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.. Ou
Trang 1The 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-subcomplex 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 mutants 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 is truncated at the C-terminus of the
targeting sequence; the mutants are null mutants for the
ESSS subunit There is no detectable complex I assembly
and activity in the absence of the ESSS subunit as revealed by blue native polyacrylamide gel electrophoresis (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 mtDNA-encoded subunits (ND1-6,4 L), it is incorporated into complex I only in presence of compatible complex I subunits from the same species Keywords: complex I; ESSS protein; mitochondria; NADH-ubiquinone oxidoreductase; respiration-deficient mutants
NADH-ubiquinone oxidoreductase (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 found in the prokaryotic complex
capable of 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 largely 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 to ubiquinone
A major challenge is to understand how 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 genetic studies, largely carried out with the bovine complex I [1,2,19–22] Complex
I lags behind, largely because a similar complex does not exist in the common yeasts Saccharomyces cerevisiae and Schizosacchoromyces pombe Genetic studies with Neuros-pora crassa[11], and more recently with the yeast 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 of a challenge A systematic investigation of human patients suffering 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 severe 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 Section, University of California, San Diego, CA 92093–0322,
USA Fax: + 1 858 5340053, Tel.: + 1 858 5342741,
E-mail: ischeffler@ucsd.edu
Abbreviations: BN/PAGE, blue native polyacrylamide gel
electro-phoresis; MBS, maleimidobenzoyl N-hydroxysuccinimide ester;
TMPD, tetramethylphenylene diamine.
(Received 10 March 2004, revised 10 June 2004,
accepted 18 June 2004)
Trang 2X-linked NDUFA1 gene (encoding the MWFE protein) was
identified by our laboratory [7,9] Until recently it 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 gene in
humans and mouse [36] Most of these subunits have also
been identified in the human enzyme [37] The present
manuscript describes the characterization of Chinese
ham-ster mutant cells from a second complementation group in
our collection in which the gene for the ESSS protein was
found to be mutated The ESSS protein is a relatively small
protein (123 amino acids in the mature form) It is predicted
to have a single transmembrane helix, and it has been
purified from the integral membrane b-subcomplex [2] As
there is no homologous protein in the prokaryotic complex,
it had previously been grouped among the ancilliary
proteins, also referred to as accessory proteins Our present
studies establish 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 G8,
V79-G18 and V79-G35 cells were from a different parental cell
line, V79 (CCL93, American Type Culture Collection)
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)1glucose (DME-Glu) to sustain
glycolysis, and a supplement of nonessential amino acids
Substitution of glucose with 1 mgÆmL)1galactose
(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, respectively) Cells were
harvested by trypsinization after one wash with TD buffer
(0.3% Tris, 0.8% NaCl, 0.038% KCl, 0.025% Na2
H-PO4Æ12H2O, brought to pH 7.4 with HCl)
Plasmids and genes
A polycistronic pTRIDENT-14 neo vector with an EF1a
promoter expressing various cDNAs in the 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 cDNA (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 modified pTRIDENT-14neo vectors such that either the HA or the HIS epitope tag was added to the end of the ESSS ORF The forward primer was: 5¢-ACga atccGATCTCCGACCCA-3¢; the reverse primer was: 5¢-ATgctagcCTCATCTTCTGGTAACTGG-3¢ Small bold letters refer to the restriction sites added to the tide primers for directional cloning The same oligonucleo-tides were used 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–mutant cells (5· 105) were seeded 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 marked 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 from a transgene at variable positions in the genome
Measurement of respiratory activities The respiratory chain activities of various 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 mMHepes, pH 7.1, 250 mM sucrose and 10 mMMgCl2) at a density of 2· 107cellsÆmL)1 Cells were permeabilized by digitonin (100 lgÆmL)1) for
5 min at 4 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· 107cellsÆmL)1 The total protein content was measured
by Bradford microassay, and 1 mg of cell suspension was used per assay Oxygen consumption was measured polaro-graphically with a Clark oxygen electrode in metabolic chamber with a water jacket maintained at 37C (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· 109cells were washed twice with
TD buffer and harvested by trypsinization The pellets were suspended in 5 mL SM buffer (50 mM Tris/HCl, pH 7.4, 0.25M sucrose, 2 mM EDTA) and homogenized using
a tightly fitting Dounce homogenizer (30–35 up/down strokes) The homogenate was centrifuged twice at 625 g for
Trang 310 min at 4C in order to remove unbroken cells and
nuclei The supernatant was centrifuged at 10 000 g for
20 min at 4C The mitochondrial pellet was suspended 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 to Immobilon-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 on the immunoblots were
detected using an Enhanced Chemiluminiscence system
(ECL+ Plus from Amersham)
The anti-MWFE serum was developed as described
previously [7] B Ackrell (University of California, San
Francisco, CA, USA) provided antiserum against the
SDHC subunit of complex 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 mM
6-aminohexanoic acid, 50 mM imidazole/HCl (pH 7.0),
50 mMNaCl, and 10% glycerol To the solubilized 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 was carried out as
described [7,47] Gel slices were incubated at room
tem-perature in 2 mM Tris/HCl (pH 7.4), 0.1 mgÆmL)1 of
NADH and 2.5 mgÆmL)1of 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
in two and suspected in the third [33,35] When the ESSS
protein was added to the list of complex I subunits, and its
gene was localized on the X chromosome in mammals, it
became a candidate for the mutated gene in one of the two
unidentified complementation groups
The construction 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 DME-Glu medium containing 800 lgÆmL)1 G418 Several colonies were marked on the 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, the 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 that ESSS cDNA can complement the muta-tions in the cell line V79-G18 but not V79-G8 Furthermore,
HA or HIS epitope 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 corresponding ESSS cDNAs from wild type (GenBank accession number AY649405), and 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 signal sequence; the third mutant allele (G35) encodes a truncated protein missing 25 amino acid residues from the C-terminus (Fig 1) Two of these mutants are
Fig 1 Sequences of Chinese hamster cDNA and wild-type Chinese hamster ESSS precursor protein (A) Complete sequence of the Chinese hamster cDNA, with the open reading frame indicated in capital letters (GenBank accession number AY649405) (B) The sequence of the wild-type Chinese hamster ESSS precursor protein, with the signal sequence and a proposed cleavage site based on the sequence 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.
Trang 4therefore effectively null mutants, with no residual,
recog-nizable ESSS protein expected
The ESSS protein is found in the b-subcomplex and is
predicted to be an integral membrane protein with a single
transmembrane segment [2,36] From a comparison with
the sequence of the mature bovine 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 residues 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), and wild-type and mutant cells stably
transfect-ed with the complementing ESSS-HA were fractionattransfect-ed
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 shown in Fig 3A,
the mutant 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 sequence is indicated in bold (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 mutant stably
trans-fected with the di-cistronic vector expressing hamster ESSS-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 nitroblue tetrazolium as electron acceptor Bottom panel:
West-ern analysis with anti-HA Ig, anti-NDUFB6 (complex I), anti-Rieske
protein (complex III), and anti-SDHC (complex II).
Trang 5accumulated 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 result was observed
with ESSS-His6, or when the mutant was complemented
with wild-type hamster ESSS without a tag
Next, mitochondria from wild-type, mutant and
comple-mented mutant cells were solubilized by sodium dodecyl b-D
maltoside (DDM) and protein complexes were fractionated
by Blue Native gel electrophoresis The 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 3B, 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 reveal the
presence of any complex I-specific subunits at that position
We believe that the band may represent a nonspecific
NADH dehydrogenase activity Complex II activity could
be measured on the same gels using the same electron
acceptor with succinate as the substrate (results not shown)
The gels were also used in a Western analysis with antisera
against the HA epitopes It is noteworthy that the epitope
tags do not interfere significantly with the incorporation of
the tagged ESSS subunit into a functional complex I
(Fig 3B, right panel) The signal from the 18 kDa subunit
of complex I (NDUFB6) served as another identification of
the complex at the position of 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 wild-type cells expressing ESSS-HA, in 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 the activity of the downstream portions of the electron
transport chain was established with succinate and
glycerol-3-phosphate as substrates Complex II activity was
deter-mined after addition of succinate, followed by inhibition by
malonate, and complex III activity was measured after the
addition of excess glycerol 3-phosphate followed by addition
of antimycin A typical set of traces from the oxygen
electrode is shown in Fig 4A, and the results are summarized
in Fig 4B Consistent with the observations with BN/
PAGE, complex I activity was restored by ESSS, ESSS-HA,
ESSS-His6, but the activity was lower than that in wild-type
mitochondria, especially in the case of the HA tag It is
possible that the HA-tagged subunit, while 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 of 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 function 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 quite 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 His6, but a precise quantitative difference between these two tags remains to be established
We believe that such discrepancies, especially with the untagged ESSS, are due to clonal 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 V79-G35 and 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 together, the cyanide-sensitive respiration rates were somewhat lower in the mutant cells, and partially restored in complemented cells It is tempting to speculate 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 the mitochondrially encoded subunits ND1-6, and ND4L Furthermore, such inter-actions are quite species-specific and affect the stability 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 selected 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 activity 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 ESSS protein seems to be
Trang 6stable, and it is found in a diffuse series of bands (500–
800 kDa) by 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
poison subunit The diffuse bands may represent a mixture
of partially assembled-subcomplexes or breakdown
prod-ucts of an unstable-subcomplex This result prompted us to
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 that
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 cross-linked by MBS to another unidentified protein
to yield a new species migrating with a mobility of a
35 kDa protein This includes wild-type hamster cells expressing ESSS-HA from the transgene, V79-G18 cells in which ESSS-HA restores complex I activity, the various hamster mutant cell lines (V79-G8, V79-G7, CCL16-B2), and significantly, the human HT1080 cells in which hamster ESSS-HA is expressed and 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 cells permeabilized by digitonin were determined by polarography Arrows on the side of the tracings represent the following consecutive additions: (a) glutamate/malate; (b) rotenone; (c) succinate; (d) malonate; (e) glycerol 3-phosphate; (f) antimycin; (g) TMPD-ascorbate and (h) cyanide (Details are given in Experimental procedures.) (B) The activities were normalized with respect to total cellular protein concen-trations The activity of wild-type cells was set at 100% The asterisk indicates activity indistinguishable from background The results represent the average of a minimum of four experiments.
Trang 7factor 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 others Two integral 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 subunits The most variable behavior is exhibited by the MWFE subunit, 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 of ESSS subunit, although these two subunits have been localized in different subdomains of the complex This suggests an interaction between these subdomains that is facilitated by the ESSS subunit
Discussion
A novel series of Chinese hamster cell mutants in a single complementation group with a complete defect in the NADH-ubiquinone oxidoreductase (complex I) is des-cribed The mutations have been identified in the X-linked gene encoding the ESSS protein, a subunit that was recently added to the list of complex I subunits [1,36] The subunit is
an integral membrane protein outside of the group of core 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 can 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 and accumulated in mitochondria, but the hamster ESSS-HA protein is 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 comparative 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 most likely already associated in a heteropolymeric-subcomplex We also found that these subunits are in every 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 was found in human mitochondria (SDS/PAGE; A), but not in
the active complex I (BN/PAGE; B) Lane 1 was loaded with
solubi-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 to complex I; the two lower bands
represent complex II and its dimer, detected by antiserum against 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 complex I-deficient
Chi-nese hamster cell lines For a description of these mutants 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
51
kDa
30
kDa TYKY PSST B8
39 kDa MWFE ESSS B17
B2 + + + + + + – – + +
G18 + + + – + + – – – +
Trang 8association 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 missing 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 failure 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 of all ND subunits (V79-G7) [7]) In other words, the
synthesis, assembly, and accumulation of integral membrane
subunits are integrated and interdependent process On the
other hand, when hamster ESSS-HA is expressed in 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 heterologous subunit (Fig 5A) The
identi-fication of precomplexes in mitochondria of human patients
has been claimed [49], although the observed-subcomplexes
could also have resulted from the dissociation of the intact
complex I with mutated subunits 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 identifiable 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 the Muscular Dystrophy Association to
I E S.
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