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

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[email protected]
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

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

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

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).

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

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

, 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

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

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-

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

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

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.
References
1. Carroll, J., Fearnley, I.M., Shannon,R.J.,Hirst,J.&Walker,J.E.
(2003) Analysis of the subunit composition of complex I from
bovine heart mitochondria. Mol. Cell Proteomics 2, 117–126.
2. Hirst, J., Carroll, J., Fearnley, I.M., S hannon, R.J. & W alker, J.E.
(2003) The nu clear encoded subunits of complex I from bovine
heart mitochondria. Biochim. Biophys. Acta 1604, 135–150.
3. Yagi, T. & Matsuno-Yagi, A. (2003) The proton-translocating
NADH-quinone oxidoreductase in the respiratory chain: th e
secret u nlocked. Biochemistry 42, 2 266–2274.
4. Matsuno-Yagi, A. & Yagi, T. (2001) Introduction: Complex I – an
l-shaped black box. J. Bioenerg. Biomembr. 33, 155–157.
5. Yagi, T ., Se o, B.B.,DiB ernardo, S.,Nakamaru-Ogiso, E.,K ao,M
C. & M atsuno-Yagi, A. (20 01) N ADH dehydrogenases: from basic
science to biomedicine. J. Bioenerg. Biomembr. 33, 233–242.
6. Friedrich, T. & S cheide, D. (2000) The r espiratory complex I of
bacteria, archea and eucharya and its module common with
membrane bound multisubunit hydrogenases. FEBS Lett. 479,
1–5.
7. Yadava, N., Potluri, P., Smith, E., Bisevac, A. & Scheffler, I.E.
(2002) Species-specific a nd m utant MWFE proteins. Their e ffect
on the assembly of a functional mammalian mitochondrial com-

mitochondrial NA DH: ubiq uinone o xidoreductase (complex I).
J. Mol. Bio l. 265, 4 09–418.
16. Videira, A. (1998) Complex I from the fungus Neurospora crassa.
Biochim. Biophys. Acta 1364, 89–100.
17. Finel, M. (1998) O rganization a nd evolution of s tructural elements
within complex I . Biochim. Biophys. Acta 1364, 112–121.
18. Bottcher, B., Scheide, D., Hesterberg, M., Nagel-Steger, L. &
Friedrich, T. (2002) A novel, enzymatically active conformation of
the Escherichia coli NADH: ubiquinone oxidoreductase (complex
I). J. Biol. Chem. 277, 17790–17977.
19. Sazanov,L.A.,Peak-Chew,S.Y.,Fearnley,I.M.&Walker,J.E.
(2000) Resolution of the membrane d omain of b ovine complex I
into-subcomplexes: implications for the structural organization of
the enzyme. B i oche mistr y 39, 7229–7235.
20. Walker, J.E., Skehel, J.M. & Buchanan, S.K. (1995) Structural
analysis of NADH: ubiquinone oxido reductase from bo vine heart
mitochondria. Methods Enzymol. 260, 14–3 4.
21. Pilkington, S .J., Arizmendi, J.M.,Fearnley,I.M.,Runswick,M.J.,
Skehel, J.M. & Walker, J.E. (1993) Structural organizat ion of
complex I from bovine mitochondria. Biochem. Soc. Trans. 21 ,
26–31.
22. Walker, J.E. ( 1992) The NADH: ubiquinone oxidoreductase
(complex I) of respiratory c hains. Q Rev. B iophysics 25, 253–324.
23. Kerscher, S., Drose, S., Z wicker, K., Zickermann, V. & Brandt, U.
(2002) Yarrowia lipolytica, a yeast genetic system to study
mitochondrial complex I. Biochim. Biophys. Acta 15 55,83.
24. van Lis, R., Atteia, A., Mendoza-Hernandez, G. & Gonzalez-
Halphen, D. ( 2003) Identification of novel m itochond rial protein
components of Chlamydomonas reinhardtii: a proteomic
approach. Plant Phy siol. 132, 3 18–330.

34. Breen, G.A.M. & S cheffler, I.E. (1979) R espiration-deficient
Chinese hamster cell mutants: biochemical characterization.
Somatic Cell Mol. Genet. 5, 441–451.
35. Day, C. & Scheffler, I .E. (1982) Mapping of the genes for some
components of complex I of the electron transport chain on the X
chromoso me of mammals. Somatic Cell G enet . 8, 691–707.
36. Carroll, J., Shannon, R.J., Fearnley, I.M., Walker, J.E. & Hirst, J.
(2002) Definition of the nuclear encoded protein composition of
bovine h eart m itochondrial complex I: ide ntification of two new
subunits. J. Biol. Chem. 277, 50311–50317.
37. Murray, J., Zhang, B., Taylor, S.W., Oglesbee, D., Fahy, E.,
Marusich, M.F., Ghosh, S.S., Capaldi, R.A. (2003) The subunit
composition of the hu man, NADH, dehydroge nase obtained by a
rapid one-step immu nopurificatio n. J. Biol. Chem. 278, 13619–
13622.
38. Ditta, G.S., Soderberg, K. & Scheffler, I.E. (1976) The selection of
Chinese hamster ce lls d eficient i n oxida tive energy metabolism.
Somatic Cell Mol. Genet. 2, 331–344.
39. Au, H .C. & Scheffler, I.E. (1997) A r espiration-deficient C hinese
hamster cell line with a defect in mi tochondrial protein synthesis:
rapid turnover of some mitochondrial transcripts. Somatic Cell
Mol. Genet. 23 , 27–35.
40. Burnett, K.G. & Scheffler, I.E. (1981) Integrity of mitochondria in
a mammalian cell mutant defective in mitochondrial protein
synthesis. J. Cell Biol. 90, 108–115.
41. Ditta, G .S., Soderberg, K. & S cheffler, I .E. ( 1977) Chine se h am-
ster cell mutant with defective mitochondrial protein synthesis.
Nature 268, 64–67.
42. Frohman, M.A., Dush, M.K. & Martin, G.R. (1988) Rapid
production of full-leng th cDNAs from rare transcripts: amplifi-

Ó FEBS 2004 Mammalian cells with severe complex I deficiency (Eur. J. Biochem. 271) 3273