Human mitochondrial complex I assembly: A dynamic and versatile process
Rutger O. Vogel, Jan A.M. Smeitink⁎, Leo G.J. Nijtmans
Nijmegen Centre for Mitochondrial Disorders, Department of Pediatrics, Radboud University Nijmegen Medical Centre,
Geert Grooteplein 10, PO BOX 9101, 6500 HB, Nijmegen, The Netherlands
Received 28 June 2007; received in revised form 24 July 2007; accepted 26 July 2007
Available online 9 August 2007
One can but admire the intricate way in which biomolecular structures are formed and cooperate to allow proper cellular function. A prominent
example of such intricacy is the assembly of the five inner membrane embedded enzymatic complexes of the mitochondrial oxidative
phosphorylation (OXPHOS) system, which involves the stepwise combination of N80 subunits and prosthetic groups encoded by both the
mitochondrial and nuclear genomes. This review will focus on the assembly of the most complicated OXPHOS structure: complex I (NADH:
ubiquinone oxidoreductase, EC 22.214.171.124). Recent studies into complex I assembly in human cells have resulted in several models elucidating a thus
far enigmatic process. In this review, special attention will be given to the overlap between the various assembly models proposed in different
organisms. Complex I being a complicated structure, its assembly must be prone to some form of coordination. This is where chaperone proteins
come into play, some of which may relate complex I assembly to processes such as apoptosis and even immunity.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Mitochondria; Oxidative phosphorylation; NADH:ubiquinone oxidoreductase; Complex I; Assembly; Chaperones
1.1. Complex I function
The cornerstone of cellular energy production is the mito-
chondrial oxidative phosphorylation (OXPHOS) system. It
produces energy via the concerted action of five membrane
embedded enzyme complexes, ultimately coupling oxidation of
substrate molecules NADH and FADH2to phosphorylation of
ADP to ATP. The largest of the five OXPHOS complexes is
complex I (NADH:ubiquinone oxidoreductase; EC 126.96.36.199;
hereafter referred to as CI). This complex binds and oxidizes
NADH to free electrons via a noncovalently bound flavine
mononucleotide (FMN). The electrons are subsequently
transferred via a cascade of up to nine iron–sulfur clusters to
electron acceptor ubiquinone and then through the OXPHOS
system to reduce molecular oxygen at complex IV. The energy
released during this process is used to drive proton translocation
across the mitochondrial inner membrane. Hence, together with
the proton translocation at complexes III and IV, a proton
gradient is formed which is used by complex V, an ATP
synthase, to generate ATP from ADP and inorganic phosphate.
The redox action of CI can be summarized in the following
overall reaction scheme [1,2]:
How electron transfer is coupled to proton translocation is
subject to debate [3,4]. It could be directly coupled via close
proximity of the ubiquinone binding site to proton translocation,
or indirectly via conformational changes of the enzyme complex.
Recent data obtained for bacterial and Yarrowia lipolytica CI
provide support for the latter option [5–9]. In addition, CI is
eukaryotes depending on temperature, pH and the presence of
bivalent cations [10–14].
1.2. Complex I structure
Complex I is L-shaped, consisting of two perpendicular arms:
a hydrophobic membrane arm which resides in the mitochondrial
Biochimica et Biophysica Acta 1767 (2007) 1215–1227
⁎Corresponding author. Tel.: +31 24 3619470; fax: +31 24 3616428.
E-mail address: J.Smeitink@cukz.umcn.nl (J.A.M. Smeitink).
0005-2728/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
protrudes into the mitochondrial matrix [15–17] (see Fig. 1).
Three-dimensional electron microscopy of Y. lipolytica CI re-
cently demonstrated detailed contours and protrusions of the
dynamic structure . At a higher resolution, the crystal struc-
ture of the peripheral arm of Thermus thermophylus CI recently
elucidated the exact arrangement of the iron–sulfur clusters
within the complex [8,19,20].
Bacterial CI consists of the 14 most conserved subunits and
is considered to be the ‘mimimal’ structure required for
functionality of the enzyme. A typical example of bacterial CI
is Escherichia coli NDH-1 [22,23]. By analogy to its core
structure, three functional modules can be distinguished for
human CI. The first is the dehydrogenase module, which is
responsible for the oxidation of NADH and consists of at least
the NDUFV2, NDUFV1 and NDUFS1 subunits (homologues
of the nuoE, F and G subunits of bacterial NDH-1) (see Table 1
for an overview). The second is the hydrogenase module,
which guides the released electrons to electron acceptor
ubiquinone and consists of at least the NDUFS2, NDUFS3,
NDUFS7 and NDUFS8 subunits (homologues of the nuoD, C,
B and I subunits of NDH-1). Finally, the third is the proton
translocation or transporter module, which consists of at least
the ND1, ND2, ND3, ND4, ND4L, ND5 and ND6 subunits
(homologues of the nuoH, N, A, M, K, L and J subunits of
Currently, 45 subunits have been described for human CI, an
addition of 31 supernumerary subunits to the functional “core”
structure of 14 subunits. Their topology was inferred by frac-
N-dimethyldodecylamine N-oxide [24–26] (see Fig. 1). The
function of most of the supernumerary subunits is yet unclear.
damage inflicted by reactive oxygen species (ROS). Further-
more, at least several of these subunits may have an additional
One such a function is in apoptosis. For example, the
NDUFA13 (GRIM-19, or Gene associated with Retinoid-IFN
induced Mortality in bovine CI) subunit is also a cell death
regulatory protein induced by interferon-beta and retinoic acid
and was demonstrated to be released from the mitochondrion
upon apoptosis [27–29]. Recently, GRIM-19 was shown to
associate with the pro-apoptotic serine protease HtrA2 to
promote cell death . An apoptotic function is also described
for subunit NDUFS1, as caspase mediated cleavage of this
subunit is a requirement for the mitochondrial changes asso-
ciated with apoptosis .
Another is a function in fatty acid biosynthesis. The Neur-
ospora crassa and bovine SDAP subunits (homologues of the
human NDUFAB1 subunit) are closely related to the acyl-
carrier proteins involved in bacterial fatty acid biosynthesis
[32–35]. Interestingly, disruption of the gene in N. crassa
resulted in a 4-fold increase in the amount of lysophospholipids
in the mitochondrial membranes, suggestive of a function for
this subunit in lysophospholipid recycling [36,37]. Whether the
N. crassa situation also applies to other organisms is subject to
debate, as recent studies for bovine heart and Arabidopsis
thaliana mitochondria show that most of the of mitochondrial
acyl-carrier protein is present in the mitochondrial matrix and
not associated to CI [35,38].
For several other subunits some features are known, but the
exact additional function is yet unclear. The NDUFA9 subunit
is known to harbour a NADH/NADPH binding site [39–42].
The importance of this binding site for CI stability was
recently demonstrated by mutagenesis of the Y. lipolytica
homologue, which resulted in CI destabilization, presumably
by destabilization of the structural fold of the subunit .
Fig. 1. CI subunit topology (adapted from ). CI is an L-shaped enzyme complex that can be dissected into several fragments, Iα, Iβ, Ik, and Iγ. The composition of
these fragments allows a basic arrangement of the 45 subunits that comprise CI.
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The NDUFS4 subunit was described to be phosphorylated by
a cAMP-dependent protein kinase (PKA), possibly indicating a
function in regulation of CI activity [44–46]. However, this
later proved to be the ESSS subunit (NDUFB11 in humans)
. Additional studies demonstrated phosphorylation of the
bovine homologues of the NDUFC2, NDUFA1, NDUFA7,
NDUFA10 and GRIM-19 subunits [47–50]. Based on the
phylogenetic distribution of different CI subunit orthologs, the
NDUFA2 and NDUFA10 subunits were shown to belong to a
L43 and S25, respectively . Finally, the NDUFA11 subunit
was found to be paralogous to the TIM17/22 family of proteins
2. Complex I assembly
Homology searches have revealed a high degree of
conservation ofcertain modulesofthecomplexbetweenvarious
organisms. Archaeal, cyanobacterial, bacterial and mammalian
CI share great structural resemblance, although the electron
input device (NADH dehydrogenase in mammalian CI) varies.
Such phylogenetic studies have led to models describing the
modular evolution of CI [22,53–57]. In these models, CI is
proposed to have originated by fusion of pre-existing protein
assemblies constituting modules for electron transfer and proton
transport. In more detail, CI is proposed to have originated from
an ancestral soluble nickel–iron hydrogenase (sharing homol-
ogy with the NDUFS2 and NDUFS7 subunits). This hydrog-
enase has gained a quinone binding site and has become
membrane bound upon acquisition of a protein of unknown
function (NDUFS3) and ferrodoxin-type (NDUFS8), ion
translocating (ND5) and quinone-binding (ND1) subunits.
This structure was subsequently expanded by triplication of
proton translocating subunits (ND2 and ND4). After the
complex has lost its nickel–iron active site and its ability to
react with molecular hydrogen, finally, membrane subunits
(ND3, ND4L and ND6) and the NADH dehydrogenase module
(NDUFS1, NDUFV1 and NDUFV2) are acquired.
It has been suggested that the co-evolutionary structural rela-
tionship between CI subunits may be reflected by the order of
assembly and composition of assembly intermediates [58,59]. If
so, how does the current model for human CI assembly relate to
groups of subunits is partially mirrored by the human assembly
system, would one not expect to see similarities in other orga-
A useful starting point for the comparison between CI
assembly mechanisms in different organisms is the structure of
is that the basic framework for the CI assembly process in all
organisms is represented by the combination of its most
conserved structural components. Through the years, the
assembly process of CI has been investigated either indirectly
in mutants or directly using conditional assembly systems, in
various organisms such as bacteria (E. coli, Paracoccus
denitrificans), the green alga Chlamydomonas reinhardtii, the
fungus N. crassa, higher plants (Zea mays, Nicotiana sylvestris,
A. thaliana) and mammals (Cricetulus griseus, Homo sapiens).
Fig. 2B summarizes the assembly models proposed for these
organisms, simplified by only showing assembly of the 14
‘minimal’ CI subunits. Details of each assembly scheme are
given below, always referring to the NDH-1 homologue of each
2.1. Complex I assembly in bacteria
In E. coli CI assembly, incorporation of the NuoE, F and G
subunits (forming the NADH dehydrogenase module) requires
the presence of NuoB, C and D (forming the hydrogenase
module) . It is yet unclear whether this combination takes
place before or after the addition of membrane arm subunits.
Regarding membrane arm assembly, certain point mutations in
the NuoH subunit of E. coli and P. denitrificans CI result in
severely disturbed assembly . In addition, disruption of the
NuoJ gene did not result in disturbed assembly, suggesting that
this membrane subunit is added to the complex at a final stage
. Finally,the distal location of the NuoM and NuoL subunits
makes it likely that these subunits are added at a late stage in
2.2. Complex I assembly in C. reinhardtii
Another organism in which CI assembly has been studied is
C. reinhardtii. In this organism, frameshift mutations for ND1/
ND6 resulted in a failure to detect the 950-kDa holo-CI [65,66].
Using the same strategy, absence of ND4 or ND4/5 resulted in
accumulation of a 700-kDa subcomplex. Consequently, it was
proposed that, as opposed to the ND1 and ND6 subunits, the
ND4 and ND5 subunits (NuoM and L homologues) are incor-
porated at a late stage in assembly . Recent investigation of
the role of ND3 and ND4L (NuoA and K homologues) in
assembly resulted in the first assembly model for this organism,
CI subunit nomenclature
CI subunit Fraction Module
E. coli H. sapiens B. taurus
This table lists the nomenclature, distribution after fractionation (bovine CI, see
also Fig. 1), and allocation in the three functional modules of the 14 most
conserved CI subunits from Escherichia coli, Homo sapiens and Bos taurus.
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in which a nuclear encoded precomplex of 200 kDa, containing
the 76-kDa (NuoG homologue) and 49 kDa (NuoD homologue)
subunits, is membrane anchored by combining with ND1 (NuoH
homologue), ND3, ND4L and ND6 (NuoJ homologue) and
subsequently expanded to result in holo-CI with the addition of
ND4 and ND5 .
Fig. 2. Schematic representation of existing CI assembly models. (A) Topology of the bacterial NDH-1 subunits. Bacterial NDH-1 is composed of the 14 most
conserved CI subunits, which are termed NuoA-N (see also Table 1). (B) Assembly of the basic building blocks by analogy to bacterial NDH-1 shown for bacteria
(E. coli, P. denitrificans), C. reinhardtti, N. crassa, higher plants (Z. mays, N. sylvestris, A. thaliana), Chinese hamster (C. griseus) and human (H. sapiens). (C) A
consensus assembly model can be extracted in which a core of nuclear DNA-encoded subunits is expanded to form a large peripheral arm intermediate. This
subcomplexis then anchored to the mitochondrial inner membrane and expanded to form fully assembled CI. It is unclear via which stepsthe peripheral arm is formed.
For details see text.
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2.3. Complex I assembly in N. crassa
Complex I assembly in the fungus N. crassa has been tho-
roughly studied by systematic introduction of mutations in CI
genes (for extensive reviews see [41,58,68–71]). In this orga-
nism, peripheral and membrane assembly intermediates are
formed independently . In turn, the membrane arm of
N. crassa CI is assembled from a large and small intermediate
[41,58,68–71]. The small intermediate contains the ND2 and
ND5 subunits (NuoN and NuoL homologues), whereas the
largeintermediate contains ND1, 3, 4, 4L and 6 (NuoH, A, M, K
and J homologues) [58,73]. Generally, mutations in subunits of
the one arm do not adversely affect the assembly of the other
[72,74,75]. Exceptions to this rule are loss of the acyl carrier
protein (NDUFAB1 or SDAP homologue), which disturbs for-
mation of both peripheral and membrane arms, and loss of the
11.5-kDa protein (NDUFS5 homologue), which results in accu-
mulation of membrane arm intermediates and failure to detect
an active peripheral arm [36,37,76]. Altogether, these studies
have resulted in one of the first models of CI assembly in a
2.4. Complex I assembly in higher plants
Insights into the assembly of higher plant mitochondrial CI
were mostly provided with mutant studies in maize (Z. mays),
tobacco (N. sylvestris) and Arabidopsis (A. thaliana). In addi-
tion to NAD1–6 and NAD4L, the plant mitochondrial genome
encodes NAD7 and NAD9, which are homologues of the NuoD
(NDUFS2) and NuoC (NDUFS3) subunits, respectively . In
the maize NCS2 (nonchromosomal stripe 2) mutant, the nad4
and nad7 genes are fused and the nad4 gene lacks the fourth
exon resulting in severe impairment of CI activity . Blue-
native analysis demonstrated the occurrence of a loosely mem-
brane bound subcomplex, which displays in-gel activity and
contains at least peripheral arm subunits 51 kDa (NuoF homo-
logue), 75 kDa (NuoG homologue), 40 kDa (NDUFA9 homo-
logue), NAD7 and NAD9 . In addition, proteins with sizes
of membrane arm subunits NAD1, 2, 3, 4 and 4L were not
detected. Therefore, it seems that absence of NAD4 (NuoM
homologue) results in the assembly of a loosely membrane
bound peripheral arm of CI. The tobacco mutant NMS1 dis-
plays a splicing defect of the first nad4 intron . Although
mitochondrial genes are unaffected and transcription seems
normal (except for nad4), numerous polypeptides (NAD7,
NAD9, 23 kDa (NuoI) and 38 kDa (NDUFA9) subunits) are
present only in trace amounts, demonstrating the importance of
NAD4 for the stability of these proteins. Immuno-purification of
CI resulted in a near-completely assembled CI, albeit in only 1–
20% of the wild-type amount. This subcomplex contains at least
NAD7 and NAD9, in line with what was found for the maize
NCS2 mutant. In the tobacco CMSI and CMSII (cytoplasmic
male sterile I and II) mutants, the NAD7 subunit is absent due to
a large mitochondrial deletion including the last two exons of the
nad7 gene (and the upstreamregionofnad1) [81–83]. CMSIand
CMSII mutants display similar alterations in phenotype. In the
CMSII mutant, besides NAD7, also mitochondrial subunits
NAD1 (NuoH homologue), NAD9 and nuclear DNA-encoded
subunits 23 kDa (NuoI) and 38 kDa (NDUFA9) are missing or
present in very low amounts [83–85]. NAD9-purification in the
CMSII mutant did demonstrate low abundant (10%) presence of
the nonintegrated subunit is subject to proteolysis. This does not
seem to occur for NAD2 (NuoN homologue) and NAD3 (NuoA
homologue), which contrary to NAD1 were detectable in the
mutant . Complementation of the defect by nuclear
expression of NAD7 in the CMSII mutant results in restoration
of CI assembly . It thus appears that NAD7 plays a key role
peripheral and membrane arm assembly in tobacco. A final
mutant concerns the Arabidopsis deletion mutant of the gamma
carbonic anhydrase gene . The anhydrase was demonstrated
be present in purifications of CI [87,88] as an integral membrane
protein facing the matrix side part of the CI membrane arm .
Knockout of the protein reduces CI and CI/CIII supercomplex
levels with about 80% . The 20% remaining CI seems to be
assembled without the anhydrase, suggesting a putative
facilitating or stabilizing role during assembly. The exact
function of the anhydrase is unclear and physiological evidence
of its carbonic anhydrase activity could not be demonstrated, but
a role in pH regulation is proposed .
2.5. Complex I assembly in mammals
Although (bovine heart) CI stability, subunit composition
and topology have been extensively studied using various
methods to fractionate the complex, the assembly process of
mammalian CI has long remained enigmatic [24,25,54,90–96].
The use of compounds that inhibit either mitochondrial or
nuclear translation and assembly studies in several rodent and
human ND-subunit mutant cell lines have demonstrated that
subassemblies of nuclear DNA-encoded CI subunits could be
formed in the absence of mtDNA-encoded subunits [97–100].
These findings had the important implication that CI subunits
are not incorporated during assembly one by one (in a
sequential manner), but that discrete assembly intermediates
consisting of several subunits occur which are combined during
assembly (in a semi-sequential manner). A useful model system
for subsequent mammalian CI assembly studies proved to be C.
griseus (Chinese hamster) [101–106]. By using (inducible)
complementation of the MWFE and ESSS subunits (homologues
of the human CI NDUFA1 and NDUFB11 subunits) it was
demonstrated that the stability of Chinese hamster homologues of
peripheral arm subunits NDUFS1, 2, 3, 7, 8 and NDUFV1, 2
(NuoB, C, D, E, F, G, I homologues) was unaffected by the
absence of MWFE, although holo-CI was not assembled. These
data strongly suggest that the peripheral arm can be assembled
without the presence of a membrane arm, analogous to assembly
in N. crassa and in higher plants. Furthermore, incorporation of
MWFE is proposed to require membrane arm subunits and the
subunit may serve as a membrane anchor to which membrane
subunits are attached during CI assembly. Likewise, the ESSS
subunit was shown only to be incorporated into CI when
membrane subunits are available .
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2.6. Complex I assembly in humans
Initial investigations for CI assembly in humans and other
higher eukaryotes have mainly contributed data concerning the
requirement of mtDNA-encoded subunits . Immunoprecip-
itation studies in an ND4 cybrid cell line demonstrated that the
membrane arm was not assembled when ND4 is disrupted .
Further investigations demonstrated that cells lacking either ND4
or ND5 display no in-gel CI activity, and immunoprecipitation of
NDUFS3 revealed a subcomplex consisting of at least the
NDUFS2, NDUFS3 and NDUFS8 subunits . This shows
that the absence of ND4 or ND5 still allows the formation of a
peripheral arm subcomplex. Other studies underline the impor-
tance of ND5 for CI stability and/or activity rather than for
assembly [99,109]. The requirement of ND6 for assembly was
demonstrated in a mouse ND6 frameshift mutant cell line ,
later confirmed for human CI assembly in a CI deficient patient
does not lead to a great disturbance of assembly [111,112]
whereas ND2 disruption results in disturbed assembly with accu-
a severe reduction of fully assembled CI .
Valuable information about the assembly of peripheral arm
subunits has predominantly come from assembly studies in CI
deficient patients. In 2001, Triepels and colleagues classified CI
deficient patients by their assembly profiles by comparison of
role for subunits NDUFA9 and NDUFS3 . In two patients
harbouring a mutation in the NDUFS6 subunit, CI assembly was
severely impaired and accumulation of a large 750 kDa sub-
addition, mutations in subunits NDUFS1, NDUFS4 and
NDUFV1 lead to accumulation of a subcomplex slightly smaller
than the fully assembled complex [117–120]. Whether this sub-
the (near) absence of CI activity and the hypothesized co-loca-
their mutation may result in similar assembly/stability defects.
Recently, Ogilvie and colleagues demonstrated association of
assembly chaperone B17.2L with this complex and performed
chaperone to identify associated proteins in wild type mitochon-
dria . Although the 830-kDa intermediate was not detected
in wild type mitochondria, co-elution was observed of the ND1,
NDUFS1–4, NDUFV1 and NDUFA13 subunits, suggesting the
existence of (an) assembly intermediate(s) with this composition.
implicated in disturbed assembly and inability to detect a 20-kDa
subunit (presumably the NDUFB8 subunit) in the holo-complex
An attempt to coherently incorporate the available assembly
data into a model was first made in 2003, when Antonicka and
colleagues published their human CI assembly model, based on
the occurrence of CI subcomplexes in a cohort of CI deficient
patients. Differentgenetic defects resultinginCIdeficiency
can result in the accumulation of similar subcomplexes, which
were argued to be illustrative of assembly intermediates. In the
model, membrane and peripheral arm assembly does not occur
anchoring of preformed nuclear DNA-encoded scaffold of sub-
ND1. Addition of another peripheral fragment containing at least
NDUFS4, NDUFS7 and NDUFV2 precedes completion of the
membrane arm and hence of holo-CI. This model provided a
useful framework for future assembly studies, although the ob-
served subcomplexes were derived from CI deficient patient cell
assembly, disturbed assembly, CI instability or degradation. In
2004, this study was followed by another based on a conditional
assembly system . In this system, the dynamics of assembly
intermediate formation could be followed in time by removing a
block in mitochondrial translation. It appeared that assembly
occurs via combination and expansion of two parallel lines of
assembly: that of the peripheral arm (via assembly of NDUFS2,3
and 4, NDUFA9 and NDUFV2) and that of the membrane arm
(via assembly of at least ND1, ND6 and B17.2L). Although this
still have pre-formed, it allowed studying of the dynamics of
subcomplex formation in time. In contrast to the 2003 study, it
showed several similarities with the N. crassa model, in that
membrane and peripheral arms could seemingly be assembled
independently via distinct substructures.
In an additional study in 2007, NDUFS3 containing sub-
complexes were made visible at high resolution using leakage
expression of an inducible NDUFS3-GFP expression system in
HEK293cells.Itconfirmedseveral ofthe detectedsubcomplexes
and demonstrated the presumable entry-point of mitochondrial
DNA-encoded subunits into the assembly process . In the
peripheral arm assembly was no longer as black and white as
described in the previous study and for N. crassa. In fact, apart
from the absence of NDUFA9 in early assembly intermediates,
colleagues. In both studies, an early peripheral arm assembly
intermediate containing NDUFS2 and NDUFS3 is membrane
anchored by ND1 prior to expansion with additional membrane
arm and peripheral arm assembly modules. Recent confirmation
of this sequence of events is provided by Lazarou and colleagues
NDUFS2 and NDUFS7 is membrane anchored to a subcomplex
ND subunits are part of the membrane anchor to which the
preformed peripheral arm fragment is attached. A smaller sub-
complex containing at least contain ND1 is detected, to which
ND2, 3 and 6 are presumably added. Upon assembly of both
with membrane arm subunits NDUFA9, NDUFA10 and
NDUFB8 and the dehydrogenase part is added with subunits
NDUFV1,2,3, NDUFS4 and NDUFS6.
To summarize, four models have been described for human
CI assembly [113,123–125]. Although different at points, the
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models agree in that a peripheral arm scaffold containing at least
subunits NDUFS2 and NDUFS3 is anchored to the membrane
by at least subunit ND1 prior to addition of remaining mem-
brane subunits and remaining hydrogenase and NADH dehy-
drogenase module subunits (Fig. 3). In Fig. 3, the depicted 400/
500 kDa membrane bound intermediates most likely also
contain subunits NDUFS1, NDUFS7 and NDUFS8, and may
also contain subunits ND2, ND3 and ND6. Added to the 800/
850 kDa intermediate most likely are also NDUFV1, NDUFV3
and NDUFS6. However, the exact allocation of these subunits
differs between the models, and for clarity purposes only the
subcomplexes and subunits are shown which consistently appear
at the same stages.
2.7. A general concept for complex I assembly
The investigated organisms are part of different evolutionary
lineages and assembly of either the membrane or peripheral CI
the other. Furthermore, methods to detect membrane arm hydro-
phobicsubunitsemployed so far are not sufficient,and the rateof
proteolysis (owing to specific proteases in various organisms) or
the kineticsof assembly/disassemblyof individualsubcomplexes
in different organisms may not be the same.
Nevertheless, a general concept can be extracted (Fig. 2C).In
this generalized model, a nuclear scaffold containing at least the
NuoC and NuoD subunits forms the starting point of peripheral
arm assembly. Addition of NuoB, I, E, F and G results in a large
peripheral arm subcomplex, which is anchored to the mitochon-
drial inner membrane via several of its transporter subunits (at
least NuoH). Subsequently, this membrane anchored peripheral
complex is expanded with additional membrane subunits to
theoretical data predicting subunit topology by evolutionary
conservation except in that the NuoE, F and G subunits are not
incorporated at the final stage of assembly.
The detailed human CI assembly pathway shown in Fig. 3 is
basically similar to the consensus model for assembly in other
organisms in Fig. 2C in the sense that a nuclear DNA-encoded
scaffold is attached to the membrane prior to further expansion.
Contrastingly, in the human CI assembly model, at least several
subunits of the NADH dehydrogenase module are added at a
final stage of assembly. This makes the human model even more
compatible with the modular evolution scheme than the con-
sensus model for other organisms.
2.8. Complex I assembly, future challenges
Altogether, these studies have set the basic outline of CI
stoichiometry were left mostly uninvestigated. The most recent
assembly studies support that assembly is not necessarily a static
process in which subassemblies are sequentially combined, but
rathera dynamic process inwhichsubunitsorsubcomplexesmay
berecycled duringassembly. Forexample, the originof one early
assembly intermediate is rather enigmatic, as it appears both after
breakdown and during assembly (subcomplex 1, ). In addi-
NDUFS3 appear in an equal ratio during assembly, suggesting
that the formation of these subcomplexes is tightly linked (sub-
complexes 2 and 3, ). Recently, Lazarou and colleagues
CI are incorporated into CI much faster than mtDNA-encoded
subunits , confirmative of what was observed for rat hepa-
toma cell lines by Hall and Hare in 1990 . Interestingly, none
of the radiolabeled nuclear DNA-encoded CI subunits seemed
limiting for assembly, which led to the proposal that newly
synthesized nuclear DNA-encoded subunits may be exchanged
with existing (incorporated) subunits. The possibility of subunit
exchange during assembly is an interesting subject for future
assemblystudies, in which turnover rates of subunits and kinetics
of the assembly process should be investigated.
The latter study addressed another interesting topic: does the
formation of individual OXPHOS complex occur prior to super-
complex formation or not? Supercomplexes are combinations of
OXPHOS complexes I,III and IV,observedafter mildpurification
colleagues demonstrated that assembly chaperone B17.2L associ-
ates to an 830-kDa intermediate of CIand to a combination of this
intermediate with a complex III dimer . B17.2L is then
released from this partially assembled supercomplex intermediate,
allowing CI assembly to be completed. Although this demands
further experimentation, the possibility that supercomplex assem-
bly occurs in conjunction with assembly of the individual
OXPHOS complexes is very interesting. It may help in explaining
Fig. 3. Generalized model for human complex I assembly. A basic assembly scheme is constructed based on existing models for human CI assembly [113,123–125].
Subunits and subcomplex sizes are denoted only when consistently observed throughout the studies. For details see text.
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why the stability of one complex in the supercomplex depends on
the stability of the other and subsequently how instability of one
OXPHOS complex leads a combined OXPHOS deficiency [131–
Although several assembly intermediates were observed for
nuclear-DNA encoded subunits during human CI assembly, clear
small and large membrane arm subcomplexessuchas thosefound
studies report the existence of subcomplexes containing multiple
ND subunits [125,136]. Further investigation of these and other
membrane arm intermediates is required for confirmation and
characterization of the key step in which a preformed nuclear
Mba1 that link mitochondrial translation to the membrane
insertionofmtDNA-encoded CI subunits is a promising endeavor
[137,138]. In addition, one can imagine that both iron–sulfur
cluster containing preformed subassemblies and hydrophobic
membrane subassemblies would require chaperones for stabiliza-
tion and combination withoutaggregationor productionofradical
species. An ongoing search for these chaperones is required for a
better understanding of the assembly process.
3. Coordination of the assembly process
To assemble a fully functional human mitochondrial CI is to
combine 38 subunits encoded by the nuclear genome and seven
subunits encoded by the mitochondrial genome. In addition to just
the combination of these subunits, it encompasses nuclear and
membrane insertion, stabilization and activation, includingnumer-
ous feedback mechanisms required to coordinate the process.
When viewed from this perspective, the requirement of chaperone
proteins for coordination of the CI assembly process becomes
3.1. The classical definition of a chaperone
when several genes, conserved from bacteria to mammals, were
found activated upon transientheat stressinDrosophila, encoding
the later termed heat-shock proteins (HSPs) [139,140]. Later, a
transiting through the ER prior to their assembly into macromo-
(fora reviewsee).Ingeneral,a chaperone is anyproteinthat
binds to an unfolded or partially folded target protein to prevent
facilitate the target protein's proper folding. A more detailed
definition was proposed by John Ellis:
molecular chaperones are currently defined infunctional terms as
a class of unrelated families of protein that assist the correct non-
covalent assembly of other polypeptide-containing structures in
when they are performing their normal biological functions. The
term assembly in this definition embraces not only the folding of
newly synthesized polypeptides and any association into
oligomers that may occur, but also includes any changes in the
degree of either folding or association that may take place when
proteins carry out their functions, are transported across
membranes, or are repaired or destroyed after stresses such as
heat shock [144–147].
3.2. Human CI chaperones
In 1998, Kuffner and colleagues used essentially the same
definition  to term Complex I Intermediate Associated pro-
teins CIA30 and CIA84 chaperones for N. crassa CI assembly
. Disruption of the 21.3-kDa nuclear DNA-encoded subunit
(homologue of the human NDUFS8 subunit) resulted in accu-
mulation of a large membrane arm intermediate, to which CIA30
and CIA84 were found associated. Metabolic labeling experi-
ments demonstrated that CIA84 cycles between a bound and
unbound state to this intermediate. Additionally, knockout of
the cia genes resulted in a membrane arm subunit knockout
phenotype.Inconclusion, the authors statedthatthe CIA proteins
component of the final functional structure, hence fitting the
definition of chaperone .
Both human orthologues of the CIA proteins have been found.
CIA84 orthologue PTCD1 was identified in a bioinformatics
screen, but its function in human CI assembly remains
uninvestigated . CIA30 orthologue NDUFAF1 was found in
humans in 2002 and, in line with what is described for N. crassa,
has animportantrole inhumanCIassembly .Itsknockdown
usingRNA interference resulted inimpairedCI assembly/stability
. NDUFAF1 associates to CI subunits NDUFB6, NDUFA6,
NDUFA9, ND1, NDUFS3 and NDUFS7 . In addition,
mtDNA-encoded subunits revealed transient association to
subunits ND1, ND2 and ND3. In the same paper, the first patient
withamutationinthe NDUFAF1gene was identifiedanddemon-
strated to be specifically CI deficient, a defect that was comple-
together, these data strongly suggest that NDUFAF1 transiently
associates to early intermediates of CI assembly to aid the
assembly process. It is yet uncertain whether its function in CI
assembly is represented by its association to the 500–850 kDa
the presence of NDUFAF1 in these complexes .
A paralogue of CI subunit B17.2, termed B17.2-like
(B17.2L), was predicted and confirmed to be a chaperone for
human CI assembly in 2005 [51,119]. In line with the above
description of a chaperone it is required for CI assembly but not
part of the final structure. The protein is visible in a subcomplex
of about 830 kDa, especially upon mutation of the NDUFV1 or
NDUFS4 subunits of CI, but also in low amounts in control skin
fibroblasts as demonstrated by incorporation of radiolabeled
B17.2L . With the use of immunoprecipitation, B17.2L
was found associated to CI subunits ND1, NDUFS2, NDUFS3,
NDUFV1, NDUFV2, NDUFS4 and GRIM-19 .
1222R.O. Vogel et al. / Biochimica et Biophysica Acta 1767 (2007) 1215–1227
The most recently identified protein demonstrated to be
essential for CI assembly is Ecsit (Evolutionarily Conserved
Signaling Intermediate in Toll pathways) . Ecsit is pre-
dominantly cytosolic, but a small amount is recruited to the
mitochondrion via its N-terminal targeting sequence. Once
imported, this mitochondrial Ecsit is incorporated into the same
three high-molecular weight chaperone complexes of 500–
850 kDa as NDUFAF1. Although only a relatively small
amount is mitochondrial, as demonstrated by siRNA knock-
down, Ecsit is required for stable mitochondrial presence of
NDUFAF1, CI assembly/stability and normal mitochondrial
physiology . Hence, being required for CI assembly but
not a component of the final functional structure, also Ecsit fits
the definition of a chaperone.
3.3. Complex I assembly chaperones, versatile proteins
Only a few chaperones found for an approximately 1 MDa
enzyme complex is a rather meagre score. By analogy to the
other OXPHOS complexes, many more must surely exist, but
why have they not been detected? One obvious answer is that
the Saccharomyces cerevisiae toolbox of genetics that has
proven so fruitful for studying e.g. CIV assembly is not avail-
able for CI, as it does not have CI. Secondly, many yet undis-
covered chaperones that can associate to CI assembly
intermediates (such as B17.2L) may not be detectable in their
high-molecular weight associations under normal circum-
stances as their binding is transient. For example, the CIA
proteins in N. crassa were only found after introduction of a
mutation that resulted in accumulation of a large membrane arm
assembly intermediate .Finally, chaperones may function in
additional processes rather than being confined to a function in
CI assembly,which makes them harder to find from a CI-limited
That assembly chaperones may have functions additional to
their requirement for CI assembly is in line with the growing
awareness that mitochondria are more than just a powerhouse
that provides ATP: they are plastic organelles, entangled with
various (sub) cellular processes such as the cell cycle, apoptosis
and development, via signaling cascades that include kinases
and phosphatases that ultimately regulate mitochondrial meta-
bolic activity .
One example of such functional versatility is provided with
B17.2L. This protein was initially described as Mimitin, the so-
was shown to be directly stimulated by c-myc and its levels were
found elevated in esophageal squamous cell carcinoma (ESCC)
tumors. As its suppression using RNA interference led to
decreased cell proliferation in several tissue types, a role in
c-myc mediated cell proliferation was proposed. It was shortly
later that Ogilvie and colleagues described its requirement for
human CI assembly .
Another is Apoptosis Inducing Factor (AIF). Loss of this
protein in mice results in specifically impaired CI assembly/
stability [155,156]. The AIF protein is known as a signaling
molecule in apoptosis and has an N-terminal mitochondrial
localization sequence. Upon apoptosis-induced mitochondrial
outer membrane permeabilisation it translocates to the nucleus,
chaperoned by HSP70. Once in the nucleus, it performs a role in
chromatin condensation . Loss of AIF leads to increased
ROS production and AIF knockdown desensitizes different cell
types to different apoptotic stimulants. In addition to this role in
apoptosis, AIF knockout results in a drop in the mouse homolo-
gues of CI subunits NDUFA9, NDUFB6, NDUFS7 and
GRIM19, CI activity and embryonic lethality, demonstrating
its requirement for CI integrity . Whether the effect of AIF
is confined to OXPHOS complex CI alone is debated, as its S.
cerevisiae homologue knockout exhibit reduced growth on non-
fermentable carbon sources, and siRNA for AIF additionally
resulted in a slight CIII defect. Whichever the exact mechanism,
a protein such as AIF shows that a protein can be a signaling
molecule in apoptosis and be specifically required for CI as-
sembly or stability. Such chaperones may represent signaling
nodes between various subcellular processes and the assembly
of the OXPHOS system.
A promising final example is the Ecsit protein . This
signaling protein in the (cytosolic) Toll-pathway mediates com-
munication between ligand binding at the plasma membrane to
activation of transcription of pro-inflammatory genes
[158,159]. The dual function of this protein in both mitochon-
drial CI assembly and (cytosolic) immunity is intriguing.
Although yet speculative, Ecsit may extend the cascade of the
immune response to the inner-mitochondrial level, e.g. to
control the amount of ATP production or to induce apoptosis
4. Closing remarks
Fed by recent discoveries, CI assembly is no longer seen as
an obligate-unidirectional pathway but rather as a highly dyna-
mic process with multiple entry-points in which existing and
newly synthesized subunits can be exchanged. Furthermore,
there is increasing support for the idea that a fully assembled
and stable CI is a key player in various immune and apoptotic
pathways. Characterization of existing and novel chaperones
and regulatory proteins may reveal additional connections bet-
ween CI assembly/stability and mitochondrial and cellular
function, with which CI assembly studies have gained a new
and exciting dimension. Altogether, CI assembly not only pro-
ves to be a dynamic but also a versatile process. Hopefully, new
insights into the process will aid the understanding of the broad
spectrum of clinical phenotypes associated with mitochondrial
(grant number OP05-04) and the European Community's sixth
Framework Programme for Research, Priority 1 “Life sciences,
genomics and biotechnology for health” (contract number
LSHMCT-2004-503116). The investigations were (in part) sup-
with the financial aid from the Netherlands Organization for
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