Cells Lacking Rieske Iron-Sulfur Protein Have a Reactive Oxygen
Species-Associated Decrease in Respiratory Complexes I and IV
Francisca Diaz,aJosé Antonio Enríquez,b,cand Carlos T. Moraesa
Department of Neurology, University of Miami, Miller School of Medicine, Miami, Florida, USAa; Centro National de Investigaciones Cardiovasculares Carlos III, Melchor
Fernández Almagro, Madrid, Spainb; and Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, Pedro Cerbuna,
known as complex III (CIII), from the electron transport chain
(ETC). CIII contains two other catalytic subunits, cytochrome b
and cytochrome c1. The active enzyme is a homodimer that cata-
lyzes the transfer of electrons from ubiquinol (coenzyme Q) to
cytochrome c in a bifurcated mechanism mediated by RISP (62).
In the last few years, structural evidence indicates that the
mitochondrial complexes of the oxidative phosphorylation
lar structures in the inner mitochondrial membrane named su-
percomplexes. Their association into megacomplexes was pro-
posed to form a functional unit known as the “respirasome” (7,
60, 69). Because the electron transport chain is one of the major
contributors to free radicals in the cell, respirasomes could mini-
mize the generation of reactive oxygen species (ROS) by allowing
the complexes, therefore avoiding the diffusion of reactive inter-
mediates (29, 42).
of organisms, including bacteria, plants, fungi, and mammals, al-
Mammalian supercomplexes are composed mainly by CI, CIII,
and CIV in different stoichiometries (I/III, I/III2, I2III2, I/III/IV,
Perez et al. (2) investigated the composition and functionality of
the different mammalian supercomplexes. The authors showed
that some of these structures contained coenzyme Q and cyto-
chrome c. Moreover, they demonstrated that some supercom-
plexes were able to “respire” by transferring electrons from
NADH to oxygen (2).
relies on the stability of its components. Respiratory complex in-
terdependence has been observed in numerous cases. Generally,
he Rieske iron-sulfur protein (RISP) is one of the catalytic
subunits of ubiquinol-cytochrome c oxidoreductase, also
chain. Cells with defects in CIII or CIV assembly have decreased
levels of CI (1, 17, 20, 43), whereas cells lacking cytochrome c
showed defects in CIV and CI (64). Likewise, defects in CV have
been associated with defects in CIII and CIV in both yeast and
mammalian cells (55, 59).
In the last few years, studies have attempted to elucidate the
role of deranged mitochondrial supercomplexes and their patho-
supramolecular architecture of OXPHOS complexes have been
observed in the rat cortex during aging, and decreased respira-
some levels have been reported in animal models of severe heart
failure (26, 54). Whether these alterations result in significant
physiological changes is still unclear.
in mouse fibroblasts deficient in RISP and uncovered a ROS-
MATERIALS AND METHODS
Cell culture. A primary culture lung fibroblast line was produced from a
knock-in mouse homozygous for the floxed UQCRFS1 gene (exon 2)
encoding the Rieske iron-sulfur protein (RISP) (28). The introduction of
activities from tissues derived from wild-type or floxed mice were com-
parable. Cells were grown at 37°C in a 5% CO2atmosphere in high-
Received 3 August 2011 Returned for modification 1 September 2011
Accepted 10 November 2011
Published ahead of print 21 November 2011
Address correspondence to Francisca Diaz, FDiaz1@med.miami.edu.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0270-7306/12/$12.00Molecular and Cellular Biologyp. 415–429mcb.asm.org
derived cell line was immortalized by transduction with a G418-resistant
retrovirus expressing E6/E7 oncogenes of type 6 papillomavirus (kindly
provided by E. Shoubridge, McGill University) (45), and G418-resistant
clones were obtained by ring cloning. These immortalized cell lines con-
taining the floxed gene were used as control cell lines from which knock-
out (KO) cells were derived. To ablate the floxed UQCRFS1 gene, one of
the immortalized clones was transfected with a hygromycin-resistant
plasmid expressing P1 Cre recombinase (kindly provided by J. Marth,
University of California San Diego). The control cell lines used in this
whereas the knockout cells deficient in RISP were derived from clone 8
and are referred to as clones 8.2, 8.4, 8.5, and 8.17.
Multiplex PCR. Cre-mediated recombination was determined by
multiplex PCR using 3 primers allowing the simultaneous amplification
of the floxed, wild-type, and deletion alleles, as shown in Fig. 1A. The
primer sequences were as follows: (i) forward, 5=TTCCCTCCTCAGGCT
TCACTTGAC3=; (ii) reverse, 5=GATTGGGAAGACAATAGCAGGCAT
relative positions and orientations of the primers are depicted in Fig. 1A
Mitochondrial preparation and enzyme activities. Mitochondria
were obtained from 8 confluent T-75 flasks by nitrogen cavitation as de-
scribed previously (20) and used for determination of the enzyme activi-
ties of different respiratory complexes and citrate synthase as described
previously (3). Total cell respiration was determined by polarography
with a Clark oxygen electrode (Hansatech Instruments, Norfolk, United
Kingdom) as described in reference 20.
Protein determination. Protein concentrations were determined us-
ing bovine serum albumin as the standard and either the Bradford or the
bicinchoninic acid (BCA) (detergent-compatible) protein assay kit from
Bio-Rad and Pierce, respectively.
BNGE. To identify individual respiratory complexes, mitochondrial
blue native gel electrophoresis (BNGE) in 4 to 13% acrylamide gradient
gels (8, 19). For Western blot analysis, amounts of about 10 to 25 ?g of
membrane (Bio-Rad), and immunodecorated sequentially with antibod-
ies against several subunits of the different mitochondrial respiratory
complexes. For in-gel activity assays, amounts of about 40 ?g of proteins
were separated by BNGE and gels were incubated with solutions contain-
ing the respective substrates for complex I or IV as described before (19).
For supercomplex analysis, mitochondrial preparations were treated
FIG 1 Ablation of RISP leads to CIII deficiency. (A) Diagram shows the floxed allele containing 3 loxP sites (triangles) flanking exon 2 of the UQCRFS1 gene
activities of CIII, CIV, and citrate synthase (CS) determined spectrophotometrically. Values represent the means and standard deviations of specific activities.
Statistical significance is shown by an asterisk (P ? 0.05). (D) Western blot of mitochondrial proteins and OXPHOS complex subunits (CI, NDUFA9; CIII,
Core1, Core2, and RISP; and CIV, Cox1). VDAC1 and Tim23 were used as mitochondrial loading controls.
Diaz et al.
mcb.asm.orgMolecular and Cellular Biology
3 to 12% precast blue native gels (Invitrogen).
For analysis of respiratory complexes by 2 dimensional (2D) BNGE,
BN gel strips from the first dimension corresponding to each lane were
4% stacking gel (20). Proteins were separated in the second dimension,
transferred to PVDF membranes, and blotted sequentially with different
For experiments that included different cell treatments (pharmaco-
?l phosphate-buffered saline (PBS) containing protease inhibitors and
adding 70 ?l of 8 mg/ml digitonin (Sigma) for 10 min on ice. Subse-
quently, samples were diluted with 1 ml PBS and centrifuged at 21,000 ?
g for 5 min. Membranes were washed with PBS, and pellets were either
stored at ?80°C until needed or resuspended in 100 ?l of 1.5 M amino-
caproic acid, 50 mM Bis-Tris (pH 7.0) buffer (8). Supercomplexes were
extracted from these preparations with highly purified digitonin (Calbi-
by BNGE (20).
Western blotting. Mitochondrial samples were separated by either
SDS-PAGE in 4 to 20% acrylamide gradient gels (Bio-Rad) or by BNGE
and 2D-BNGE and transferred to PVDF membranes. Membranes were
with specific primary antibodies. Secondary antibodies conjugated to
horseradish peroxidase (Cell Signaling) were used, and the reaction was
detected by chemiluminescence using SuperSignal West reagent (Pierce,
Rockford, IL) or RapidStep ECL reagent (EMD).
The following antibodies were used to identify individual respiratory
complexes: complex I, NDUFA9 and NDUFS3 (Mitosciences); complex
II, SDHA (succinate dehydrogenase subunit A) flavoprotein (Molecular
Probes); complex III, Core1, Core2 (Mitosciences), and Rieske iron-
complex V, ATP synthase ? (Molecular Probes). Additionally, premade
antibody cocktails from Mitosciences specific for BNGE (NDUFA9,
SDHA, Core2, Cox IV, and ATPase ?) or for SDS-PAGE (NDUFB8,
the inner mitochondrial membrane; Molecular Probes) were used as mi-
tochondrial loading controls.
To detect HIF1-?, cells were immediately lysed after hypoxia in ice
using 2? Laemmli buffer without bromophenol blue. Cells were scraped
off the tissue culture plate, and lysate heated at 96°C for 20 min and
acrylamide gels and transferred to PVDF membranes, and HIF1-? was
detected with a specific antibody obtained from Novus Biologicals.
Signal intensities were quantified using the free NIH software Image J
available for download at http://rsb.info.nih.gov/ij/. Fold changes in pro-
tein levels annotated in the text were calculated as the ratio between the
protein signal and the corresponding loading control (in the same mem-
Recombinant lentivirus. Wild-type RISP was amplified from mouse
cDNA and cloned into the pLenti-MP2-MCS-IRES-EGFP lentiviral vec-
expression of both RISP and green fluorescent protein. A mutant form of
the RISP was created by site-directed mutagenesis. The mutant RISP had
two point mutations in positions 716 and 717 to change the wild-type
acid change from histidine to arginine that corresponded to amino acid
239 in the murine sequence. Lentiviruses expressing green fluorescent
protein (GFP) and wild-type or mutant RISP were produced by the Viral
Core Facility at the University of Miami.
lentiviruses expressing GFP, wild-type RISP-GFP, or mutant RISP-GFP.
High-GFP-expressing cells were sorted out by fluorescence-activated cell
sorting (FACS) in an Aria-IIu cell sorter (Becton Dickinson) and main-
tained as a cell line for subsequent experiments.
cloned in the pWXIRESpuro vector) were used to transduce RISP-
deficient cells as described above. Lentivirus expressing SOD2 contained
puromycin resistance. Antibiotic-resistant stable cell lines were used in
Pharmacological inhibition of mitochondrial respiratory com-
plexes. The pharmacological inhibition of CIII was achieved by growing
cells in the presence of 2 nM antimycin A or 2 ?M myxothiazol in com-
plete medium for 3 days. The inhibition of CI, CIV, and CV was achieved
by growing cells in the presence of 100 nM rotenone, 400 ?M potassium
cyanide (KCN), or 5 ?g/ml oligomycin, respectively, for 3 days in com-
daily to avoid highly acidified medium. All OXPHOS inhibitors were ob-
tained from Sigma.
immediately placed under conditions of normoxia or hypoxia. A hypoxic
struments) directly connected to a nitrogen-CO2mixture tank (Medi-
blend clinical blood gas mixture, 5% CO2plus nitrogen) was placed in a
ditions (21% O2, 5% CO2) at 37°C. In some experiments, cells were
treated with different concentrations of MitoQ or decyltriphenylphos-
normoxia or hypoxia for 4 h.
Assessment of oxidative stress. To measure superoxide levels, cells
(3 ? 105/well in 6-well plates) were plated, and the next day, the medium
was replaced with fresh medium containing 10 ?m oligomycin (positive
control) or dimethyl sulfoxide (DMSO; vehicle) for 1.5 h at 37°C. Then,
either 2.5 ?M MitoSox (Invitrogen) or 2.5 ?M dihydroethidium (DHE,
harvested by trypsinization, washed, and resuspended in Hanks balanced
solution. Fluorescence was detected by FACS in an Aria-IIu flow cytom-
of 396/580 for MitoSox and 518/605 for DHE.
To measure hydrogen peroxide production in normoxia/hypoxia
experiments, cells were plated in 96-well plates at a density of 104
cells/100 ?l/well and incubated with 8 ?M H2DCF-DA (2=,7=-
dichlorodihydrofluorescein diacetate) in 200 ?l medium (56) prior to the
24 h. At the end of the incubation period, DCF fluorescence was measured
immediately with a plate reader (Victor II; Perkin Elmer). The fluorescence
values were corrected for the fluorescence of the medium alone. Cells were
incubated with 100 ?M tert-butyl hydroperoxide (tBOOH) and 2 mM
n-acetyl cysteine (NAC) as positive controls for the assay. Values represent
Analysis of cellular antioxidant system during normoxia/hypoxia
dismutases (SOD1 and SOD2) were determined by Western blot analysis
and by in-gel activity assay as described previously (68). To determine
steady-state levels of these enzymes by Western blotting, we used an anti-
SOD1 antibody from Calbiochem and an anti-SOD2 antibody from Up-
state. Glutathione (GSH) levels were determined using the GSH-Glo glu-
tathione assay kit from Promega, and glutathione peroxidase (GPx)
activity was measured with the GPx assay kit from Cayman Chemicals,
following the manufacturer’s instructions.
Statistical analysis. Values represent the means and standard devia-
tions of at least 3 independent measurements. Statistical significance was
RISP Knockout Decreases Levels of Complexes I and IV
January 2012 Volume 32 Number 2mcb.asm.org 417
determined by using the two-tailed unpaired Student t test. A P value of
?0.05 was considered statistically significant.
Creation and characterization of RISP KO fibroblasts. Primary
cultures of lung fibroblasts from a knock-in mouse (28) homozy-
gous for the floxed UQCRFS1 gene were immortalized by E6/E7
expression. The knock-in mouse contained loxP sites flanking
exon 2 of the UQCRFS1 gene and a selection cassette at the 3=
untranslated region (UTR) (Fig. 1A) (28). The UQCRFS1 gene
subunits of complex III (CIII). Two immortalized clones harbor-
ing the floxed UQCRFS1 (clones 8 and 14) were used as control
cell lines in this study.
To obtain CIII-deficient cells, the floxed clone 8 was trans-
fected with a plasmid expressing the Cre recombinase. The effi-
detecting the floxed, deletion, and wild-type alleles simultane-
ously (Fig. 1A). PCR of the RISP knockout (KO) clones (clones
8.2, 8.4, 8.5, and 8.17) only amplified the band corresponding to
only amplified in the control cells, clones 8 and 14. These results
indicate a complete recombination of the floxed gene (Fig. 1B).
The RISP KO clones were unable to consume oxygen and
quickly acidified the medium, indicating increased production of
lactate due to impaired respiration (not shown). Biochemical
sured spectrophotometrically showed, as expected, that deletion
of RISP completely abolished CIII enzymatic activity (Fig. 1C).
RISP deletion was also associated with a decrease in complex IV
the parental cell line (Fig. 1C). Citrate synthase (CS) activity was
increased in all the KO fibroblasts compared to the activity in the
parental clone 8. We found a marked variation in CIV activity
parental cell line. Control cell lines 8 and 14 showed variation in
CIV and CS (Fig. 1C), possibly related to genome instability in
Western blotting (Fig. 1D) showed no detectable levels of RISP in
the KO clones, confirming the deletion of the gene. Other CIII
subunits (UQCRC1 and UQCRC2, also known as Core1 and
Core2) were decreased in the KO clones (1.6- to 2-fold and 7- to
10-fold lower than control levels, respectively). The levels of CI
fold and 7- to 9.6-fold lower than control levels, respectively).
Likewise, the levels of the CIV subunit Cox1 were decreased in
those clones with decreased CIV activity (2- to 7-fold lower than
control levels). In contrast, CII and CV subunits (SDHA and
ATPase-?, respectively) were increased in the RISP KO clones
of other mitochondrial proteins, such as cytochrome c, VDAC1
(voltage-dependent anion-selective channel protein 1), and
Tim23 (inner mitochondrial protein import complex compo-
nent) were comparable in all samples analyzed (Fig. 1D).
and the levels of newly synthesized Cox1 in the KO cells were
comparable to control levels (not shown). These results suggest
that Cox1 was destabilized posttranslationally.
of assembled OXPHOS complexes using blue native gel electro-
detect individual complexes. The deletion of RISP significantly
levels of CIV were decreased in those KO clones with lower CIV
the presence of isolated CI, CIII, and supercomplexes in the con-
trol cell line when analyzed by 2-dimensional blue native gel elec-
trophoresis (2D-BNGE) (Fig. 2A). Still, only by overexposing or
loading more protein into the gels were we able to observe low
the RISP KO clones (Fig. 2A and B).
Although all the steps for CIII assembly in mammals are un-
known, the final steps in yeast appear to be the incorporation of
RISP and the Qcr10 subunits. RISP incorporation is mediated by
the chaperone Bcs1. In mammals, the homologue subunits are
termed RISP and UQCRC11, respectively, and the homologue
chaperon is BCS1L. The fully assembled CIII is a dimer with a
molecular mass of about 550 kDa (16, 70). The CIII observed in
lacks RISP and UQCR11 (pre-CIII). Under the conditions used,
assembled CIII and pre-CIII from control and RISP KO cells,
in Fig. 3A (GFP lanes, probed with Core2-specific antibody).
of assembly intermediates already occurred to form the pre-CIII
subassembly/degradation intermediate containing Core2 but not
Core1 in the KO clones (Fig. 2B).
Respiratory complex and supercomplex instability has been
attributed, in some instances, to low levels of phospholipids, such
as cardiolipin (47, 52, 67). However, analysis of phospholipids by
thin-layer chromatography did not reveal major differences be-
tween control and RISP KO cells (data not shown).
stitution experiments introducing RISP back into the KO clones
ing GFP (control infection), mouse wild-type RISP (WT-RISP),
or the mutant RISP (MUT-RISP). We mutated the mouse histi-
dine 219 residue to an arginine that corresponded to the histidine
181 in yeast. We chose this particular mutation because the yeast
H181R mutant had the highest levels of RISP protein among the
collection of no-activity mutants (32).
Wild-type RISP was incorporated into CIII in RISP KO cells
of CIII in KO clones. In fact, we were unable to detect the mutant
RISP protein (Fig. 3C). It is possible that the mouse mutant pro-
tein, unlike the yeast mutant H181R, was not stable and it was
degraded. The levels of other CIII subunits, Core1 and Core2,
Diaz et al.
mcb.asm.org Molecular and Cellular Biology
were increased in KO clones expressing WT-RISP compared to
the levels in the clones expressing either GFP or MUT-RISP (Fig.
3C). Not surprisingly, these results suggest that the fully assem-
bled CIII containing wild-type RISP is more stable than the
WT-RISP was able to restore CIII activity in KO clones only
partially (perhaps due to lower-than-normal expression from the
integrated transgene). The improvement in CIII activity ranged
activity (not shown).
Wild-type RISP is required for CI, CIV, and supercomplex
assembly/stability. The pleiotropic CI defect due to ablation of
cells. Fully assembled CI levels were increased to control levels in
KO clones transduced with the WT-RISP (Fig. 3A, NDUFA9
panel). Moreover, analysis by BNGE followed by in-gel activity
cells expressing GFP or mutant RISP. Similarly, only WT-RISP
improved the levels and activity of CIV (Fig. 3B). The increase in
CIV in-gel activity was only observed in those clones that initially
spectrophotometric measurements (not shown). The increase in
CIV was accompanied by higher steady-state levels of the CIV
subunits Cox1 and Cox4 (Fig. 3C). The levels of other subunits,
such as ATPase-? from CV or SDHA from CII, were unchanged
Analysis of reconstituted KO cells by 2D-BNGE showed that
WT-RISP restored supercomplexes. Figure 4 shows color-coded
Images of individual antibodies added sequentially to the same
membrane were superimposed. Each color reflects an antibody.
NDUFA9 (yellow); and for CIV, we used Cox1 (green). RISP KO
clones transduced with the WT-RISP lentivirus contained higher
levels of CI compared to those observed in cells transduced with
GFP lentivirus (Fig. 4, yellow spots). Moreover, CI was also de-
tection of CIV with Cox1 antibody (green spots) showed that its
levels were increased in those cells expressing WT-RISP.
RISP KO cells are able to induce HIF1-? in hypoxia. Free
radicals generated by CIII through RISP have been proposed to
stabilize the hypoxic transcription factor HIF1-? during hypoxia
by inhibiting the prolyl hydroxylase that constantly targets
provided by RISP knockdown experiments (6, 33).
The availability of our bona fide RISP knockout cells allows us
to examine the levels of HIF1-? in hypoxic conditions. We found
marked differences in the levels of HIF1-? depending on the time
in hypoxia. Figure 5A shows that, when exposed to hypoxia (1%
O2) for 24 h, the RISP KO cells were able to stabilize HIF1-?.
Moreover, the levels of the transcription factor were higher in the
RISP KO cells than in control cells (clones 8.4 and 8.5 had higher
levels than control cells, with a 4- and 2.7-fold increase, respec-
FIG 2 Ablation of RISP reduced the levels of CI, CIII, and supercomplexes. (A) Two-dimensional BNGE (2D-BNGE) and Western blot of mitochondrial
proteins. Membranes were sequentially blotted with different antibodies, and signals obtained are indicated with an arrow in each panel. NDUFS3 antibody
showed a strong unknown signal (?). Cox1 signal is circled with a white dotted line to distinguish it from the unknown signal from NDUFS3. Positions of
KO clones. (C) Western blot of VDAC1 as protein loading control.
RISP Knockout Decreases Levels of Complexes I and IV
January 2012 Volume 32 Number 2mcb.asm.org 419
tively). Additionally, HIF1-? stabilization was also induced when
cells were exposed to CoCl2, a prolyl hydroxylase inhibitor, al-
though the chemical stabilization of HIF1-? was less robust than
with hypoxia (Fig. 5A).
19.7 times depending on the clone) than that of the control cells
(Fig. 5B), in agreement with previous observations (4, 6, 12, 33).
However, unlike previous observations (4, 57), MitoQ, a mito-
the control compound dTPP (not shown). At 2.5 ?M MitoQ, we
observed a slight decrease in the levels of HIF1-? in some of the
57). The levels of HIF1-? were still detectable and only decreased
1.4- and 5.7-fold in the control and KO clone 8.4, whereas it re-
mained unchanged in the other clones (Fig. 5B). Similar results
were obtained with the reconstituted clones expressing WT-RISP
(Fig. 5C). In these cells, HIF1-? levels only decreased 1.8- and
2.9-fold in the presence of 2.5 ?M MitoQ but did not change in
the presence of 5 ?M MitoQ (Fig. 5C).
percomplexes in RISP-deficient cells. In the course of the hyp-
oxia experiments, we also tested whether the decrease in oxygen
cells to 1% O2for 24 h since that is the time frame required to
lines (Fig. 6A). In particular, the levels of CI and CIV were mark-
edly increased in the KO clone 8.5 under low-oxygen conditions.
normoxic activity (not shown).
Analysis of the steady-state levels of OXPHOS subunits
showed that they were slightly increased in the hypoxia-treated
increased 1.5- and 1.6-fold, respectively, during hypoxia, whereas
its levels were unchanged in the control cell line. The highest
changes were observed for Core2, with 4.3- and 3.5-fold the nor-
moxia levels in the KO clones 8.4 and 8.5, respectively, and a
1.9-fold increase for control cells. Likewise, the levels of SDHB in
KO clones 8.4 and 8.5 were 2.4- and 1.3-fold higher than in nor-
moxia. The levels of ATPase-? increased 1.7-fold after hypoxia
VDAC1, remained unaltered (Fig. 6B).
An increase in reactive oxygen species affects supercomplex
stability. From the results described above, we inferred that the
levels of OXPHOS complex subunits by Western blotting.
FIG 4 Expression of wild-type RISP increased levels of CI and CIV and restored
supercomplex assemblies. Two-dimensional BNGE and Western blotting were
performed to assess supercomplex assembly. Antibodies were added sequentially
ity. For identification of CIII, we used antibodies against Core2, RISP, and the
assembly factor Bcs1L; for CI, NDUFA9; and for CIV, Cox1. The positions of
Diaz et al.
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stability/assembly of respiratory complexes might be affected by
the production of free radicals. We reasoned that under low oxy-
gen levels (hypoxia), fewer free radicals should be produced, and
should improve. To test this hypothesis, we treated control cells
with different OXPHOS inhibitors (many known to produce
ROS) and determined their effects on the assembly/stability of
OXPHOS complexes and supercomplexes.
Control cells were grown in the presence of either rotenone,
antimycin A, KCN, or oligomycin (CI, CIII, CIV, and CV inhibi-
tors, respectively) for 3 days, and OXPHOS complexes analyzed
by BNGE after digitonin extraction. Treatment with oligomycin
had a profound effect on CI assembly/stability and completely
disrupted the supercomplexes containing CI (Fig. 7A, NDUFA9
panel). Analysis of steady-state levels of the respiratory complex
subunits showed that oligomycin treatment strongly affected the
also affected the levels of Core2 and Cox1 from CIII and CIV,
respectively (Fig. 7B, Cocktail panel).
Treatment with antimycin A had an effect on CI stability/as-
milder (Fig. 7A). Conversely, CI was not affected when cells were
treated with either rotenone or KCN. In fact, KCN treatment
mainly affected CIV subunit levels and, in turn, also affected su-
percomplexes containing CIV (Fig. 7A and B, respectively). Pre-
edly increase ROS generation, whereas KCN does not (44).
We further tested the effects of antimycin A and myxothiazol
on supercomplex assembly and stability since the results of phar-
macological inhibition of CIII presumably should resemble more
FIG 5 RISP is not required for HIF1-? stability during hypoxia. (A) Steady-
state levels of HIF1-? in cells exposed to either hypoxia (1% O2) or CoCl2(50
?M) for 24 h. (B) Levels of HIF1-? in cells preincubated with 2.5 ?M MitoQ
KO cells expressing wild-type RISP. Tubulin was used as loading control.
FIG 6 Hypoxia increased the levels of OXPHOS complex and supercomplex
24 h, and supercomplexes (SC) analyzed by BNGE. (B) Steady-state levels of
VDAC1 was used as loading control. LDH, lactate dehydrogenase.
RISP Knockout Decreases Levels of Complexes I and IV
January 2012 Volume 32 Number 2mcb.asm.org 421
and CIV compared to the levels in untreated cells (Fig. 7C). Sim-
the pharmacological inhibition, this association was disrupted
and most of the complex appeared in its dimeric form (Fig. 7C,
Core2 panel). Likewise, the association of CIV with other com-
plexes was also disrupted by both CIII inhibitors (Fig. 7C, Cox1
panel). The instability of CI upon CIII inhibition was also re-
to 6-fold lower levels than in untreated cells) (Fig. 7D, Cocktail
panel). Other subunits of the electron transport chain complexes
(ATPase ?, Cox1, Core2, and SDHB) or mitochondrial mem-
(Fig. 7D). Incubation with antimycin A and myxothiazol inhib-
ited CIII activity and did not have any significant effect on CIV or
citrate synthase activity measured spectrophotometrically (not
RISP-deficient cells had increased levels of free radicals. Be-
cause antimycin A and oligomycin are potent generators of reac-
tive oxygen species (ROS), we investigated whether instability/
assembly of CI, CIV, and supercomplexes in the RISP-deficient
cells was related to increased levels of free radicals in these cells.
We measured the levels of superoxide by flow cytometry with
the fluorescent probe MitoSox. Figure 8A shows that clone 8.4
contained higher levels of superoxide and clone 8.5 had only a
slight increase in the levels of mitochondrial superoxide, presum-
ably due to its lower mitochondrial membrane potential (not
shown). Oligomycin increased ROS production in all cell lines,
unknown, but it suggests that clone 8.5 produced larger amounts
levels of total cellular superoxide using another dye, dihydro-
ethidium (DHE), confirming that the RISP KO clones produced
more ROS than the parental cell line (Fig. 8B).
Antioxidants stabilized OXPHOS complexes/supercom-
plexes. To test whether the stability of OXPHOS complexes, par-
ticularly CI and supercomplexes, is affected by increased free rad-
were grown for 3 days in the presence of either a hydrogen perox-
ide scavenger, n-acetylcysteine (NAC), or with a superoxide dis-
mutase mimetic compound, MnTBAP. The results in Fig. 9A
show that NAC had a beneficial effect in CI and supercomplex
but did not stabilize supercomplexes in the RISP-deficient fibro-
ity was preserved in the RISP KO (Fig. 9A).
We also tested the effects of both antioxidants in control cells
treated with a CIII inhibitor. Low NAC concentrations (1 mM)
could not block the detrimental effect of antimycin A on CI and
supercomplex stability. At higher concentrations (10 mM), NAC
slightly protected supercomplex stability from antimycin A. Sim-
ilarly, higher concentrations of MnTBAP improved the super-
complex levels in cells treated with antimycin A (Fig. 9B).
Taken together, these results showed that OXPHOS compo-
nents, especially CI and supercomplexes, are vulnerable to in-
creased levels of ROS. It also showed that by scavenging superox-
ide (but not hydrogen peroxide), we were able to partially rescue
this instability. To confirm these results, we transduced the fibro-
blasts with lentivirus expressing mitochondrial SOD2 and ob-
tained stable cell lines. Figure 9C shows that the SOD2 expression
lentiviral transduction. This increase in SOD2 levels completely
FIG7 Pharmacological inhibition of OXPHOS complexes affected supercomplex assembly/stability. (A) BNGE and Western blot of control fibroblasts treated
with rotenone (Rot; 100 nM), antimycin A (AA; 20 nM), KCN (400 ?M), and oligomycin (Olig; 5 ?g/ml). Ctrl, untreated control. (B) Steady-state levels of
Tim23 signals were used as loading controls.
Diaz et al.
mcb.asm.orgMolecular and Cellular Biology
rescued the instability of CI and supercomplexes in the RISP-
deficient clone. An improvement of CI and supercomplex stabil-
ity/assembly was also observed in clone 8.5 but to a much lesser
extent, since SOD2 was only increased 1.3-fold after lentiviral
transduction (Fig. 9C). We also analyzed cells with a more-than-
20-fold increase in SOD2 levels, but the high levels of expression
were detrimental for cell growth and OXPHOS complexes (not
Antioxidant defenses were increased in RISP KO cells. The
production of free radicals under hypoxia remains controversial
and unresolved. Therefore, we tested whether exposure of cells to
low levels of oxygen resulted in increased free radical production
exposed to normoxia or hypoxia (1% O2) for 24 h. We were un-
hypoxia compared to the levels under normoxia (Fig. 10A). In
with tert-butyl hydroperoxide (tBOOH) produced a significant
of NAC under both normoxic and hypoxic conditions (Fig. 10A).
Using the same assay, we were unable to detect changes in the
levels of H2O2in RISP KO cells during hypoxia compared to the
normoxic levels, indicating that our cells did not produce ROS
when exposed to hypoxia (Fig. 10B).
We also examined the antioxidant defense system under
normoxia and hypoxia. The results in Fig. 10C show that the
RISP clone 8.5 had higher steady-state levels of both SOD1 and
SOD2 than the control and clone 8.4. The increased steady-
state levels of these two proteins also correlated with increased
levels in their enzymatic activity as assessed by in-gel activity
flow cytometry as described for panel A. Values from representative experiments are shown. Vertical lines indicate the mean fluorescence values for untreated
RISP Knockout Decreases Levels of Complexes I and IV
January 2012 Volume 32 Number 2mcb.asm.org 423
assays (not shown). When cells were exposed to hypoxia (1%
and clone 8.5 (0.4- and 0.6-fold decrease, respectively), al-
though in clone 8.4, the levels remained unchanged (Fig. 10C).
After hypoxia, the levels of SOD1 in the control and clone 8.4
were unchanged, whereas clone 8.5 had a slight decrease of
SOD1 levels of about 0.6-fold (Fig. 10C). Reduction of the
levels of superoxide scavenger enzymes during hypoxia sug-
gests less production of free radicals. Since SOD2 is an induc-
ible enzyme, its high levels suggest increased levels of superox-
ide, particularly in the RISP KO clone 8.5. This could explain
why we could not detect higher levels of mitochondrial super-
oxide with MitoSox in this clone (Fig. 8A). Higher levels of
superoxide dismutase could counterbalance the mitochondrial
ROS that is being produced.
The levels of GPx were significantly increased in the RISP KO
clone 8.4 compared to levels in the control cell line (Fig. 10D).
Exposure of cells to hypoxia did not alter the levels of GPx in any
of the cells tested. However, the levels of total glutathione were
significantly increased in RISP KO cells compared to the levels in
the control in normoxia, in agreement with alterations in oxida-
diminished significantly in all cell lines, suggesting a reduction in
free radical production (Fig. 10D).
FIG 9 Scavenging superoxide radicals increases CI and supercomplex stability. (A) BNGE and Western blot of cells treated with either NAC (10 mM) or
MnTBAP (100 ?M). (B) BNGE of control cells treated with antimycin A (20 nM) and the effects of different concentrations of either NAC or MnTBAP on
supercomplex (SC) stability. (C) BNGE of control and RISP KO fibroblasts infected with lentivirus expressing mitochondrial SOD2. Antibodies used are
indicated beside each panel. The levels of SOD2 were determined by SDS-PAGE. VDAC1 and Tim23 were used as protein loading controls.
Diaz et al.
mcb.asm.orgMolecular and Cellular Biology
The present study characterized the effect of the deletion of the
Rieske iron-sulfur protein on CIII assembly and showed that the
interdependence and supramolecular organization of OXPHOS
complexes are associated with the increased levels of ROS.
The complete assembly process of CIII is still under investiga-
tion, but work in yeast suggested that it starts with the formation
bly process, these three intermediates form a precomplex, pre-
be inserted (70, 71). In yeast, three chaperones, Cbp3, Cbp4, and
Bcs1, aid in the assembly process (16, 39). The addition of RISP is
catalyzed by Bcs1 in an ATP-dependent manner, and it appears
that the structural dimerization of the complex occurs before
RISP is incorporated (16). The mammalian CIII contains an ad-
is after its addition into the complex by BCS1L (human homolog
of Bcs1). Interestingly, the Core1 and Core2 subunits display
mitochondrion-processing peptidase activity (18).
bility of the fully assembled CIII and that of the one lacking RISP.
and the formation of the pre-CIII assembly intermediate take
structurally unstable, as we observed low levels of pre-CIII and
subassembly intermediates/degradation products in the RISP KO
clones. These results are in agreement with observations made in
Bcs1L patients, where little incorporation of RISP into the pre-
CIII occurs (25, 31, 36, 49).
also the levels of CI, CIV, and supercomplexes. Several possibili-
ties that could contribute to the interdependence of respiratory
complexes include the existence of specific threshold levels of re-
spiratory complexes (17), specific subunits and lipid interactions
(47, 52), or as we showed in this study, increased levels of ROS.
Mitochondria, in particular the electron transport chain
(ETC), are major generators of ROS (reviewed in reference 41).
The established sites for mitochondrial ROS production are CI,
CII, and CIII. Complexes I and II produce superoxide within the
mitochondrial matrix, whereas CIII releases the radicals into ei-
ther the matrix or the mitochondrial intermembrane space (30,
35, 41, 72).
Analysis of RISP KO clones showed increased levels of ROS
associated with destabilization of CI and supercomplexes. Com-
plex I instability has been shown previously in both human and
mouse cell lines with mutations in the cytochrome b gene that
impeded the assembly of CIII (1). Studies in NDUFS4 KO mice
containing CIII in the absence of the CI subunit NDUFS4 (9).
Our results suggest that CI stability is affected by factors other
than the “physical support” conferred by CIII to assemble into
per se is not required for CI stability, since the exposure of RISP
KO fibroblasts to hypoxia and, presumably, low ROS was suffi-
cient to increase CI to the levels observed in control cells. This
indicates that the partial assembly of CIII into the pre-CIII in the
FIG 10 Ablation of RISP leads to an increase in cellular antioxidant defenses.
(A) H2O2levels were determined by DCF-DA fluorescence in control cells
with either 100 ?M tBOOH or a mixture of 2 mM NAC and 100 ?M tBOOH
to increase or suppress ROS production. AU, arbitrary units. (B) DCF-DA
A and B represent the means and standard deviations of fluorescence in trip-
(C) Steady-state levels of the superoxide scavenger enzymes SOD1 and SOD2
in cells exposed to either normoxia or hypoxia for 24 h. (D) Total glutathione
and hypoxia for 24 h. Values represent means and standard deviations. ?, P ?
0.05 for KO cells compared to control in normoxia; ??, P ? 0.05 for hypoxia
compared to normoxia.
RISP Knockout Decreases Levels of Complexes I and IV
January 2012 Volume 32 Number 2 mcb.asm.org 425
RISP KO cells was enough to stabilize CI and supercomplex for-
mation. Unfortunately, the previous studies on CI stability men-
tioned above did not investigate the effect of increased free radi-
cals in stability/assembly (1, 9).
To test whether increased levels of ROS affected OXPHOS
complex stability, we exposed control cells to different OXPHOS
inhibitors known to increase free radicals. Studies in submito-
coenzyme Q to its reduction site in CI, permitting the release of
electrons directly to oxygen at the N2 center and forming super-
oxide radicals (24). The generation of free radicals by CIII is inti-
mately linked to its catalytic mechanism. During the Q cycle,
ubiquinol is oxidized and two electrons enter CIII in a bifurcate
fashion. According to this model, inhibition of CIII with antimy-
heme bLand does not interfere with ubiquinol binding; in this
used, CIII and CV inhibitors known to be ROS generators dis-
played the most detrimental effect on CI and supercomplex sta-
levels of CI in mouse control cells derived from the L929 line
(subcutaneous aerolar and adipose tissue) (1). However, the de-
crease that they observed in CI levels was not as severe as the one
we observed in this study. This difference could be attributed to
variations in cell type or drug treatment time. It is possible that
culture adapted to the inhibition or that the “less fit” cells were
selected against in growing cultures.
RISP is thought to be required for the production of reactive
observed in our RISP KO fibroblasts are probably generated by
showed that the levels of free radicals produced by mitochondria
from patients with Björnstad syndrome (Bcs1L mutations) and
other CIII deficiencies were increased by 50 to 80%. The authors
determined that the increased levels of H2O2detected in the pa-
tients were produced by CI and not by CIII (36). More recently,
of ROS-scavenging enzymes in fibroblasts derived from patients
with different mutations in the BCS1L gene. Remarkably, the lev-
els of ROS in these patients correlated with the severity of the
disease (49). These results support our findings of increased ROS
in the RISP KO cells.
The production of free radicals under hypoxia is still contro-
versial. In this paradox, researchers supporting hypoxia-induced
ROS contend that at low oxygen levels, the flow of electrons
through the electron transport chain slows down, increasing the
contrast, other reports showed decreased ROS during hypoxia
(23, 48, 63). This controversy might rely on the nature of the free
radical species measured. As pointed out by Poyton’s group, not
only reactive oxygen species but also reactive nitrogen species
(RNS) can be generated during hypoxia (10, 53). Low levels of
both ROS and RNS act as signaling molecules during physiologi-
cal conditions, and only excessive amounts, usually generated
during a pathological state, produce oxidative stress (34). A po-
tential reconciliation of this controversy came from the studies
measuring the oxidation state of redox-sensitive GFP (roGFP)
probes in different cellular compartments. Waypa et al. (66)
found that hypoxia increased the oxidation of roGFP in the cyto-
sol and intermembrane space, whereas it decreased oxidation in
the mitochondrial matrix (66). Decreased mitochondrial matrix
ROS supports our observations of increased OXPHOS complex
stability/assembly during hypoxia.
It has been proposed that CIII is the mitochondrial oxygen
sensor that regulates cellular responses during hypoxia and that
the ROS generated by CIII is responsible for stabilizing the tran-
scription factor HIF1-? that initiates the hypoxic signaling cas-
cade (reviewed in reference 11). This hypothesis is based in nu-
merous studies performed in different cell types, including rho0
b and cytochrome c mutants, specific OXPHOS inhibitors, mito-
chondrially targeted antioxidants, and knockdown studies (4, 6,
RISP knockdown on HIF1-? stability. Brunelle et al. (6) showed
that transient transfection of two RISP small interfering RNA se-
quences led to a reduction in the stabilization of HIF1-? in
Hek293 cells exposed to hypoxia (1.5% O2) for 2 h (6). Stable
knockdown of RISP in 143B cells showed similar results, and
when exogenous H2O2was added, hypoxic HIF1-? stabilization
was restored, suggesting that ROS is required for stability of the
transcription factor (33). Hypoxic abrogation of HIF1-? stabili-
have been reported in other cell types also. Interestingly, recent
studies implicate another CIII subunit (UQCRB) in the oxygen-
sensing role during hypoxia (37).
The use of MitoQ, a mitochondrially targeted antioxidant,
supported the hypothesis that mitochondrial ROS was required
for hypoxic signaling. Control and cytochrome b mutant cybrids
pretreated with MitoQ prior to hypoxic exposure failed to stabi-
lize HIF1-? (4). This concept has been recently challenged by
bition of the electron transport chain decreased the HIF1-? half-
inhibited and that there was no increase in ROS production dur-
ing hypoxia (14). The authors proposed that, rather than requir-
ing ROS produced by CIII, HIF1-? stability during hypoxia is
related to the intracellular concentrations of oxygen, which are
determined by the rate of mitochondrial respiration.
Similar to results of the RISP knockdown studies described
above, we were able to detect a marked decrease in the levels of
increased, reaching levels that were even higher than the ones in
control fibroblasts. Moreover, reducing mitochondrial ROS with
ity in the RISP KO cells during hypoxia may be independent of
ROS production, as has been suggested (14). The RISP KO cells
had increased levels of superoxide, and antioxidant defenses were
upregulated. Presumably, in our cells, ROS is generated by means
other than CIII. However, we did not observe an increase in hy-
drogen peroxide during hypoxia.
We were surprised to find that hypoxia also increased the sta-
bility/assembly of CI, CIV, and supercomplexes. Although the re-
sults of the experiments described above do not rule out the par-
ticipation of HIF1-? in this process, they showed that ROS is an
active mediator of complex/supercomplex stability. Interestingly,
Diaz et al.
mcb.asm.orgMolecular and Cellular Biology
there are two forms of CI: one form, called the A-form, is catalyt-
are in the reduced state. During hypoxia, a deactivation of CI was
the deactivation of CI could be a protective mechanism for a po-
tential burst of free radicals during reoxygenation (27). Unfortu-
nately, they did not investigate the stability of the CI and super-
complexes during hypoxia.
CI instability and affected the levels of supercomplexes and, con-
versely, that conditions of lower oxygen and, presumably, lower
levels of free radicals preserved supercomplexes even in
OXPHOS-defective cells prompts us to propose a possible model
for regulation of OXPHOS complex interactions into supercom-
plexes. Figure 11 illustrates this model: in wild-type cells,
OXPHOS complexes are able to form stable supercomplexes. Al-
terations in OXPHOS function can produce increased free radi-
and supercomplexes disassembled. Under conditions of low oxy-
gen or increased superoxide scavengers (SOD2 or MnTBAP), less
ROS is produced, resulting in the restoration of a safe environ-
ment for the respiratory complexes and supercomplexes to reas-
semble. In addition, supercomplex stability/assembly could be
subunits of the respiratory chain and could be mediated by
Our results also addressed a previously puzzling observation.
Mouse tissues defective in CIV (21, 22) or CIII (F. Diaz, unpub-
observed in cultured cells. However, the oxygen concentration in
tissues is 3 to 6%, which is markedly lower than the 21% concen-
speculate that increases in tissue oxygenation (physiological or
pathological) may lead to a signaling pathway that controls the
levels of CI and supercomplexes to avoid further exacerbation of
the oxidative stress, as postulated in our model.
levels at the mitochondrial inner membrane affect the assembly/
stability of CI and supercomplexes. CI appears to be particularly
sensitive to this oxidant environment effect. This system may be
physiologically relevant for the control of respiration and ROS
and Ana LaTorre for their help with flow cytometry and technical assis-
tance, to T. Wenz for insightful discussions, and to A. Barrientos and E.
Perales-Clemente for critical review of the manuscript. We also thank
Mike Murphy and Rob Smith for providing MitoQ and dTPP.
We are indebted to the following funding agencies for supporting our
work: the James and Esther King Biomedical Research Program, Florida
Department of Health, for grant 08KN-01 (F.D.), PHS for grants
NS041777, CA085700, and EY10804 (C.T.M.), and the Spanish Ministry
of Education for DGA-B55 grants SAF2009-08007 and CSD2007-00020
(J.A.E.). The CNIC is supported by the Spanish Ministry of Science and
Innovation and the Pro-CNIC Foundation.
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