Acyl-Co dehydrogenase 9 (ACAD9) was thought to play a role in fatty acid oxidation. Nouws et al. (2010) reveal a novel and essential role for this enzyme in mitochondrial complex I assembly. A mutation in ACAD9 causes an isolated complex I deficiency in a subset of patients with mitochondrial disease

Scheffler, Immo E.

Acyl-Co dehydrogenase 9 (ACAD9) was thought to play a role in fatty acid oxidation. Nouws et al. (2010) reveal a novel and essential role for this enzyme in mitochondrial complex I assembly. A mutation in ACAD9 causes an isolated complex I deficiency in a subset of patients with mitochondrial disease

Scheffler, Immo E

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Assembling Complex I with ACAD9

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Assembling Complex I with ACAD9 

Cell Metabolism
PreviewsAssembling Complex I with ACAD9Immo E. Scheffler1,*
1Division of Biology, Molecular Biology Section, University of California, San Diego, La Jolla, CA 92093-0322, USA
*Correspondence: ischeffler@ucsd.edu
DOI 10.1016/j.cmet.2010.08.008
Acyl-Co dehydrogenase 9 (ACAD9) was thought to play a role in fatty acid oxidation. Nouws et al. (2010)
reveal a novel and essential role for this enzyme in mitochondrial complex I assembly. A mutation in
ACAD9 causes an isolated complex I deficiency in a subset of patients with mitochondrial disease.Mitochondrial diseases due to partial
deficiencies in themitochondrial oxidative
phosphorylation system have been rec-
ognized for more than 20 years, and
they constitute a substantial fraction of
metabolic defects responsible for neurop-
athies, myopathies, and related dysfunc-
tions (Wallace et al., 2010). Among
patients, those with ‘‘isolated complex I
deficiencies’’ have presented a particular
challenge because many did not carry
mutations in any of the known complex I
structural genes. The function of complex
I, or NADH-ubiquinone oxidoreductase, is
to reoxidize NADH produced from the
Krebs cycle, and it also uses the free
energy released from this oxidation to
pump protons out of the mitochondrial
matrix to establish the proton gradient
that drives ATP synthesis. The mamma-
lian complex I contains 45 subunits, of
which 38 are encoded by nuclear genes
and 7 by mitochondrialDNA (mtDNA).
The analysis of these structural genes
in a substantial number of patients with
isolated complex I deficiencies has not
revealed any obvious mutations, strongly
suggesting that factors encoded by other
genes, commonly referred to as assembly
factors (or chaperones), must be involved
in the biogenesis and assembly of a fully
functional complex I.
In this issue, Nouws et al. (2010) identify
Acyl-Co dehydrogenase 9 (ACAD9) as a
novel assembly factor critical for complex
I biogenesis and establish that a defect in
this factor is responsible for the exercise
intolerance, cardiomyopathy, lactic aci-
dosis, and related symptoms in two unre-
lated patients with isolated complex I
deficiency. ACAD9 had previously been
cloned and identified as a member of
the acyl-CoA dehydrogenase family, ex-
hibiting such enzymatic activity in vitro
(Zhang et al., 2002). One prior publication
on patients with ACAD9 mutations haddescribed a new genetic disorder in mito-
chondrial fatty acid oxidation (He et al.,
2007). Noews et al. found that, in vivo,
however, ACAD9 does not have signifi-
cant b oxidation activity or function and
instead plays an essential role in the
assembly of functional complex I.
The initial attention on ACAD9 was the
result of a successful ‘‘fishing expedition’’
using the known assembly factors
NDUFAF1 and Ecsit as ‘‘baits’’ in copreci-
pitation experiments (Vogel et al., 2007a).
The proposed interactions were con-
firmed by mitochondrial membrane frac-
tionations and colocalization of this pro-
tein with assembly intermediates (Vogel
et al., 2007a, 2007b). Knockdown by
RNA interference showed not only the
role of these proteins in complex I
assembly, but also a role for ACAD9 in
affecting NDUFAF1 and Ecsit protein
levels, with NNDUFAF1 knockdown af-
fecting ACAD9 levels. Complex formation
between these three proteins, perhaps in
combination with complex I subunits,
may therefore be required for their stabili-
zation and accumulation.
Though the participation of these
assembly factors in complex I biogenesis
is becoming well established, and their
association with characterized assembly
intermediates has been demonstrated,
much remains to be learned. Although
they are essential for assembly, they are
not present in the final active complex.
Thus, there must be amechanism for their
release and recycling. This raises another
question, as the mammalian complex I
contains 31 subunits that are referred to
as ‘‘supernumerary’’ or ‘‘accessory,’’ but
no definite function has been assigned
to any of them. They have been specu-
lated to function in assembly, in stabiliza-
tion of complex I, in its incorporation
into supercomplexes, in its activity, or
perhaps in its involvement with otherCell Metabolism 12, Smetabolic pathways. It should be kept in
mind that the distinction between an
assembly factor and a subunit may be
artificially created by the conditions used
for solubilizing the complex from intact
mitochondria.
Unexpectedly, ACAD9 is closely related
to the well-characterized very long-chain
fatty acid dehydrogenase enzyme
(VLCAD), with both being membrane
associated and localized to the mitochon-
dria. Extensive sequence homology with
VLCAD and a phylogenetic analysis sug-
gest that ACAD9 arose from a gene
duplication of the ACADVL gene (encod-
ing VLCAD) at the time of the origin of
vertebrates. From molecular modeling
and a comparison with the known crystal
structure of VLCAD, it is suggested that
sequence changes in ACAD9 are associ-
ated with two external a helices in the
ACAD9 dimer that might represent new
interaction domains important for its
recently acquired role in complex I
assembly. Further investigation should
examine whether ACAD9 has any role at
all in fatty acid metabolism in vivo.
The crystal structure of the prokaryotic
complex I, including its peripheral mem-
brane and integral membrane domains,
has recently been published, and it is fully
functional with the 14 subunits that
constitute the ‘‘core’’ of the mammalian
complex I (Efremov et al., 2010). De-
scribing a detailed function for the
assembly factors and for the other 35 sub-
units remains a future challenge. The fact
that they are essential has been demon-
strated in several cases by studies with
mutants in tissue culture (Scheffler et al.,
2004) and by the existence of patients
suffering isolated complex I deficiencies
(Potluri et al., 2009; Fernandez-Moreira
et al., 2007). The studies with fibroblasts
from patients and cell culture studies
clearly establish ACAD9’s function as aeptember 8, 2010 ª2010 Elsevier Inc. 211
Cell Metabolism
Previewscomplex I assembly factor in vertebrates;
it should be noted, however, that similar
multisubunit complex I structures exist
in most eukaryotic mitochondria, and
ACAD9 is absent in all lineages below
vertebrates.
More than 50 years ago at the dawn of
molecular biology, Salvador Luria stated
that ‘‘it is always better to have a mutant.’’
In our time, patients with genetic diseases
pose such a challenge. The successful
molecular genetic characterization of
such patients nowadays facilitates a de-
finitive diagnosis, offers the possibility of
a cure or amelioration, and provides a
solid foundation for genetic counseling.
The study by Nouws and colleagues
(2010) is also a splendid illustration of
how the analysis of patients/mutants can212 Cell Metabolism 12, September 8, 2010 ªprovide fundamental new insights into
very basic phenomena in biology, specif-
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Assembling Complex I with ACAD9

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