Tài liệu Báo cáo khoa học: The role of the ESSS protein in the assembly of a functional and stable mammalian mitochondrial complex I (NADH-ubiquinone oxidoreductase) pptx

The role of the ESSS protein in the assembly of a functional and stable
mammalian mitochondrial complex I (NADH-ubiquinone
oxidoreductase)
Prasanth Potluri, Nagendra Yadava and Immo E. Scheffler
Division of Biology, Molecular Biology Section, University of California, San Diego, California, USA
The ESSS protein is a recently identified subunit of mam-
malian mitochondrial complex I. It is a relatively small
integral membrane protein (122 amino acids) found in the
b-subco mplex. Genomic sequence database searches reveal
its localization to the X-chromosome in humans and mouse.
The ESSS cDNA from Chinese hamster cells was cloned and
shown to complement one complementation group of our
previously described m utants with a proposed X-linkage.
Sequence analyses of the ESSS cDNA in these mutants
revealed chain termination mutations. In two of these
mutants the protein i s truncated at the C-terminus of the
targeting sequence; the m utants are null mutants for the
ESSS subunit. There is no detectable complex I assembly
and a ctivity in the absence of the ESSS subunit as revealed by
blue n ative polyacrylamide gel e lectrophoresis (BN/PAGE)
analysis and polarography. Complex I activity can be re-
stored with ESSS subunits tagged with either hemagglutinin
(HA) or hexahistidine (His6) epitopes at the C-terminus.
Although, the accumulation of ESSS-HA is not dependent
upon the presence of m tDNA-encoded subunits (ND1-
6,4 L ), it is incorporated into complex I only in presence o f
compatible co mplex I subunits from the same species.
Keywords: complex I; ESSS protein; mitochondria; NADH-
ubiquinone oxidoreductase; respiration-deficient mutants.
NADH-ubiquinone oxidored uctase (complex I) is the first
enzyme in the mitochondrial electron transport chain

responsible for the oxidation of NADH. The complex I
from bovine heart is composed of 46 distinct subunits, of
which 14 have been assigned to the core complex, as
homologous subunits are f ound in the p rokaryotic complex
capable o f carrying out the same known functions: NADH
oxidation and establishment of a membrane potential by
proton translocation [1–6]. The precise role of the other 32
subunits is lar gely unknown, although some of these
(MWFE, the acyl carrier protein) have been shown to be
absolutely essential for assembly and function of the
complex [7–14].
No crystal structure is available for complex I; its overall
boot-shaped conformation has been deduced from low-
resolution electron microscopic studies [15–18]. In the
bovine complex a large subdomain is made up of  20
integral membrane proteins contributing > 60 transmem-
brane segments. Some of these must be intimately involved
in proton pumping. Another large subdomain is attached to
the membrane-subcomplex via a narrower neck-shaped
domain. This peripheral-subcomplex contains a flavin
mononucleotide and at least seven iron sulfur centers
involved in electron transport from NADH t o ubiquinone.
A major challenge is to understand h ow electron transport
is coupled to proton pumping.
Structure–function analyses of electron transport com-
plexes have in the past been advanced considerably by a
combination of biochemical and g enetic studies, largely
carried out with the bovine complex I [1,2,19–22]. C omplex
I lags behind, largely because a similar complex does not
exist in t he common yeasts Saccharomyces cerevisiae and

Schizosacchoromyces pombe. Genetic studies with Neuros-
pora crassa [11], and more recently with t he ye ast Yarrowia
lipolytica [23] and the unicellular algae Chlamydomonas
[24,25] have provided some notable insights.
Finding mutations in mammalian systems affecting
complex I has been even more o f a challenge. A systematic
investigation of human patients suffer ing from mitochond-
rial diseases has led to the characterization of human cell
lines with partial complex I deficiency. Such cell lines can be
subdivided into those with mutations in the mitochondrial
genome [26], and those with mutations in nuclear genes
[27–30].
Our laboratory has described a series of respiration
deficient Chinese hamster cell mutants with very s evere or
complete defects in complex I activity [31–34]. A genetic
analysis by somatic cell hybridization has revealed the
existence of several complementation groups, and it has
been proposed that more than one of these genes are
X-linked [35]. These early conclusions were confirmed for
one complementation group in which a defect in the
Correspondence to I. E. Scheffler, Division of Biology, Molecular
Biology Se ction, University of California, Sa n Diego, CA 92093–0322,
USA. Fax: + 1 858 5340053, Tel.: + 1 858 5342741,
E-mail: ischeffl
Abbreviations: BN/PAGE, blue native polyacrylamide gel electro-
phoresis; MBS, maleimidobenzoyl N-hydroxysuccinimide ester;
TMPD, tetramethylphenylene diamine.
(Received 1 0 March 2004, revised 10 June 2 004,
accepted 18 June 2004)
Eur. J. Biochem. 271, 3265–3273 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04260.x

X-linked NDUFA1 gene (encoding the MWFE protein) was
identified b y our laboratory [ 7,9]. Until recently i t was the
only X-linked structural gene known. An exhaustive
biochemical analysis of the composition of complex I from
bovine heart has revealed the existence of two additional
subunits (bringing the total to 46), and one of these, the
ESSS protein, is also encoded by an X-linked g ene in
humans and mouse [36]. Most of these subunits have also
been identified in the human enzyme [37]. T he present
manuscript describes the characterization of Chinese ham-
ster mutant cells from a second complementation group in
our collection in w hich the gene for the ESSS protein was
found to be mutated. The ESSS protein i s a relatively small
protein (123 amino acids in the mature form). It is predicted
to have a s ingle transmembrane helix, and it has been
purified from the integral membrane b-subcomplex [2]. As
there is no homologous protein in the prokaryotic com plex,
it had previously been grouped among the ÔancilliaryÕ
proteins, also referred to as ÔaccessoryÕ proteins. Our present
studies estab lish that the ESSS protein is another essential
subunit for assembly of an active mammalian mitochondrial
complex I.
Experimental procedures
Cell lines and cell culture
The isolation and preliminary biochemical and genetic
characterization of a series of respiration-deficient Chinese
hamster mutant cell lines has been described [7–9,32–35,38].
The CCL16-B11 mutant cell line was derived from the
CCL16-B10 cells after an additional selection in thioguanine
to select for HPRT deficiency (parental cells CCL16,

American Type Culture Collection). The V79-G8, V79-
G18 and V79-G35 cells were from a different parental cell
line, V79 (CCL93, American Type Culture C ollection).
V79-G7 cells are also respiration-deficient (res
–
)hamster
cells with almost no measurable mitochondrial protein
synthesis [39–41]. The res
–
cells grow normally in DME
medium with 4.5 mgÆmL
)1
glucose (DME-Glu) to sustain
glycolysis, and a supplement of nonessential amino acids.
Substitution of glucose with 1 mgÆmL
)1
galactose (DME-
Gal) represents the nonpermissive condition for res
–
cells
[38]. Routinely, the medium contained 10% fetal bovine
serum, and the antibiotics gentamicin and fungizone
(50 mgÆmL
)1
and 2 .5 mgÆmL
)1
, re spectively). C ells were
harvested by t rypsinization after one wash with TD buffer
(0.3% Tris, 0.8% NaCl, 0.038% KCl, 0.025% Na
2

H-
PO
4
Æ12H
2
O, brought to pH 7.4 with HCl).
Plasmids and genes
A polycistronic pTRIDENT-14 neo vector with an EF1a
promoter expressing various cDNAs i n t he first cistron has
been described [7]. This vector was further modified to allow
fusion of C-terminal HA- or HIS-epitope tags to the
encoded proteins. Unique EcoRI and NheI sites permit
directional in-frame cloning. For the present study the
complete cDNA/ORF for the ESSS protein [1,36] from
hamster was obtained as follows. Primers from the available
mouse and human cDNA sequences were used in PCR
to obtain an almost complete hamster cDNA sequence
(R. Janssen, NCMD, Nijmegen, the Netherlands). Using
specific primers for the hamster, 5¢-RACE [42,43] was
performed to obtain the complete hamster c DNA (including
the 5¢-UTR) for sequencing. Subsequently two primers were
used to amplify an ESSS coding sequence (ORF) flanked by
EcoRI and NheI sites for cloning into the unique EcoRI and
NheI sites of the m odified pTRIDENT-14neo vectors s uch
that either the HA or t he HIS epitope tag was added t o the
end of t he ESSS ORF. The forward primer was: 5¢-ACga
atccGATCTCCGACCCA-3¢; the reverse primer was:
5¢-ATgctagcCTCATCTTCTGGTAACTGG-3¢. Small bo ld
letters refer to the r estriction sites added to the oligonucleo-
tide primers for directional cloning. The same oligonucleo-

tides were u sed for RT-PCR and sequencing of ESSS cDNAs
from various mutant cell lines.
Transfections
Cells were transfected with DNA using 5–10 lL Super-
Fect reagent essentially as described [7], and according to
the manufacturer’s instructions (Qiagen). The res
–
mutan t
cells (5 · 10
5
) w ere s eeded in a six-well tissue culture plate
overnight and then transfected with the polycistronic
vector (0.5–2.0 lg). Forty-eight hours later, 800 lgÆmL
)1
geneticin (G418) was added to select stable transfectants.
After 2 weeks, visible resistant colonies were marke d on
the plate and exposed to DME-Gal. Survival and further
growth was evidence for complementation [38]. For
further analysis many surviving colonies were pooled to
represent a population in which the ESSS protein is
expressed f rom a transgene at variable positions in the
genome.
Measurement of respiratory activities
The respiratory chain activities of v arious cells were meas-
ured as described [7,44]. The cells were harvested by
trypsinization, collected by centrifugation (350 g) and resus-
pended in 1 · HSM buffer (20 m
M
Hepes, pH 7.1, 250 m
M

sucrose and 10 m
M
MgCl
2
) at a density of 2 · 10
7
cellsÆmL
)1
.
Cells were permeabilized by digitonin (100 lgÆmL
)1
)for
 5minat4°C, the cell suspension was diluted 10 fold with
HSM buffer, and the cells were harvested by centrifugation.
Subsequently, after one wash, cells were resuspended at
3 · 10
7
cellsÆmL
)1
. The total protein content was measured
by Bradford microassay, and  1 mg of cell s uspension was
used per assay. Oxygen consumption was measured polaro-
graphically with a Clark oxyge n electrode in metabolic
chamber with a water jacket maintained at 37 °C(Hansa-
tech, Norfolk, UK). Substrates, inhibitors, etc. could be
added via a capillary opening using microsyringes as
described previously [7].
Isolation of mitochondria and mitochondrial fractions
Mitochondria were isolated from cells essentially according
to [45]. Approximately 1 · 10

9
cells were washed twice with
TD buffer and harvested by t rypsinization. The pellets were
suspended in 5 mL SM buffer (50 m
M
Tris/HCl, pH 7.4,
0.25
M
sucrose, 2 m
M
EDTA) and homogenized using
a tightly fitting Dounce h omogenizer (30–35 up/down
strokes). The homogenate was centrifuged twice at 625 g fo r
3266 P. Potluri et al. (Eur. J. Biochem. 271) Ó FEBS 2004
10 min a t 4 °C in order to remove unbroken cells and
nuclei. The supernatant was centrifuged at 10 000 g for
20 min at 4 °C. The mitochondrial pellet w as s uspended in
0.1 mL of the SM buffer. This fraction is designated as the
mitochondrial fraction.
Immunochemical assays and antibodies
Mitochondrial protein samples (between 50 and 100 lg)
were separated by SDS/PAGE and BN/PAGE and trans-
ferred t o I mmobilon-P (0.2 l) membranes. Anti-HA and
anti-porin sera were used at 1 : 5000 dilution whereas the
anti-MWFE and anti-18 kDa sera were used at 1 : 1000
dilution. Horseradish peroxidase-conjugated secondary
antibodies (anti-rabbit or anti-mouse) were used at
1 : 5000 dilution, and signals o n the immunoblots were
detected using an Enhanced Chemiluminiscence system
(ECL+ Plus from Ame rsham).

The anti-MWFE serum was developed as described
previously [7]. B. Ackrell (University of California, San
Francisco, CA, USA) provided antiserum against the
SDHC sub unit o f c omplex II. Sources of other antibodies
were as follows: anti-porin from Calbiochem, anti-HA from
Covance BabCo, anti-mouse and anti-rabbit secondary
antibodies from Bio-Rad Laboratories and Amersham
Pharmacia Biotech, respectively. Antibodies against the
Rieske protein, PSST, and 18 kDa were purchased from
Molecular Probes (Eugene, OR, USA).
Blue native polyacrylamide gel electrophoresis
(BN/PAGE)
Mitochondrial respiratory complexes were separated by
BN/PAGE essentially as described [46]. Mitochondrial
pellets equivalent to 400 lg of protein were solubilized
with 800 lg of dodecyl-b-
D
-maltoside (Sigma) in 5 m
M
6-aminohexanoic acid, 50 m
M
imidazole/HCl (pH 7.0),
50 m
M
NaCl, and 10% g lycerol. To the solubilize d samples
Coomassie Brilliant Blue G-250 (Serva) was added at a dye/
detergent ratio of 1 : 5 (w/w). A 4–13% acrylamide gradient
gel was used for electrophoresis.
The NADH dehydrogenase assay w as carried out as
described [7,47]. Gel slices were incubated at room tem-

perature in 2 m
M
Tris/HCl (pH 7 .4), 0.1 mgÆmL
)1
of
NADH and 2.5 mgÆmL
)1
of nitroblue tetrazolium (Sigma)
for 2–4 h.
Other reagents
All other reagents were of the highest grade available.
Results
Identification of ESSS as essential accessory subunit
Three complementation groups of complex I-deficient
Chinese hamster cell mutants had been characterized and
X-linkage of the corresponding genes had been established
intwoandsuspectedinthethird[33,35].WhentheESSS
protein was added to the list of complex I s ubunits, a nd its
gene was localized on the X chromosome in mammals, it
became a candidate for t he mutated gene in one of the two
unidentified complementation groups.
The c onstruction of the di-cistronic vector expressing
hamster ESSS with either HA or HIS epitope tags at the
C-terminus is described in Experimental procedures. The
mutant cell lines V79-G8 (group II), and V79-G18 (group
III) were transfected with these vectors, and stable colonies
were selected over a period of  2 weeks in D ME-Glu
medium containing 800 lgÆmL
)1
G418. Several colonies

were marked on th e bottom of the plate and tested for their
ability to grow/survive after a shift to DME-Gal medium. In
parallel, a selection was also performed directly in DME-
Gal. In contrast to the original mutant cells, t he transfected
cells from group III, but not from group II were able to
proliferate under conditions where the rate of glycolysis is
severely reduced, and respiration (oxidative phosphoryla-
tion) becomes essential for survival. The results clearly
established t hat ESSS cDNA can complement the muta-
tions in the cell line V79-G18 but not V79-G8. Furthermore,
HA or HIS e pitope tags at the C-terminus did not interfere
seriously with the ability of the ESSS protein to complement
the growth in DME-GAL medium. Many colonies were
pooled for the subsequent experiments.
Characterization of other mutants within same
complementation group
To characterize the independently isolated mutations within
the same complementation group (group III), we sequenced
the c orresponding ESSS cDNAs from w ild type (GenBank
accession number AY649405), a nd from each of the three
mutant cell lines, CCL16-B11, V79-G18, V79-G35. They
were amplified by RT-PCR using primers from the 5¢-and
3¢-untranslated regions and sequenced directly in both
directions. Each of the mutants was found to have a
premature chain-termination codon within the open reading
frame. In two of the mutants (CCL16-B11 and V79-G18)
the predicted protein is truncated at a position very close to
the end of the s ignal sequence; the third mutant allele (G35)
encodes a truncated protein m issing 25 amino acid residues
from the C-terminus (Fig. 1). Two of these mutants are

Fig. 1 . Sequences of Chinese hamster cDNA a nd wild-type Chinese
hamster ESSS precursor protein. (A) C omplete sequence of t he Chinese
hamster c DNA, with the open reading frame indicated in capital l etters
(GenBank accession number AY649405). (B) The sequence of the
wild-type Chinese hamster ESSS precursor protein, with the signal
sequence and a p r oposed c leavage site ba sed o n the se quen ce of the
mature bovine ESSS protein. The truncated proteins in the three
Chinese hamster mutant cell lines CLL16-B11, V79-G18, V79-G35 are
also indicated.
Ó FEBS 2004 Mammalian cells with severe complex I deficiency (Eur. J. Biochem. 271) 3267
therefore effectively null mutants, with no residual, recog-
nizable ESSS protein expected.
The ESSS protein is found in the b-subcomplex and is
predictedtobeanintegralmembraneproteinwithasingle
transmembrane segment [2,36]. From a comparison with
the sequence o f the mature bo vine protein the hamster
protein has a mitochondrial targeting sequence of 29
residues that is removed, presumably by the metallo-
protease in the matrix. The mature hamster protein has
122 r esidues of which 55 at the N -terminus are predicted to
form a domain on the matrix side, and 36 form a domain
extending into the intermembrane space. In the third
mutant (V79-G35) one might expect a protein to be inserted
into the inner membrane, but it is missing a major portion
of the domain localized in the intermembrane space. A
comparison of all the known mammalian ESSS sequences is
presented in Fig. 2. The protein is highly conserved,
especially near the C-terminus. The sequences in bold
represent the predicted transmembrane domain.
Analysis of complex I assembly and activity

The first step in the analysis was to analyze mitochondria by
SDS/PAGE. Mitochondrial extracts from mutant cells
(V79-G18), a nd wild-type and mutant cells stably transfect-
ed with the complementing ESSS-HA were fractionated.
Western blots were used to show the presence of the epitope
tagged ESSS, and two other complex I subunits (MWFE
and the PSST) in the mitochondria. As s hown in Fig. 3A,
the m utant mitochondrial extract has no ESSS-HA (as
expected), and no signal for the MWFE and PSST subunits.
We have described previously, that the MWFE subunit is
Fig. 2. Sequence alignments of mammalian ESSS proteins. (A) Pre-
dicted mature protein sequences based on the bovine protein. The
predicted transmembrane se quence i s indicated in bo ld. ( B) Putative
mitochondrial targeting presequences.
Fig. 3. Results from SDS/PAGE, Western blot and BN/PAGE ana-
lyses. (A) SDS/PAGE and Western analysis of mitochondria from
wild-type cells, V79-G18 mutants, and the same mutan t stably trans-
fected with the d i-cistronic vector e xpressing hamster ESS S-HA. The
blots were probed with antisera against HA, with anti-porin, and with
two other antisera against complex I proteins (MWFE and PSST).
(B) BN/PAGE. Top panel: histochemical assay for NADH oxidation
with n itroblu e tetrazolium as electron acceptor. Bottom p anel: West-
ern analysis w ith anti-HA Ig, anti-NDUFB6 (complex I ), anti-Rieske
protein ( complex III), and a nti-SDHC (complex II).
3268 P. Potluri et al. (Eur. J. Biochem. 271) Ó FEBS 2004
accumulated only when a stable complex I is formed [7,48].
Clearly, expressing ESSS-HA in the mutant cells restores the
MWFE and PSST signals. A similar r esult was observed
with ESSS-His
6

, or when the mutant was complemented
with wild-type hamster ESSS without a tag.
Next, mitochondria from wild-type, m utant and comple-
mented mutant cells were solubilized by sodium dodecyl b-
D
maltoside (DDM) a nd protein complexes were fractionated
by Blue Native gel electrophoresis. T he ESSS -HA was also
expressed in wild-type cells, i.e. in the presence of the
endogenous ESSS protein. The gels were first used in a
histochemical assay which detects the reduction of nitroblue
tetrazolium dye by NADH (Fig. 3 B, left panel). No activity
was detectable in extracts from mutant cells, while the
complemented mutant extracts clearly showed activity at
the position of the wild-type complex ( 900 kDa). Com-
plex I activity was restored, but the levels appeared to be
somewhat variable from different complemented cells and
even from experiment to experiment. We did not see any
reproducible NADH-NBT oxidoreductase activity in the
mutant lane at positions that would correspond to partially
assembled complex I. A relatively strong signal seen half
way down the gel was intriguing, but subsequent Western
blotting with available antisera [anti-51 kDa, anti-TYKY,
anti-30 kDa, anti-18 kDa (NDUFB6)] failed to r eveal the
presence of any c omplex I-specific subunits at that position.
We believe that the band may represent a nonspecific
NADH dehydrogenase activity. Complex I I activity could
be measur ed on the same gels using the same electron
acceptor with succinate as the substrate (results not shown).
The gels w ere also u sed i n a Western analysis with antisera
against th e HA epitopes. It is noteworthy that the epitope

tags do not interfere s ignificantly w ith the incorporation o f
the tagged ESSS subunit into a functional complex I
(Fig. 3 B, right panel). The signal from the 18 kDa subunit
of complex I (NDUFB6) served as a nother identification of
the complex at the position o f the histochemical stain in the
left panel. Antisera against the SDHC subunit of complex II
and against the Rieske protein of complex III revealed the
presence of these complexes in all cells (Fig. 3B, right panel).
Rates of respiration were determined in wild-type parental
cells (V79-G3), in w ild-type cells express ing ESSS-HA, i n the
V79-G18, CCL16-B11 and in V79-G35 mutant cells com-
plemented with the hamster ESSS, or with HA- or HIS-
tagged hamster ESSS. Complex I activity was measured as
the malate/glutamate-induced, rotenone-sensitive activity,
and th e activity of the downstream portions of the e lectron
transport chain was established with succinate and glycerol-
3-phosphate as substrates. Complex II activity was deter-
mined after addition of succinate, followed by i nhibition by
malonate, and complex III activity was measured after the
addition of exces s glycerol 3-phosphate followed by addition
of antimycin. A typical set of traces from the oxygen
electrode is shown in Fig. 4A, and t he results a re summarized
in Fig. 4B. Consistent with the observations with BN/
PAGE, complex I activity was restored by ESSS, ESSS-HA,
ESSS-His
6
, but the activity was lower than that in wild-type
mitochondria, especially in the case of the HA tag. It is
possible t hat the HA-tagged subunit, wh ile functional, does
not function as well as the native ESSS protein. It is the only

subunit present in the complemented null mutants. In
transfected wild-type cells the ESSS-HA protein competes
with the endogenous ESSS protein, but the fraction o f
complex I with the modified subunit has lower activity. At
this point it is not yet completely confirmed that the epitope
tags exert a negative effect on assembly or fun ction of
complex I. The ESSS protein without the epitope tag
was subsequently also expressed in the mutant cells, and
activity was restored, but not quite to the level of the parental
cells (Fig. 4B). The C-terminal domain is q uite short
(36 residues) and it is likely that it interacts with other
hydrophilic domains of surrounding integral membrane
subunits in the b-subcomplex. Thus, the addition of these
charged epitope tags may constitute a measurable perturba-
tion. HA is less charged than His
6
, but a precise quantitative
difference between these two tags remains t o be established.
We believe that such discrepancies, especially with the
untagged E SSS, are due to clon al variations that have been
observed in a different context in the past [7]. The cells are
tumor cells subject to variations in gene expression, and it is
still unclear how the level of a complex of 46 subunits is
determined.
The activities of complex II and the downstream complex
III of the electron transport chain were measured and found
to be near normal in the V79-G18 mutant and various
transfected derivatives. Similar results were observed for the
mutants V 79-G35 a nd CCL16-B11 (results not shown).
They originated from two distinct Chinese hamster parental

cell lines. Curiously, when succinate and glycerol 3-phos-
phate were added t ogether, the cyanide-sensitive respiration
rates were somewhat lower in the mutant cells, and partially
restored in complemented cells. It is t empting to spe culate
about the formation of supercomplexes and the effect of
the absence of intact complex I, but the results are too
preliminary in this regard.
Heterologous expression of the ESSS-HA subunit
The localization of the ESSS subunit in the b-subcomplex of
the integral membrane domain suggests that ESSS may
interact with one or more of th e mitochondrially encoded
subunits ND1-6, and ND4L. Furthermore, such inter-
actions are quite species-specific and affect the s tability of
the protein as shown by the behavior of the MWFE subunit
[7]. The polycistronic vector allowed expression of an
epitope-tagged ESSS in various cells, including the hetero-
logous human HT1080 cells. After transfection, stable
HT1080 cells were sele cted in G418 for two weeks. When
mitochondria from such cells were analyzed by SDS/PAGE
and Western analysis, the HA-tagged hamster ESSS protein
was found at high abundance (Fig. 5A), in contrast to our
previous results with hamster MWFE.HA in the same
human cells. It appears that the heterologous ESSS is stable
and accumulated to a significant level. Mitochondria from
the same cells were also analyzed by BN/PAGE. No
hamster ESSS-HA could be detected in the band corres-
ponding to complex I ( 900 kDa); the same band had
NADH dehydrogenase a ctivity with NBT as electron
acceptor ( Fig. 5B, left panel), and other complex I proteins
such as MWFE could be localized at the same position. The

heterologous hamster ESSS is excluded from the human
complex I just as the hamster MWFE is excluded [7].
However, in contrast to the unassembled and unstable
MWFE protein, the unassembled E SSS protein seems to be
Ó FEBS 2004 Mammalian cells with severe complex I deficiency (Eur. J. Biochem. 271) 3269
stable, and it is found in a diffuse series of bands (500–
800 kDa) b y BN/PAGE (Fig. 5B, lane 2, right panel). The
expression of the heterologous ESSS-HA did not affect the
assembly of the native complex I, i.e. it did not act as a
Ôpo ison subunitÕ. The diffuse bands may represent a mixture
of partially assembled-subcomplexes or breakdown prod-
ucts of an unstable-subcomplex. T his result prompted us t o
express ESSS-HA in all the respiration-deficient hamster
mutant cells, including V79-G7 in which no mitochondrial
protein synthesis takes place, and all the ND subunits are
missing. In all of these mutants ESSS-HA is still accumu-
lated to near normal levels (Fig. 6). This behavior contrasts
strongly with that of the MWFE subunit. It is possible t hat
the ESSS-HA subunit is stable in isolation, but there are
indications that ESSS-HA interacts with at least one other
nuclear-encoded subunit. Cross-linking studies (P. Potluri,
unpublished data) reveal that in all cells examined ESSS-HA
can be c ross-linked by MBS to another u nidentified protein
to yield a new species migrating with a mobility o f a
 35 kDa protein. This includes wild-type hamster cells
expressing ESSS-HA from the transgene, V 79-G18 cells in
which ESSS-HA restores complex I activity, the various
hamster m utant cell lines (V79-G8, V79-G7, CCL16-B2),
and significantly, the human HT1080 cells in which hamster
ESSS-HA is expressed a nd found in a series of-subcom-

plexes.
Partial complex I assembly in different respiration
deficient mutants of Chinese hamster cells
We have investigated the presence or absence of several
known subunits of complex I in several representatives of
three complementation groups with mutations in X-linked
genes [33,35]. Two of the genes have now been identified,
and for the third group (mutants V79-G8, V79-G4) the gene
is still unknown. There is at this time no other known
subunit in complex I encoded by an X-linked gene. It is
possible that these mutants are missing an assembly
Fig. 4. Rates of oxygen consumption in cells; activities were normalized with respect to total cellular protein concentrations. (A) Rates of oxygen
consumption in ce lls permeabilized by digitonin were determined by pol arography. Arrows on the side of the tracings represent the following
consecutive add itions: ( a) glutamate/ malate; (b ) rotenone ; (c) succ inat e; (d) malon ate; (e) glycerol 3-phosp hate; (f) ant imycin; (g) T MPD-ascorbate
and (h) cyanide. (Details are g ive n in Experimental procedures.) (B) The activities were n ormalized with respect to t otal cellular protein concen-
trations. The activity of wild-type cells was set at 100%. The asterisk indicates activity indistinguishable from background. T he results r epresentthe
averageofaminimumoffourexperiments.
3270 P. Potluri et al. (Eur. J. Biochem. 271) Ó FEBS 2004
factor that is only transiently involved in the biogenesis of
complex I. For comparison, the mutant V79-G7 defective in
mitochondrial protein synthesis is also included. Table 1
lists the subunits that can be detected by Western blotting
after SDS/PAGE with isolated mitochondria from these
mutant cells. The subunits in the peripheral-subcomplex k
as defined by Hirst et al. [2] appear to be present with the
exception of the PSST subunit, found in the CCL16-B2
mutant, but not in the o thers. Two i ntegral membrane
proteins in the integral membrane-subcomplex b [2] could
be monitored. The B17 protein was found in all mutants,
while the ESSS subunit was absent only in the V79-G18

mutant where the gene is mutated. Such a result may have
been unexpected in the V79-G7 mutant, suggesting that
these subunits (ESSS and B17) can be accumulated in a
stable form in the absence of any of the mitochondrially
encoded ND s ubunits. The most variable behavior is
exhibited by the MWFE subun it, localized in the c-sub-
complex that has been proposed to comprise the connecting
domain between the peripheral-subcomplex k and the
integral membrane-subcomplex b [2]. The MWFE subunit
is apparently unstable in the absence of any of the ND
subunits (V79-G7), or in the absence of the ESSS subunit
(V79-G18). Strikingly, the PSST subunit is also unstable in
absence o f ESSS subun it, although t hese two subunits have
been localized in different s ubdomains of the c omplex. This
suggests an interaction b etween these subdomains that is
facilitated by the ESSS sub unit.
Discussion
A novel series of Chinese hamster cell m utants in a single
complementation group with a complete defect in the
NADH-ubiquinone oxidoreductase (complex I) is des-
cribed. The mutations have been identified i n the X-linked
gene encoding the ESSS protein, a subunit that was recently
added to the list of c omplex I s ubunits [1,36]. T he subunit is
an integral membrane protein outside of the group of ÔcoreÕ
proteins common to prokaryotes and eukaryotes. It is
shown here that the ESSS protein is another essential
protein for the formation of a functional complex I in
mammals. The null mutants can be complemented with
ESSS proteins epitope-tagged (HA or HIS) at the
C-terminus, although it is possible that the epitopes interfere

slightly with either the assembly or the activity of the
enzyme.
The epitope-tagged proteins c an be expressed in a wild-
type background. In a homologous background the ESSS-
HA protein can compete with the endogenous ESSS for
incorporation into the complex, where it may have a slight
effect on activity. In a heterologous background the protein
is expressed a nd accumulated in mitochondria, but the
hamster ESSS-HA protein i s not assembled into the human
complex I. Inspection of the amino acid sequences of the
known mammalian ESSS proteins reveals a high degree of
conservation in the C-terminal domain (including the
transmembrane region), but a significant number of differ-
ences in the N-terminal domain (located on the matrix side).
It is likely that the N-terminal domain is involved in
protein–protein interactions with other hydrophilic domains
of neighboring integral membrane subunits, and interspecies
interactions are incompatible.
From the co mparative studies with a series of Chinese
hamster cell mutants defective in complex I activity two
preliminary conclusions emerge: (a) In the absence of one or
more integral membrane subunits the majority of the
subunits in the peripheral-subcomplex are accumulated in
a stable form, and m ost likely already associated in a
heteropolymeric-subcomplex. We also found that these
subunits are in e very case associated with the membrane
fraction of sonicated mitochondria (see also, [9]), but this
Fig. 5. Heterologous expression of hamster ESSS-HA in human
HT1080 cells. Stable, transfected cells were analyzed, and the hamster
protein w as found i n human mitoc hondria (SDS/PAGE; A), but not in

the active c omplex I (BN/PAGE; B ). Lane 1 was loaded with solu bi-
lized mitochondria (equivalent to 50 lg) from untransfected cells, lane
2 had mitochondria from the transfected cells. Left panel: the bands
represent anti-MWFE Ig bound t o complex I; the two lowe r bands
represent complex II and its dimer, detected by antiserum agai nst the
SDHC subunit. Right panel: the same blot probed with anti-HA Ig
detecting ESSS-HA.
Fig. 6. Expression of ESSS-HA in a series of c omplex I-deficient Chi-
nese hamster cell lines. F or a description of t hese mu tants see Experi-
mental procedures.
Table 1. Western analysis of mitochondria from respiration-deficient
Chinese hamster mutants with available antisera against complex I
proteins l.
Mutants
k (a) c (a) b
51
kDa
30
kDa TYKY PSST B8
39
kDa MWFE ESSS B17
WT +++ + +++ + +
B2 +++ + ++–– + +
G7 +++ – ++ – + +
G8 +++ – +++ + +
G18 + + + – + + – – – +
Ó FEBS 2004 Mammalian cells with severe complex I deficiency (Eur. J. Biochem. 271) 3271
association appears to be weak, as it does not survive the
conditions for solubilization used for blue native gel
electrophoresis. The PSST subunit (purified with the

k-subcomplex [2]) is absent in three of the mutants. Its
localization at or near the membrane may explain why its
stability and accumulation depends on one or more integral
membrane proteins that are m issing in the V79-G7, V79-G8,
and V79-G18 mutants. It must still be determined whether
its absence in the V79-G18 mutant is the result of the missing
ESSS subunit alone, or whether the absence of ESSS causes
the f ailure of other integral membrane subunits to accumu-
late or assemble properly. (b) Integal membrane subunits
such as the MWFE subunit may not accumulate because of
rapid turnover when the assembly of the integral membrane-
subcomplex is prevented, either in the absence of a single
crucial subunit (e.g. ESSS, or ND4, or ND6, or in the
absence o f a ll ND subunits (V79-G7) [7]). I n other words, the
synthesis, assembly, and accumulation of integral me mbrane
subunits are i ntegrated a nd interdependent process. On the
other hand, when hamster ESSS-HA is expressed i n human
cells, it is relatively stable, even though it is not assembled in
the mature complex. It may be protected by incorporation
into precomplexes that then fail to go further because of the
presence of the h eterologous subunit (Fig. 5A). The identi-
fication of precomplexes in mitochondria of human patients
has b een claimed [49], although t he observed-subcomplexes
could also have resulted from the dissociation of the intact
complex I with mutated s ubunits during the solubilization
for blue native gel electrophoresis. It remains to be seen
whether the 20 subunits of the integral membrane-
subcomplex also assemble via the formation of distinct
and i dentifiable assembly intermediates.
The mutants promise to be valuable tools in the

elucidation of the assembly and function of the integral
membrane-subcomplex. In the future, the consequences of
highly specific amino acid changes introduced by site-
directed mutagenesis can also be examined.
Acknowledgements
This research was supported by grants from the US Public Health
Service (GM59909) and by t he Muscular Dystrophy Association to
I. E. S.
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Ó FEBS 2004 Mammalian cells with severe complex I deficiency (Eur. J. Biochem. 271) 3273