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: ischeffler@ucsd.edu 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. 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- plex I. J . Biol. Chem. 277, 21221–21230. 8. Scheffler, I.E. & Yadava, N. (2001) Molecular genetics of the mammalian NADH-ubiquinone oxidoreductase. J. Bioenerg. Biomembr. 33, 2 43–250. 9. Au, H.C., Seo, B.B., Matsuno-Yagi, A., Yagi, T. & Scheffler, I.E. (1999) The NDUFA1 gene product(MWFEprotein)isessential for activity of complex I in mammalian mitochondria. Proc. N atl Acad. Sci. USA 96, 4354–4359. 10. Marques, I., D uarte, M. & Videira, A. (2003) The 9.8 kDa subunit of complex I, related to bacterial Na(+)-translocating NADH dehydrogenases, is required for enzyme assembly and f unctio n in Neurospora c rassa. J. Mol Biol. 329, 283–290. 11. Videira, A. & Duarte, M. (2002) From NADH to ubiquinone i n Neurospora mitochondria. Biochim. Biophys. Acta 1555, 187– 191. 12. Runswick, M.J., Fearnley, I.M., Skehel, J.M. & Walker, J.E. (1991) Pre sence of an acyl car rier protein in NADH: ubiquinone oxidoreductase from bovine heart mitoch ondria. 286., 121–124. 13. Schneider, R., Massow, M., Lisowsky, T. & Weiss, H. (1995) Different respiratory-defective phenotype s of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl ca rrier protein. Curr. Genet. 29, 10–17. 14. Triepels, R., Smeitink, A., Loeffen,J.,Smeets,R.,Buskens,C., Trijbels, F. & van den Heuvel, L. (1999) The human nuclear- encoded acyl carrier subunit (NDUFAB1) of the m itochond rial complex I in human pathology. J. Inherit. Metab. Dis. 22, 163– 173. 15. Guenebaut, V., Vioncentelli,R.,Weiss,H.&Leonard,K.R. (1997) Three-dimensional structure between bacterial and 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. 25. Cardol, P., Matagne, R.F. & Remacle, C. (2002) Impact of mutations affectin g ND mitochond ria-encode d subunits on the activity and assembly of complex I in Chlamydomonas: implication 3272 P. Potluri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 for the structural organ ization of th e e nzyme. J. Mol . Biol. 319, 1211–1221. 26. Wallace, D .C. (1999) M itochondrial diseases i n man and mouse. Science 283, 1 482–1488. 27. Niers, L.E., Smeitink, J.A., Trijbels, J.M., Sengers, R.C., Janssen, A.J. & van den Heuvel, L.P. (2001) Prenatal diagnosis of NADH: ubiquinone oxidoreductase deficiency. Prenat. Diagn. 21, 8 71–880. 28. VandenHeuvel,L.&Smeitink,J.(2001)Theoxidativephos- phorylation (OXPHOS) system: nuclear genes and human genetic diseases. Bioessays 23 , 518–525. 29. Smeitink, J., Sengers, R., Trijbels, F. & Van den Heuvel, L. (2001) Human NADH: ubiq uinone oxidoreductase. J. Bioenerg. Bio- membr. 33, 259–266. 30. Triepels, R .H., van den Heuvel, L.P., Trijbels, J.M. & S meitink, J.A. (2001) Respiratory chain complex I de ficiency. Am.J.Med. Genet. 106, 37– 45. 31. DeFrancesco, L., Scheffler, I.E. & Bissell, M.J . (197 6) A respira- tion-deficient Chinese hamste r cell line with a d efect in NADH - Coenzyme Q reductase. J. Biol. Chem. 251 , 4588–4595. 32. Soderberg, K., Mascarello, J.T., Breen, G .A.M. & Scheffler, I.E. (1979) Respiration-deficient Chinese hamster cell mutants: genetic characterization. Somatic Cell Genet. 5, 225–240. 33. Scheffler, I.E. (1986) Biochemical genetics of respiration -deficient mutants of a nima l cells. I n Carbohydrate M etabolism in Cultured Cells (Morgan, M.J., ed.), pp. 77–109. Plenum Publishing Co, London. 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- cation using a single gene-specific oligonucleotide primer. Proc. NatlAcad.Sci.USA85, 8998–9002. 43. Bahring, S., Sandig, V., Lieber, A. & Strauss, M. (1994) Mapping of transcriptional start and capping points by a modifie d 5¢RACE technique. Biotechni ques 16, 807–808. 44. Seo, B.B., K itajima-Ihara, T., Chan, E.K.L., S cheffler, I.E., Matsuno-Yagi, A. & Yagi, T. (1998) M olecular remedy of com- plex I d efects: rotenone insensitive internal N A DH-quinone oxi- doreductase of Saccharomyces cerevisiae mitochondria restores NADH oxidase a ctivity o f c omplex I-de ficient mammalian c ells. Proc.NatlAcad.Sci.USA95, 9167–9171. 45. Trounce, I.A., Kim, Y.L., Jun, A.S. & Wallace, D.C. (1996) Assessment of m itocho ndrial oxidative p hosphorylatio n in patient muscle biopsies, lymphoblasts and transmitochondrial cell lines. Methods Enzymol. 264 , 484–509. 46. Schagger, H. (2001) Blue-native gels t o isolate protein complexes from mitochondria. In Mitochondria (Pon, L.A. & Schon, E.A., eds), pp. 2 31–244. Academic Press, San Diego. 47. Dabbeni-Sala, F., Di Santo, S., Franceschini, D., S kape r, S.D. & Giusti, P. (2001) Melatonin protects against 6-OHDA-induced neurotoxicity in rats: a role f or mitochondrial complex I a ctivity. FASEB J. 15, 164–170. 48. Yadava, N., Houchens, T., Potluri, P., Scheffler, I.E. (2004) Development and characterization of a c onditional mitochond rial complex I assembly system. J. Biol. Chem. 27 9 , 12406–12413. 49. Antonicka, H., Ogilvie, I., Taivassalo, T ., Anitori, R.P ., Haller, R.G., Vissing, J., Kennaway, N.G. & Shoubridge, E.A. (2003) Identification and characterization of a c ommon set of complex I assembly inter mediates in mitochondria from patien ts with com- plex I deficiency. J. Biol. Chem. 278, 43081–43088. Ó FEBS 2004 Mammalian cells with severe complex I deficiency (Eur. J. Biochem. 271) 3273 . The role of the ESSS protein in the assembly of a functional and stable mammalian mitochondrial complex I (NADH-ubiquinone oxidoreductase) Prasanth. 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