxin has
been identified as an important component of the
Fe–S cluster assembly machinery in mammals and
other organisms [6,7,12,15–17]. In this study, we have
shown that frataxin deficiency causes a severe protein
loss for several mitochondrial Fe–S enzymes, contrib-
uting to the overall Fe–S deficit in FRDA. The
decrease in protein level at 5, 7 and 10 weeks was not
due to transcriptional regulation, as mRNA levels
showed no substantial difference between controls and
mutants. According to structural studies, Fe–S sub-
units and other proteins of complexes I and II actually
fit into each other [40,56]. The complex I hydrophobic
A
B
Fig. 5. Gene expression profile of mitochondrial Fe–S proteins and
SDHA and ND6 subunits of the respiratory chain in cardiac muscle
of wild-type and MCK mutant mice. Total RNA, which was
extracted from the hearts of control and MCK mutant mice at 3, 5
and 10 weeks of age, was used to assess mRNA expression of
genes encoding mitochondrial aconitase, ferrochelatase, NDUFS3,
SDHB and Rieske proteins (A), and ND6 and SDHA proteins (B) by
quantitative real-time PCR. The mRNA expression levels in MCK
samples were expressed as fold change over controls (assigned
the value of 1) and were normalized to 18S ribosomal RNA. Experi-

ments were performed at least three times, and data are presented
as mean ± standard deviation for three separate experiments. Sta-
tistical analysis was performed using Student’s t-test. *P < 0.05;
**P < 0.001.
Upregulation of mitochondrial proteases in FRDA B. Guillon et al.
1042 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS
subunits ND1–ND6 form a shell around the Fe–S
protein fragments [57], and SDHA flavoprotein of
complex II dimerizes with the Fe–S protein domain
(Ip), forming a soluble catalytic heterodimer [40].
Interestingly, protein levels of ND6 and SDHA were
also reduced. In contrast, protein expression of both
ATP2, the b-subunit of mitochondrial ATP synthase,
and of mitochondrial prohibitin, neither of which con-
tains Fe–S clusters or is related to Fe–S proteins, was
unaffected in mutant mice, indicating that frataxin
deficiency specifically affects Fe–S proteins and their
protein partners. These results are reminiscent of studies
showing that Yfh1p-deficient yeast cells undergo degra-
dation of mitochondrial aconitase [6] and that, in plants,
depletion of chloroplastic NifS, another important com-
ponent for Fe–S cluster biogenesis, decreases the abun-
dance of several Fe–S proteins and partners [58]. By
statistical analysis, we found that the time course of
mitochondrial Fe–S protein loss in MCK mutants sig-
nificantly correlates with the progressive increase in the
levels of both mitochondrial Lon and ClpP proteases
(Fig. S1). Taking all these data together, it is tempting
to speculate that the frataxin-dependent defect in Fe–S
cluster biogenesis leads to the formation of mitochon-

drial apoenzymes, which are recognized as misfolded or
low-stability proteins, and degraded by Lon and ⁄ or
ClpP proteases. Although it is unlikely, we cannot
exclude the possibility that aggregation of unfolded
Fe–S protein also participates in the decrease of Fe–S
protein assessed by immunoblot. Research on the devel-
opment of specific Lon inhibitors has started very
recently [59] and, when they are available, they will be
very useful in discovering whether mitochondrial prote-
ase activation is a deleterious or protective process in
FRDA. To date, reports concerning the understanding
of Lon cellular functions suggest a protective role of
Lon against aggregation and intracellular accumulation
of oxidized proteins in mitochondria [29,32]. Therefore,
increased Lon and ClpP activity in MCK mutants, by
preventing the accumulation of carbonylated proteins,
may hide increased oxidative damage in MCK mutants.
Specific inhibitors of Lon proteases will help to further
characterize the Lon and ClpP cellular functions and
identify whether Fe–S proteins are specific substrates of
Lon protease in FRDA.
Experimental procedures
Animals
MCK mutants were generated by crossing mice homozy-
gous for a conditional allele of Frda (Frda
L3 ⁄ L3
) with mice
heterozygous for the deletion of Frda exon 4 (Frda
D ⁄ +
),

which carries a tissue-specific Cre transgene under the con-
trol of the MCK promoter [5]. In this study, mice carrying
the transgene and conditional allele (L3 ⁄ L; MCK+) are
called MCK mutants. Control mice were littermates having
at least one normal frataxin-expressing allele. All methods
employed in this work are in accordance with the Guide
for the Care and Use of Laboratory Animals published by
the US National Institutes of Health (NIH Publication
No. 85-23, revised 1996).
Preparation of total protein extracts
Using a glass homogenizer (Duall 21), a tissue homogenate
was prepared from the heart of wild-type and MCK mutant
mice in 100 mm Tris (pH 7.4) and 150 mm NaCl in the pres-
ence of protease inhibitors (protease inhibitor cocktail
set III; Calbiochem, Darmstadt, Germany). After centrifu-
gation at 400 g for 10 min at 4 °C, red blood cells were lysed
by resuspending the cell pellet in 800 lL of hypotonic solu-
tion (Sigma, Saint-Quentin Fallavier, France). The resulting
white pellet was then lysed in 100 mm Tris (pH 7.4) contain-
ing 0.5% Triton X-100, with the protease inhibitor cocktail.
After centrifugation at 10 000 g for 10 min at 4 °C, the
supernatant was collected and protein content was deter-
mined before storage at )80 °C for further measurements.
Isolation of cardiac mitochondria
Hearts of wild-type and MCK mutant mice were homo-
genized in 210 mm mannitol, 5.0 mm Mops, 70 mm sucrose,
and 1.0 mm EDTA (pH 7.4). The homogenate was centri-
fuged at 500 g for 10 min at 4 °C to remove tissue frag-
ments and cell nuclei, and the supernatant was
recentrifuged at 10 000 g for 10 min at 4 °C to bring down

the mitochondrial pellet. After two washings in homoge-
nized buffer, the mitochondrial pellets were either stored at
)80 °C for subsequent immunoblot analysis or immediately
used for the measurement of ATP-dependent proteolytic
activity as well as detection of carbonylated proteins.
Immunoblot analysis
Total protein extract (20–40 lg) or mitochondrial fraction
(60 lg) was loaded on 10% or 12% SDS ⁄ PAGE gel
(depending on the expected protein size), and proteins were
transferred onto a Hybond nitrocellulose membrane. Spe-
cific proteins were detected by immunoblotting with the
indicated primary antibodies and peroxidase-conjugated
secondary antibody (DAKO Cytomation, Glostrup,
Denmark). Blots were developed with an enhanced chemilu-
minescence detection system [Super Signal Pierce (Perbio
Science, Berbie
`
res, France) or Immobilon Western (Milli-
pore, Saint-Quentin en Yvelines, France)].
B. Guillon et al. Upregulation of mitochondrial proteases in FRDA
FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1043
Immunochemical reagents
Rabbit polyclonal anti-peptide Lon protease serum was
kindly provided by L. Sweda (Case Western Reserve Uni-
versity, Cleveland, OH, USA). Mouse monoclonal IgG
against complex I subunit NDUFS3 (NADH dehydroge-
nase ubiquinone Fe–S protein), complex II subunit b (one
Fe–S subunit of complex II), Rieske Fe–S protein subunit
of complex III and the flavoprotein subunit of complex II
were from MitoSciences (Eugene, OR, USA). Monoclonal

anti-mitochondrial DNA-encoded ND6 subunit IgG (Mito-
Sciences) was kindly provided by G. Dujardin (Centre de
Ge
´
ne
´
tique Mole
´
culaire, Gif-sur-Yvette, France). Rabbit
polyclonal sera were raised against synthetic peptides corre-
sponding to amino acids 45–61 of mouse mitochondrial
aconitase and to amino acids 410–422 of mouse ferrochela-
tase. The serum against frataxin used was the 1250 poly-
clonal serum [5]. Serum against ClpP raised against the
peptide corresponding to amino acids 65–80 of human
ClpP was kindly provided by L. Sweda (Oklahoma Medical
Research Foundation, Oklahoma City, OK, USA). Sera
against vinculin (Sigma) and prohibitin (Neomarkers,
Fremont, CA, USA) were used as loading controls. Serum
against the ATP2 b1-subunit of yeast ATP synthase was
kindly provided by J. Velours (Institut de Biochimie et
Ge
´
ne
´
tique Cellulaire, Bordeaux, France).
ATP-stimulated protease activity
ATP-stimulated Lon and ClpP protease activity was deter-
mined using casein–fluorescein isothiocyanate (0.5 lgÆlL
)1

)
as substrate [31]. Mitochondrial pellets (50–150 lg) were
incubated in assay buffer containing 50 mm Tris (pH 7.9),
10 mm MgCl
2
,1mm dithiothreitol and 0.1% Triton X-100
in the presence or absence of 8 mm ATP. Proteolysis of
fluorescein isothiocyanate-labeled casein was then per-
formed for 90 min at 37 °C. At incubation times of
0–90 min, an aliquot was collected and proteins were pre-
cipitated by adding 8% trichloroacetic acid. After centrifu-
gation at 15 000 g for 30 min, supernatant containing
fluorescent peptides was recovered and neutralized by add-
ing sodium borate (0.6 m final concentration, pH 10). Fluo-
rescence was then measured with excitation ⁄ emission
wavelengths of 495 ⁄ 515 nm. Activities were expressed as
fluorescence units ⁄ min ⁄ mg protein, and a ratio between the
ATP-dependent proteolytic activities found in MCK and
wild-type mice was calculated.
Carbonylated protein detection
Carbonylated proteins were detected using the OxyBlot
protein oxidation detection kit according to the manufac-
turer’s protocol (Chemicon International, Temecula, CA,
USA). Briefly, mitochondrial pellets were lysed in 100 mm
Tris (pH 7.5), 0.5% Triton X-100 and 50 mm dithiothreitol.
After centrifugation, clear lysate was denatured by adding
6% SDS, and the carbonyl groups in proteins were deriva-
tized to 2,4-dinitrophenylhydrazone (DNP) using 1· dini-
trophenylhydrazine solution for 15 min at 25 °C. After
neutralization of the reaction, DNP-derivatized proteins

were loaded on a 10% SDS ⁄ PAGE gel, transferred to a
nitrocellulose membrane, and detected with a primary anti-
body specific to the DNP moiety. Carbonylated proteins in
the mitochondrial fractions from hearts of control and
MCK mutant mice were measured using the protein car-
bonyl ELISA kit according to the manufacturer’s instruc-
tions (BioCell Corp., Auckland, New Zealand).
Quantitative real-time PCR analysis
Total RNA was extracted using the SV Total RNA Isolation
System kit (Promega, Charbonnie
`
res-les-bains, France), and
4 lg of total RNA was reverse-transcribed using the High
Capacity cDNA Archive Kit (Applied Biosystems, Courta-
boeuf, France) according to the manufacturer’s instructions.
Quantitative real-time PCR was performed using the Roche
Light Cycler system and the FastStart DNA master plus
SYBR green I kit (Roche Applied Sciences, Meylan, France).
Lon protease (forward, 5¢-AGGATCTTGCCTTGTGT
GGA-3¢; reverse, 5¢-TGGATGAGGAGCTGAGCAAG-3¢)
ClpP (forward, 5¢-CACACCAAGCAGAGCCTACA-3¢;
reverse, 5¢-CCCAGCAGAGGAAGTTTCAG-3¢), mitochon-
drial aconitase (forward, 5¢-AGGAGTTTGGCCCTGTA
CCT-3¢; reverse, 5¢-GCCTTGAATGGTCAGCTTGT-3¢),
NDUFS3 (forward, 5¢-CTGTGGCAGCACGTAAGAAG-3¢;
reverse, 5¢-ACTCATCAAGGCAGGACACC-3¢), SDHB
(forward, 5¢-GGAGGGCAAGCAACAGTATC-3¢; reverse,
5¢-GCGTTCCTCTGTGAAGTCGT-3¢), Rieske (forward,
5¢-TGGTCTCCCAGTTTGTTTCC-3¢; reverse, 5¢-GCAGC
TTCCTGGTCAATCTC-3¢) and SDHA (forward, 5¢-CAG

AAGTCGATGCAGAACCA-3¢; reverse, 5 ¢-CGACCCGCA
CTTTGTAATCT-3¢) sequence-specific primers were
designed to span intron–exon boundaries to generate ampli-
cons of approximately 100 bp. Values were normalized to
the relative amounts of 18S ribosomal cDNA (forward,
5¢-CTGAGAAACGGCTACCACATC-3¢; reverse, 5¢-CGCT
CCCAAGATCCAACTAC-3¢). Sequences of mitochondrial
gene primers are listed in Table S2.
Acknowledgements
We wish to thank Alexandre Diet, Aurelien Bayot,
Agnieszka Malinowska and Fabienne Pierre for techni-
cal support, and Laurence Reutenauer for the genera-
tion and genotyping of all the mice for this project.
This work was supported by funds from the French
National Agency for Research (ANR-05-MRAR-013-
01) and the French Medical Research Foundation
Upregulation of mitochondrial proteases in FRDA B. Guillon et al.
1044 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS
(H. Puccio). M. Wattenhofer-Donze
´
is an ATER at
the Colle
`
ge de France.
References
1 Puccio H & Koenig M (2002) Friedreich ataxia: a para-
digm for mitochondrial diseases. Curr Opin Genet Dev
12, 272–277.
2 Campuzano V, Montermini L, Molto MD, Pianese L,
Cossee M, Cavalcanti F, Monros E, Rodius F, Duclos

F, Monticelli A et al. (1996) Friedreich’s ataxia: autoso-
mal recessive disease caused by an intronic GAA triplet
repeat expansion. Science 271, 1423–1427.
3 Puccio H (2007) Conditional mouse models for
Friedreich ataxia, a neurodegenerative disorder associ-
ating cardiomyopathy. Handb Exp Pharmacol 178,
365–375.
4 Rotig A, de Lonlay P, Chretien D, Foury F, Koenig
M, Sidi D, Munnich A & Rustin P (1997) Aconitase
and mitochondrial iron–sulphur protein deficiency in
Friedreich ataxia. Nat Genet 17, 215–217.
5 Puccio H, Simon D, Cossee M, Criqui-Filipe P, Tiziano
F, Melki J, Hindelang C, Matyas R, Rustin P &
Koenig M (2001) Mouse models for Friedreich ataxia
exhibit cardiomyopathy, sensory nerve defect and Fe–S
enzyme deficiency followed by intramitochondrial iron
deposits. Nat Genet 27, 181–186.
6 Chen OS, Hemenway S & Kaplan J (2002) Inhibition of
Fe–S cluster biosynthesis decreases mitochondrial iron
export: evidence that Yfh1p affects Fe–S cluster synthe-
sis. Proc Natl Acad Sci USA 99, 12321–12326.
7 Muhlenhoff U, Richhardt N, Ristow M, Kispal G &
Lill R (2002) The yeast frataxin homolog Yfh1p plays a
specific role in the maturation of cellular Fe ⁄ S proteins.
Hum Mol Genet 11, 2025–2036.
8 Zhang Y, Lyver ER, Knight SA, Pain D, Lesuisse E &
Dancis A (2006) Mrs3p, Mrs4p, and frataxin provide
iron for Fe–S cluster synthesis in mitochondria. J Biol
Chem 281, 22493–22502.
9 Muhlenhoff U, Gerber J, Richhardt N & Lill R (2003)

Components involved in assembly and dislocation of
iron–sulfur clusters on the scaffold protein Isu1p.
EMBO J 22, 4815–4825.
10 Ramazzotti A, Vanmansart V & Foury F (2004)
Mitochondrial functional interactions between frataxin
and Isu1p, the iron–sulfur cluster scaffold protein, in
Saccharomyces cerevisiae. FEBS Lett 557, 215–220.
11 Yoon T & Cowan JA (2003) Iron–sulfur cluster biosyn-
thesis. Characterization of frataxin as an iron donor for
assembly of [2Fe–2S] clusters in ISU-type proteins.
J Am Chem Soc 125, 6078–6084.
12 Stehling O, Elsasser HP, Bruckel B, Muhlenhoff U &
Lill R (2004) Iron–sulfur protein maturation in human
cells: evidence for a function of frataxin. Hum Mol
Genet 13, 3007–3015.
13 Seznec H, Simon D, Bouton C, Reutenauer L, Hertzog
A, Golik P, Procaccio V, Patel M, Drapier JC, Koenig
M et al. (2005) Friedreich ataxia: the oxidative stress
paradox. Hum Mol Genet 14, 463–474.
14 Martelli A, Wattenhofer-Donze M, Schmucker S,
Bouvet S, Reutenauer L & Puccio H (2007) Frataxin
is essential for extramitochondrial Fe–S cluster
proteins in mammalian tissues. Hum Mol Genet 16,
2651–2658.
15 Anderson PR, Kirby K, Hilliker AJ & Phillips JP
(2005) RNAi-mediated suppression of the mitochondrial
iron chaperone, frataxin, in Drosophila. Hum Mol Genet
14, 3397–3405.
16 Layer G, Ollagnier-de-Choudens S, Sanakis Y & Fonte-
cave M (2006) Iron–sulfur cluster biosynthesis: charac-

terization of Escherichia coli CYaY as an iron donor
for the assembly of [2Fe–2S] clusters in the scaffold
IscU. J Biol Chem 281, 16256–16263.
17 Busi MV, Maliandi MV, Valdez H, Clemente M, Zabal-
eta EJ, Araya A & Gomez-Casati DF (2006) Deficiency
of Arabidopsis thaliana frataxin alters activity of mito-
chondrial Fe–S proteins and induces oxidative stress.
Plant J 48, 873–882.
18 Gerber J, Muhlenhoff U & Lill R (2003) An
interaction between frataxin and Isu1 ⁄ Nfs1 that is
crucial for Fe ⁄ S cluster synthesis on Isu1. EMBO Rep
4, 906–911.
19 Foury F, Pastore A & Trincal M (2007) Acidic residues
of yeast frataxin have an essential role in Fe–S cluster
assembly. EMBO Rep 8, 194–199.
20 Wang T & Craig EA (2008) Binding of yeast frataxin to
the scaffold for Fe–S cluster biogenesis, Isu. J Biol
Chem 283, 12674–12679.
21 Powell SR (2006) The ubiquitin–proteasome system in
cardiac physiology and pathology. Am J Physiol Heart
Circ Physiol 291, H1–H19.
22 Koppen M & Langer T (2007) Protein degradation
within mitochondria: versatile activities of AAA prote-
ases and other peptidases. Crit Rev Biochem Mol Biol
42, 221–242.
23 Ngo JK & Davies KJ (2007) Importance of the lon pro-
tease in mitochondrial maintenance and the significance
of declining lon in aging. Ann NY Acad Sci 1119, 78–
87.
24 de Sagarra MR, Mayo I, Marco S, Rodriguez-Vilarino

S, Oliva J, Carrascosa JL & Castan JG (1999) Mito-
chondrial localization and oligomeric structure of
HClpP, the human homologue of E. coli ClpP. J Mol
Biol 292, 819–825.
25 Santagata S, Bhattacharyya D, Wang FH, Singha N,
Hodtsev A & Spanopoulou E (1999) Molecular cloning
and characterization of a mouse homolog of bacterial
ClpX, a novel mammalian class II member of the
Hsp100 ⁄ Clp chaperone family. J Biol Chem 274,
16311–16319.
B. Guillon et al. Upregulation of mitochondrial proteases in FRDA
FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1045
26 Kang SG, Ortega J, Singh SK, Wang N, Huang NN,
Steven AC & Maurizi MR (2002) Functional proteo-
lytic complexes of the human mitochondrial ATP-
dependent protease, hClpXP. J Biol Chem 277,
21095–21102.
27 Mettert EL & Kiley PJ (2005) ClpXP-dependent prote-
olysis of FNR upon loss of its O2-sensing [4Fe–4S] clus-
ter. J Mol Biol 354, 220–232.
28 Major T, von Janowsky B, Ruppert T, Mogk A &
Voos W (2006) Proteomic analysis of mitochondrial
protein turnover: identification of novel substrate pro-
teins of the matrix protease pim1. Mol Cell Biol 26,
762–776.
29 Bota DA & Davies KJ (2002) Lon protease preferen-
tially degrades oxidized mitochondrial aconitase by
an ATP-stimulated mechanism. Nat Cell Biol 4, 674–
680.
30 Majeran W, Wollman FA & Vallon O (2000) Evidence

for a role of ClpP in the degradation of the chloroplast
cytochrome b(6)f complex. Plant Cell 12, 137–150.
31 Bakala H, Delaval E, Hamelin M, Bismuth J, Borot-
Laloi C, Corman B & Friguet B (2003) Changes in rat
liver mitochondria with aging. Lon protease-like reac-
tivity and N(epsilon)-carboxymethyllysine accumulation
in the matrix. Eur J Biochem 270, 2295–2302.
32 Bulteau AL, Lundberg KC, Ikeda-Saito M, Isaya G &
Szweda LI (2005) Reversible redox-dependent modula-
tion of mitochondrial aconitase and proteolytic activity
during in vivo cardiac ischemia ⁄ reperfusion. Proc Natl
Acad Sci USA 102, 5987–5991.
33 Bulteau AL, Dancis A, Gareil M, Montagne JJ, Cama-
dro JM & Lesuisse E (2007) Oxidative stress and prote-
ase dysfunction in the yeast model of Friedreich ataxia.
Free Radic Biol Med 42, 1561–1570.
34 Seznec H, Simon D, Monassier L, Criqui-Filipe P,
Gansmuller A, Rustin P, Koenig M & Puccio H (2004)
Idebenone delays the onset of cardiac functional alter-
ation without correction of Fe–S enzymes deficit in a
mouse model for Friedreich ataxia. Hum Mol Genet 13,
1017–1024.
35 Davies KJ (1993) Protein modification by oxidants and
the role of proteolytic enzymes. Biochem Soc Trans 21,
346–353.
36 Fu GK & Markovitz DM (1998) The human LON
protease binds to mitochondrial promoters in a single-
stranded, site-specific, strand-specific manner. Biochem-
istry 37, 1905–1909.
37 Liu T, Lu B, Lee I, Ondrovicova G, Kutejova E &

Suzuki CK (2004) DNA and RNA binding by the
mitochondrial lon protease is regulated by
nucleotide and protein substrate. J Biol Chem 279,
13902–13910.
38 Lu B, Yadav S, Shah PG, Liu T, Tian B, Pukszta S,
Villaluna N, Kutejova E, Newlon CS, Santos JH et al.
(2007) Roles for the human ATP-dependent Lon
protease in mitochondrial DNA maintenance. J Biol
Chem 282, 17363–17374.
39 May BK, Borthwick IA, Srivastava G, Pirola BA &
Elliott WH (1986) Control of 5-aminolevulinate syn-
thase in animals. Curr Top Cell Regul 28, 233–262.
40 Sun F, Huo X, Zhai Y, Wang A, Xu J, Su D, Bartlam
M & Rao Z (2005) Crystal structure of mitochondrial
respiratory membrane protein complex II. Cell 121,
1043–1057.
41 Schoenfeld RA, Napoli E, Wong A, Zhan S, Reut-
enauer L, Morin D, Buckpitt AR, Taroni F, Lonnerdal
B, Ristow M et al. (2005) Frataxin deficiency alters
heme pathway transcripts and decreases mitochondrial
heme metabolites in mammalian cells. Hum Mol Genet
14, 3787–3799.
42 Kato GJ (1999) Human genetic diseases of proteolysis.
Hum Mutat 13, 87–98.
43 Grune T, Jung T, Merker K & Davies KJ (2004)
Decreased proteolysis caused by protein aggregates,
inclusion bodies, plaques, lipofuscin, ceroid, and ‘aggre-
somes’ during oxidative stress, aging, and disease. Int J
Biochem Cell Biol 36, 2519–2530.
44 Bota DA & Davies KJ (2001) Protein degradation in

mitochondria: implications for oxidative stress, aging
and disease: a novel etiological classification of
mitochondrial proteolytic disorders. Mitochondrion 1,
33–49.
45 Yu AY & Houry WA (2007) ClpP: a distinctive family
of cylindrical energy-dependent serine proteases. FEBS
Lett 581, 3749–3757.
46 Bross P, Andresen BS, Knudsen I, Kruse TA & Greger-
sen N (1995) Human ClpP protease: cDNA sequence,
tissue-specific expression and chromosomal assignment
of the gene. FEBS Lett 377, 249–252.
47 Wang N, Gottesman S, Willingham MC, Gottesman
MM & Maurizi MR (1993) A human mitochondrial
ATP-dependent protease that is highly homologous to
bacterial Lon protease. Proc Natl Acad Sci USA 90,
11247–11251.
48 Hori O, Ichinoda F, Tamatani T, Yamaguchi A, Sato
N, Ozawa K, Kitao Y, Miyazaki M, Harding HP, Ron
D et al. (2002) Transmission of cell stress from endo-
plasmic reticulum to mitochondria: enhanced expression
of Lon protease. J Cell Biol 157, 1151–1160.
49 Lodi R, Cooper JM, Bradley JL, Manners D, Styles P,
Taylor DJ & Schapira AH (1999) Deficit of in vivo
mitochondrial ATP production in patients with
Friedreich ataxia. Proc Natl Acad Sci USA 96,
11492–11495.
50 Tarabishi R, Zahedi K, Mishra J, Ma Q, Kelly C,
Tehrani K & Devarajan P (2005) Induction of Zf9 in
the kidney following early ischemia ⁄ reperfusion. Kidney
Int 68, 1511–1519.

51 Jeyaraj SC, Dakhlallah D, Hill SR & Lee BS (2006)
Expression and distribution of HuR during ATP
Upregulation of mitochondrial proteases in FRDA B. Guillon et al.
1046 FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS
depletion and recovery in proximal tubule cells. Am J
Physiol Renal Physiol 291 , F1255–F1263.
52 Zhao Q, Wang J, Levichkin IV, Stasinopoulos S, Ryan
MT & Hoogenraad NJ (2002) A mitochondrial specific
stress response in mammalian cells. EMBO J 21, 4411–
4419.
53 Suzuki CK, Rep M, van Dijl JM, Suda K, Grivell LA
& Schatz G (1997) ATP-dependent proteases that also
chaperone protein biogenesis. Trends Biochem Sci 22,
118–123.
54 Luciakova K, Sokolikova B, Chloupkova M & Nelson
BD (1999) Enhanced mitochondrial biogenesis is associ-
ated with increased expression of the mitochondrial
ATP-dependent Lon protease. FEBS Lett 444, 186–188.
55 Bota DA, Ngo JK & Davies KJ (2005) Downregulation
of the human Lon protease impairs mitochondrial
structure and function and causes cell death. Free Radic
Biol Med 38, 665–677.
56 Sazanov LA (2007) Respiratory complex I: mechanis-
tic and structural insights provided by the crystal
structure of the hydrophilic domain. Biochemistry 46,
2275–2288.
57 Chomyn A, Cleeter MW, Ragan CI, Riley M, Doolittle
RF & Attardi G (1986) URF6, last unidentified reading
frame of human mtDNA, codes for an NADH dehy-
drogenase subunit. Science 234, 614–618.

58 Van Hoewyk D, Abdel-Ghany SE, Cohu CM, Herbert
SK, Kugrens P, Pilon M & Pilon-Smits EA (2007)
Chloroplast iron–sulfur cluster protein maturation
requires the essential cysteine desulfurase CpNifS. Proc
Natl Acad Sci USA 104, 5686–5691.
59 Bayot A, Basse N, Lee I, Gareil M, Pirotte B,
Bulteau AL, Friguet B & Reboud-Ravaux M (2008)
Towards the control of intracellular protein
turnover: mitochondrial Lon protease
inhibitors versus proteasome inhibitors. Biochimie 90,
260–269.
Supporting information
The following supplementary material is available:
Fig. S1. The inverse correlation diagram of mitochon-
drial protein levels versus Lon and ClpP protease
levels.
Table S1. ATP-dependent proteolytic activity in heart
mitochondria of control and MCK mutant mice at 5,
7 and 8 weeks of age.
Table S2. Oligonucleotide primers used to amplify
mitochondria-encoded genes of the respiratory chain in
cardiac muscle of control and MCK mutant mice.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
B. Guillon et al. Upregulation of mitochondrial proteases in FRDA

FEBS Journal 276 (2009) 1036–1047 ª 2009 The Authors Journal compilation ª 2009 FEBS 1047