The complexes of the mitochondrial respiratory chain assemble into higher-order structures called supercomplexes or respirasomes that are thought to be important in channeling electron flow and controlling ROS production. A number of recent papers identify the first protein factors necessary for supercomplex assembly and stability

Shoubridge, Eric A.

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Supersizing the Mitochondrial Respiratory Chain 

Cell Metabolism
PreviewsSupersizing the Mitochondrial Respiratory ChainEric A. Shoubridge1,*
1Department of Human Genetics, McGill University/MNI, Montreal, QC H3A 2B4, Canada
*Correspondence: eric@ericpc.mni.mcgill.ca
DOI 10.1016/j.cmet.2012.02.009
The complexes of the mitochondrial respiratory chain assemble into higher-order structures called super-
complexes or respirasomes that are thought to be important in channeling electron flow and controlling
ROS production. A number of recent papers identify the first protein factors necessary for supercomplex
assembly and stability.The oxidative phosphorylation system,
which comprises the four multimeric
enzyme complexes (CI–CIV) of the respi-
ratory chain and the ATP synthase
complex (CV), drives the synthesis of
ATP in most cells. Two mobile carriers,
ubiquinone and cytochrome c, are used
to transfer electrons from NADH, pro-
duced by the oxidation of carbohydrates
and lipids, to molecular oxygen (Figure 1).
This creates a proton electrochemical
gradient that is harnessed by CV to
synthesize ATP. Although it was initially
proposed that the process of electron
transfer occurred by random collisions
between the individual enzyme com-
plexes, many biochemical studies sug-
gested the existence of higher-order
structures, or supercomplexes, consist-
ing of two or more different respiratory
chain complexes that could channel
electron flow. With the advent of native
electrophoretic gel systems that could
separate large membrane complexes
(Scha¨gger and von Jagow, 1991), super-
complexes were shown in a wide variety
of eukaryotes, including yeast, plants,
and animals. They have been variously
hypothesized to be important in in-
creasing the efficiency of electron trans-
port, controlling the production of reactive
oxygen species (a byproduct of oxidative
phosphorylation), or stabilizing the levels
of the complexes themselves (Boekema
and Braun, 2007). However, it has been
very difficult to test these ideas, as the
factors that regulate supercomplex
stability or assembly have remained a
mystery. Using bakers’ yeast as a model
system, three independent studies have
now identified some of the protein
components that regulate supercomplex
assembly and stability (Vukotic et al.,
2012; Chen et al., 2012; Strogolova
et al., 2012).As fermentative yeasts lack a functional
CI, the only supercomplexes that have
been observed are associations of a CIII
dimer with a CIV monomer or dimer
(CIII2/CIV2 or CIII2/CIV). Using a tagged
version of a CIII structural subunit, the
Rehling group affinity purified these
supercomplexes, separated them on
native gels, and analyzed the components
by mass spectrometry (Vukotic et al.,
2012). This analysis identified two small,
uncharacterized proteins that copurified
with the supercomplexes, later named
Rcf1 (for respiratory super complex
factor 1) and Rcf2. The Stuart group was
studying the association of the adenine
nucleotide carrier (the protein that trans-
ports ATP/ADP in and out of mitochon-
dria) with the supercomplexes and
discovered Rcf1 as comigrating protein.
Affinity purification of CIII using a tagged
structural subunit confirmed the associa-
tion of Rcf1with the supercomplex (Stro-
golova et al., 2012). Finally, the Rutter
group (Chen et al., 2012) identified Rcf1
in their investigations of phylogenetically
conserved but uncharacterized mito-
chondrial proteins. They affinity purified a
tagged version of Rcf1 and, using mass
spectrometry analysis, identified several
CIII and CIV structural subunits, suggest-
ing that the protein was a component of
the supercomplex (Chen et al., 2012).
All three groups found that Rcf1 was
tightly associated with CIV, but analysis
of a panel of mutants affecting either CIII
or CIV assembly showed that it could
stably associate with either complex
alone, demonstrating that it is not a
subunit of CIV and suggesting that it likely
bridges a gap between the two com-
plexes. What does Rcf11 have to do with
the assembly of the supercomplexes, and
what happens when it is missing? The
most striking molecular phenotype inCell MetabolismRcf1-deleted cells was a severe reduction
in the level of the CIII2/CIV2 supercom-
plex. This was accompanied by a specific
decrease in CIV activity, decreased
oxygen consumption, impaired growth
on nonfermentable substrates, and an
increase in the production of reactive
oxygen species. These phenotypes were
exacerbated when both factors were
deleted together (Strogolova et al., 2012).
One might have expected a complete
dissociation of CIII and CIV and not just
a decrease in one species, were Rcf1
both necessary and sufficient for super-
complex stability, and this observation
led the Rehling group to test whether in
fact all CIV monomers were created
equal. These experiments led them to
the quite unexpected observation that
RCf1 was crucial for the incorporation of
a peripheral CIV subunit (Cox13, human
COXVIa) and that monomers lacking this
subunit could not assemble into the
CIII2/CIV2 complex. Another peripheral
CIV subunit (Cox12, human COXVIb)
was also inefficiently incorporated when
supercomplex assembly was disrupted
(Strogolova et al., 2012). Thus, an addi-
tional role for supercomplexes may be to
fine-tune the cell for growth in response
to different metabolic cues through the
assembly of different protein isoforms.
Rcf1 is a member of the hypoxia induc-
ible gene 1 (HIG1) family, and it has at
least two human homologs, HIGD1A and
HIGD2A. Human HIGD2A partially sup-
pressed the phenotype caused by dele-
tion of the yeast Rcf1 (Vukotic et al.,
2012), and siRNA-mediated knockdown
of the murine homolog of HIGD2A re-
sulted in a decrease in supercomplexes
containing CIV (Chen et al., 2012), sug-
gesting that the mammalian protein is
also important in stabilizing CIV-contain-
ing supercomplexes. The organization of15, March 7, 2012 ª2012 Elsevier Inc. 271
Matrix
NADH NAD+
½O2 H2O
IMS
½O2 H2O
Respirasome
NADH NAD+
Loss of HIGD2A
Deficient supercomplex assembly
HIGD2A
Cytc
Figure 1. Mammalian Respiratory Chain Supercomplexes
The respirasome is embedded in the inner mitochondrial membrane with components facing the mito-
chondrial matrix and the intermembrane space (IMS). It contains the mobile electron carriers ubiquinone
and cytochrome c (Cytc) necessary to transfer electrons between CI and CIII and between CIII and CIV,
respectively. It can thus catalyze all of the reactions in the respiratory chain, in which electrons donated
from NADH are ultimately used to reduce molecular oxygen. The assembly of the respirasome
(C1/CIII2/CIV) requires the activity of HIGD2A, a homolog of Rcf1, which mediates CIII2/CIV2 supercom-
plex assembly in yeast. RNAi-mediated knockdown of HIGD2A leads to loss of supercomplexes contain-
ing CIV. Under those conditions, reduced Cytc must diffuse to its binding site on free CIV. CI is colored
blue, CIII2 red, and CIV green. It seems likely that additional supercomplex assembly factors, mediating
the CI/CIII interaction, will be found in mammals.
Cell Metabolism
Previewssupercomplexes inmammals is, however,
considerably more complex than in yeast,
partly because mammals have a CI and
because the relative ratios of CI:CIII:CIV
are roughly 1:3:6 (Scha¨gger and Pfeiffer,
2001). In a variety of cell lines and tissues,
CI appears in a large supercomplex with
a CIII dimer and a CIV monomer (C1/
CIII2/CIV), the so-called respirasome
(Scha¨gger and Pfeiffer, 2001; Moreno-
Lastres et al., 2012). Respiratory active
respirasomes containing ubiquinone and
cytochrome c have been isolated (Acı´n-
Pe´rez et al., 2008), and the placement of
substrate binding sites in the structure,
determined by CryoEM, permits efficient
movement of quinones between CI and
CIII and of cytochrome c between CIII
and CIV (Althoff et al., 2011; Dudkina
et al., 2011). CI has been suggested to272 Cell Metabolism 15, March 7, 2012 ª201provide the scaffold for respirasome
assembly (Moreno-Lastres et al., 2012),
and patients with mutations in CIII and
CIV assembly factors often have reduced
amounts of CI, further indicating that the
respirasome is necessary for CI stability
(Moreno-Lastres et al., 2012). On the
other hand, substantial fractions of CIII
and CIV do not assemble into the respira-
some in human cells, but appear as
smaller complexes or individual com-
plexes, and defects in CI assembly rarely
lead to pleiotropic respiratory chain
phenotypes.
What regulates respirasome formation,
and why do some complexes apparently
not assemble as supercomplexes? Are
additional factors necessary to assemble
the mammalian respirasome? Is regula-
tion tissue specific, responding to energy2 Elsevier Inc.demand or nutrient or oxygen supply?
Five of the subunits of mammalian CIV
(COXIV, VIa, VIb, VIIa, and VIII) have
tissue-specific isoforms whose function
is not well understood, and perhaps, as
in yeast, different supercomplexes could
contain different CIV isoforms. Although
many questions remain, the identification
of the first supercomplex stability factors
will now permit manipulation of these
structures in animal models to rigorously
investigate their function in health and
disease.
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Supersizing the Mitochondrial Respiratory Chain

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