Molecular genetics of complex I-deficient Chinese hamster cell lines

Biochimica et Biophysica Acta 1659 (2004) 160 – 171 http://www.elsevier.com/locate/bba

Review

Molecular genetics of complex I-deficient Chinese hamster cell lines Immo E. Scheffler*, Nagendra Yadava, Prasanth Potluri Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0322, USA Received 1 June 2004; received in revised form 28 July 2004; accepted 9 August 2004 Available online 21 August 2004

Abstract The work from our laboratory on complex I-deficient Chinese hamster cell mutants is reviewed. Several complementation groups with a complete defect have been identified. Three of these are due to X-linked mutations, and the mutated genes for two have been identified. We describe null mutants in the genes for the subunits MWFE (gene: NDUFA1) and ESSS. They represent small integral membrane proteins localized in the Ia (Ig) and Ih subcomplexes, respectively [J. Hirst, J. Carroll, I.M. Fearnley, R.J. Shannon, J.E. Walker. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim. Biophys. Acta 1604 (7-10-2003) 135–150.]. Both are absolutely essential for assembly and activity of complex I. Epitope-tagged versions of these proteins can be expressed from a poly-cistronic vector to complement the mutants, or to be co-expressed with the endogenous proteins in other hamster cell lines (mutant or wild type), or human cells. Structure–function analyses can be performed with proteins altered by site-directed mutagenesis. A cell line has been constructed in which the MWFE subunit is conditionally expressed, opening a window on the kinetics of assembly of complex I. Its targeting, import into mitochondria, and orientation in the inner membrane have also been investigated. The two proteins have recently been shown to be the targets for a cAMP-dependent kinase [R. Chen, I.M. Fearnley, S.Y. Peak_Chew, J.E. Walker. The phosphorylation of subunits of complex I from bovine heart mitochondria. J. Biol. Chem. xx (2004) xx–xx.]. The epitope-tagged proteins can be cross-linked with other complex I subunits. D 2004 Elsevier B.V. All rights reserved. Keywords: Mitochondria; Complex I; Respiration-deficient mammalian cell

1. Introduction The study of the respiratory chain (RC) and cellular energy production via oxidative phosphorylation (OXPHOS) is a problem of long standing in biochemistry [45]. Even today, many questions remain with regard to the finer details of its organization (e.g., supramolecular complexes), its biogenesis, its adaptation to mammalian cell differentiation, and its integration into other cellular activities [46]. The involvement of mitochondria in regulated cell death (apoptosis) and detection of OXPHOS deficiencies in a multitude of human pathologies (~1:10,000 living births) have reinvigorated this field in the last decade [8,50,56]. Human patients with mutations in either mitochondrial DNA (mtDNA) or nuclear genes have been found.

* Corresponding author. Tel.: +1 858 534 2741; fax: +1 858 534 0053. E-mail address: [email protected] (I.E. Scheffler). 0005-2728/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbabio.2004.08.002

The clinical disorders associated with OXPHOS deficiencies and the severity of their expression vary over a broad range. While the brain and muscles are the main tissues affected, it is still a challenge to explain many of the tissuespecific abnormalities. Isolated complex I deficiencies are relatively frequent, possibly because such a large number of genes is involved in assembly and function. The biochemical characterization of complex I from several such patients has revealed that levels of assembled complex are reduced, in contrast to finding normal amounts of the complex with a reduced specific activity. The majority of these nuclear mutations are point mutations (missense mutations), and their impact may be on the assembly or stability of the complex I [53,54,56]. The availability of crystal structures for four of the OXPHOS complexes [succinate-ubiquinone oxidoreductase (complex II), ubiquinone–cytochrome c oxidoreductase (complex III), cytochrome oxidase (complex IV), and ATP-synthase (complex V] has considerably advanced our

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understanding of their function in relation to structure [45,46]. In contrast, our understanding of the NADHubiquinone oxidoreductase (complex I) is still more limited, because no crystal structure is available as yet. The mammalian complex I is a very large enzyme with a calculated molecular mass of ~980 kDa [10,27]. The number of subunits constituting this enzyme is 46 in bovine heart mitochondria [10,27], and the composition and organization of the human and bovine complex I appear to be very similar [32]. Mitochondrial DNA (mtDNA) encodes seven subunits of complex I (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6) and 39 subunits are encoded by nuclear DNA (nDNA), synthesized in the cytosol, and then imported into mitochondria. A detailed analysis of the import of complex I subunits, especially the integral membrane proteins, is still lacking. Only ~1/3 of the nDNA-encoded subunits appear to have a recognizable mitochondrial targeting sequence. Fourteen of these subunits, known as "core" subunits, including all the mtDNA-encoded ones, have orthologues in prokaryotes [31,63,67]. This simpler prokaryotic complex of 14 subunits can perform all the currently known functions of mammalian mitochondrial complex I: (1) NADH oxidation and hydride transfer to a flavin mononucleotide and electron transfer via at least seven [Fe–S] centers to a quinone, and (2) proton/cation translocation across the membrane. This electron transport-coupled proton pumping across the membrane (cation translocation) by complex I is the major contributor towards the establishment of the electrochemical gradient (protonmotive force) used for ATP synthesis by complex V. The role of the other 32 "supernumerary" or "accessory" subunits in the mammalian complex I is presently not clear. Many, if not most, of these subunits are also present in the corresponding fungal and plant complexes. The final composition of different species with respect to the number and identity of "accessory" subunits appears somewhat variable between the fungus Neurospora crassa, Caenorabditis elegans, Drosophila melanogaster, and humans. For example, some of the accessory proteins found in N. crassa do not have their counterparts in mammalian complex I. It is possible, however, that they are not recognized due to limited sequence conservation. Several questions can be raised about the roles of "accessory" subunits in the mitochondrial complex I: (1) Are they essential for complex I biogenesis? (2) Are they required for assembly (scaffolding factors) or in the stabilization of the complex? (3) Do they regulate complex I activity? (4) Do they confer other cellular functions to complex I? Several studies have unambiguously established that some "accessory" subunits are essential components of mitochondrial complex I in lower eukaryotes [30,43,48,52,58]. Our own studies have demonstrated an absolute need for the MWFE and ESSS subunits in the biogenesis of an active mammalian complex I.

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From bacteria to humans, the overall "boot-shaped" structure of complex I is conserved, with one arm protruding into the matrix (peripheral subcomplex) and the other localized in the plane of the membrane (membrane subcomplex). This indicates clearly that the overall shape of the mammalian complex I is dictated by the organization of the "core" subunits, suggesting the possibility of protective roles for the accessory subunits by shielding the core catalytic unit. The peripheral subcomplex contains all the known prosthetic groups, whereas the function of the large membrane subcomplex is less obvious, although it must be involved in proton pumping. The ND5 subunit has some structural features related to those found in Na+/H+ antiporters, and bacterial enzymes have been described that pump Na+ ions instead of protons [21,22]. Several studies suggest that binding sites for quinone and inhibitors are located near the junction (neck) between peripheral and membrane subcomplexes. In the bacterial complex the integral membrane protein NuoM (the orthologue of the mammalian ND4 subunit) can be affinity-labeled competitively with a quinone analogue, and thus appears to be part of the CoQ binding site [23]. However, the nature of the CoQ binding site is still unclear, and there are suggestions that the complex can bind two or more quinones [18,34,66]. In this review, we describe our current understanding of mammalian complex I biogenesis from our analysis of complex I mutants in Chinese hamster fibroblasts previously described from this laboratory. All these mutants have severe or complete complex I defects. Two of these groups have mutations in homologues of X-linked complex I subunits, MWFE and ESSS. They are located in different subdomains of the integral membrane subcomplex (Fig. 1). We have shown that MWFE and ESSS subunits are essential components of complex I. Both MWFE and ESSS are required during the assembly process of complex I. A tentative model on the interdependence during assembly of a few selected subunits is presented. A third complementation group can be complemented with another X-linked gene that has not been identified and may not be a structural gene, but encodes an assembly factor required for complex I maturation.

2. The MWFE subunit 2.1. Import and orientation The MWFE protein from diverse mammalian and vertebrate species is highly conserved. In all the vertebrate species so far examined, the protein is precisely 70 residues long, with two distinct domains. A postulated transmembrane region close to the N-terminus (comprised of amino acids 5–24) is followed by a highly charged domain of ~45 amino acids (Fig. 2). A sequence of the first 27 residues including H26 and R27 is sufficient to target a chimeric

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Fig. 1. Schematic representation of complex I with the localization of the MWFE and ESSS subunits within the subcomplexes obtained by the fractionation procedures described by [27]. The size and shape of the subunits are exaggerated to emphasize their orientation (topology) in the inner membrane.

GFP construct to the mitochondria. When a shorter targeting sequence was tested without the positive charges, the reporter protein appeared in the ER. However, these positive charges are not translocated across the inner membrane [62]. The protein is not proteolytically processed upon import. The N-terminal sequence therefore acts both as a mitochondrial targeting sequence and as a membrane anchor. The highly conserved negative charge at position 4 is unusual for a mitochondrial targeting sequence. Negative charges have been implicated in the insertion of integral membrane proteins from the matrix side, since the membrane potential

would help in moving the charges from inside to outside [7,42]. The significance of this negative charge was investigated by site-directed mutagenesis. Changing the glutamate to a neutral serine (E4S), or to a hydrophobic alanine (E4A), did not prevent the import of the protein, since such constructs were capable of complementing the CCL16-B2 null mutant [62]. If the N-terminal is inserted as in a normal import pathway for mitochondrial matrix proteins, one would predict the hydrophilic domain to extend into the intermembrane space. Proteinase-K sensitivity assays of the HA-

Fig. 2. Sequence homologies in the MWFE protein for several primate, rodent and other vertebrate species. The first ~28–30 amino acids represent the mitochondrial targeting sequence, and the segment between I5 and I26 constitutes the transmembrane domain. Acidic side chains are represented in red, basic side chains are represented in blue, and hydrophobic side chains are represented in green.

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epitope of MWFE.HA protein support such an interpretation [62]. A similar conclusion was reached by Chen et al. [12] based on an in vitro phosphorylation assay. One can also consider the two distinct domains of the protein from another point of view. The N-terminus may serve strictly for import and as a membrane anchor, while the hydrophilic C-terminal domain is engaged in functional interactions with other complex I subunits. Some preliminary studies suggest that this picture is too simple. A chimeric protein with the N-terminal and transmembrane region of MVNL and the C-terminal domain of MWFE was expressed in mutant cells, but was incapable of complementing the mutation in the NDUFA1 gene [62]. The MVNL protein (MNLL in the bovine complex [27]) is another small subunit with a presumed single transmembrane domain and a short N-terminal region on the matrix side. It is likely that there are highly specific interactions not only between the hydrophilic regions of these proteins, but also between the various transmembrane domains. There are 60 or more predicted transmembrane helices in complex I, and their interactions within the plane of the membrane must clearly be considered. In this context, it is noteworthy that the introduction of a positive side chain (M12H) in the transmembrane domain did not prevent the formation of an active complex I (Yadava, unpublished observations). 2.2. Role of MWFE in complex I assembly CCL16-B2 cells have a mutation in the NDUFA1 gene that causes a splicing error, and the resulting transcript encodes a very short nonfunctional protein [3,47,60,61]. In the absence of any MWFE protein, no intact complex I is detected by polarographic measurements or by BN-PAGE/ Western blot analyses. There is no accumulation of a large (~900 kDa) complex. However, a number of subunits of the peripheral membrane subcomplex can be detected by Western blot analysis of purified mitochondrial membranes, suggesting that they are already attached to the inner membrane. [3]. This presumed intermediate appears to be relatively unstable under the conditions employed for BNPAGE. The expression of wild-type or epitope-tagged MWFE could restore complex I assembly, stability and activity. It is therefore likely that the MWFE protein is required at an intermediate step for the complete assembly of complex I. It is difficult to imagine a small protein like MWFE (representing less than 1% of the total mass) being required for stabilizing such a large complex. The role of MWFE in complex I assembly and stability was investigated by expressing HA-epitope-tagged MWFE protein (MWFE-HA) from a doxycycline-inducible promoter in CCL16-B2 cells. Stably transfected CCL16-B2 cells that lack complex I and were completely respirationdeficient (res ) could be made respiration-competent (res +) after the addition of doxycycline to the growth medium. The conditional expression of the MWFE.HA subunit led to conditional complex I assembly and respiration. This model

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system permitted more detailed analyses of the role of the MWFE protein in (1) the assembly, (2) the kinetics of assembly, and (3) the maintenance of the complex I [60]. Upon induction, the MWFE-HA protein appeared within hours and reached steady state levels after about 24 h. The most interesting finding was that the formation of a mature and functional complex I required an additional ~24 h, and the appearance of MWFE.HA in the membrane was not a rate-limiting step in the assembly pathway. These observations suggest that stable MWFE may appear in a precomplex (assembly intermediate) that was formed relatively rapidly, but its conversion into a mature complex I required more time. However, no such high molecular weight intermediate containing MWFE.HA could be detected by BN-PAGE and Western blot analyses. It may be unstable under the experimental conditions used. Experiments with a pulse of inducer (24 h) followed by a chase in the absence of inducer strongly support this idea. At the end of the pulse MWFE.HA can be seen in whole mitochondria only on SDS-PAGE, but it is incorporated during the 24-h chase into a stable, ~900-kDa complex I detectable by BN-PAGE. A priori, one might have expected that the MWFE subunit could be inserted into the membrane after synthesis, to be used later when required for assembly of the complex. As will be described below, we believe that the MWFE subunit is highly unstable in isolation, and any accumulation to measurable levels requires the presence of additional subunits which interact with it and thus protect it from turnover. After the complex has been assembled to steady state levels in the presence of inducer, the inducer can be removed and one can measure the decay of complex I as a whole (BN-PAGE, polarography) and the disappearance of MWFE from a mitochondrial preparation. The results from our studies suggest that the turnover of the complex and the turnover of the MWFE subunit are tightly linked. In other words, we did not find any indications that MWFE could escape from the complex and be degraded more rapidly than the complex as a whole. The single transmembrane helix of MWFE is likely to be deeply embedded in the large bundle of the other (~60) transmembrane helices, rather than being loosely associated at the periphery of the bundle. In agreement with previous studies by others, we find that the electron transport complexes have a relatively long half-life. A more rapid disappearance of MWFE would have clearly excluded its role in stabilization and maintenance of complex I. 2.3. Requirement for other subunits to stabilize the MWFE subunit A Chinese hamster mutant cell line (V79-G7) has been described by our laboratory in which mitochondrial protein synthesis is severely inhibited or absent [2,9,17,60]. Mitochondria from such cells lack all the ND subunits, and MWFE-HA insertion and/or stabilization in the

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inducible mutant cells (see above) is not observed. The endogenous MWFE protein that is constitutively expressed is also not detectable by Western blot analysis. Directly or indirectly, one or more of the ND subunits must be involved in the stabilization and accumulation of the MWFE subunit. Mutant cells lacking a single ND subunit have also been investigated. In the ND4 (human) and the ND6 (mouse) mutant cell lines [4,5,13,14], the endogenous MWFE appears to be equally unstable and undetectable. In an ND5 (mouse) cell line the MWFE protein is detectable, but at significantly reduced levels. All of these cell lines have a severe complex I defect. The ND4, ND5 and ND6 subunits are found in different subcomplexes (ND6 in 1a, ND4 and -5 in 1h) that have been defined from a specific purification protocol. Do these 1a and 1h subcomplexes have any relationship to assembly intermediates? Are they assembled independently? MWFE is found in the 1a subcomplex and is missing in the ND6 mutant, consistent with such a view. However, contrary to this view, MWFE is also absent in the ND4 mutant, i.e., mutants in which only the 1h subcomplex assembly would be expected to be affected. From such data it becomes apparent that the accumulation and assembly of these (and other) subunits are a coordinated, interdependent process, likely involving many more subunits. One can exclude a mechanism in which these subunits are inserted into the membrane independently and maintained there in pools until needed in complex I biogenesis. They may also be subject to rapid turnover when the maturation towards a complete complex I is disturbed by mutations or inhibitors. The ND subunits belong to the bcoreQ of the complex I as defined for prokaryotes, and MWFE is a protein that has been added to the complex during evolution of the eukaryotic lineages. It may not have been unexpected to find the membrane insertion and stabilization of MWFE subunits to be dependent on the presence of ND subunits. A further speculation is that the conversion of the pre-complex to an active complex I requires the insertion of additional subunits, including some synthesized in the mitochondrial matrix. In a study by Hall and Hare examining the mechanism of complex I assembly in rat hepatoma cells by pulse-chase experiments, mitochondrially translated proteins did not appear in an immuno-precipitable holoenzyme until after a particularly long chase, and the suggestion was made that a bscaffolding or nucleation complexQ of nuclear-encoded proteins was required before the ND proteins could be binserted into the enzymeQ. When cytosolic protein synthesis was inhibited by cycloheximide, the ND proteins were not assembled and they were apparently degraded by an ATP-dependent protease [25]. The relationship between the insertion and stabilization of MWFE and the seven ND subunits can give rise to interesting speculations that can be resolved when specific antisera against each of the ND subunits become available. Unfortunately, existing antisera do not cross-react with the corresponding hamster proteins. Are there some ND subunits that are inserted and accumulated in the absence

of MWFE, and others that can only be inserted after the assembly has progressed beyond the step requiring MWFE? Formally one can imagine that mitochondrial protein synthesis with concurrent insertion into the membrane is a continuous process, but ND subunits are unstable when complex I assembly cannot proceed in the absence of MWFE. Mitochondrial translation and membrane insertion are tightly coupled. The components necessary for membrane insertion are still poorly defined, but it is tempting to speculate that a partially assembled pre-complex containing some nuclear subunits acts as receptor or docking site for the selective, co-translational insertion of (some) ND subunits. It is not clear whether MWFE is an obligatory component of such an import mechanism, or whether the subsequent fate of these subunits depends on the presence of MWFE (possibly in another, distinct pre-complex). Carroll et al. [11] have recently reported that the nuclear-encoded B14.7 subunit (gene NDUFA11) has some homology to Tim proteins, normally involved with import of proteins from the cytosol. Studies of protein insertion from the matrix side have shown an essential role for the mitochondrial export translocase Oxa1 [26,33]. Genetic and molecular studies in yeast have demonstrated that membrane-associated and transcript-specific proteins are required for the translation and membrane insertion of mtDNA-encoded Cox subunits into cytochrome oxidase (complex IV) [24,44]. However, these proteins are true assembly factors and not found in the final complex. It is likely that similar assembly factors (chaperones) that are transiently associated during complex I assembly are also required [28,49]. An explicit assembly pathway has been proposed for complex I assembly in N. crassa [57]. In this model the peripheral subcomplex is assembled independently and associated with the membrane subcomplex at a late stage. For mammalian systems contrasting models have recently been described by Antonicka et al. [1] and by Ugalde et al. [53]. These authors reported on the status of complex I assembly in several patients with mitochondrial disease and partial complex I deficiencies. Several distinct subcomplexes with peripheral membrane subunits were identified by two-dimensional BN/SDS gel electrophoresis; a similar pattern of subcomplexes was observed in the diverse patients. It was suggested that these subcomplexes are intermediates in the assembly of the holoenzyme complex. As a note of caution, it should be mentioned that it is difficult to distinguish between assembly intermediates and breakdown products due to instability introduced by mutations (under conditions of solubilization for BN-PAGE). 2.4. Mutant and heterologous alleles of MWFE The CCL16-B2 mutant is effectively a null making a very short, nonfunctional fragment frame-shifted reading frame of an abnormally transcript. Complementation was first achieved

mutant from a spliced with a

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mouse or hamster X chromosome, but not with a human X chromosome [15]. When the corresponding NDUFA1 cDNAs were cloned into an expression vector, the mouse and hamster protein restored complex I activity, but the human protein did not. This observation may have been surprising in view of the very high degree of sequence conservation in the MWFE protein among vertebrates (see below), but it could also have been anticipated from some pioneering studies of Wallace [59] and later by Moraes [6,16,64,65] and others [64,65] that mitochondrial genomes and nuclear genomes in interspecies cybrids must be from very closely related species to make a functional electron transport chain. We speculated early on that the human MWFE protein, although also encoded by an X-linked gene, might not be functional because of its incompatibility in the complex with hamster mitochondrial (ND) proteins. Sequences from a number of vertebrate species are shown in Fig. 2; among the mammals there is 80% identity and 90% similarity, with relatively few nonconservative substitutions. The differences in the region from amino acid 39 to 46 attracted our attention in a comparison between rodents and primates. Differences in almost all other positions represent highly conservative amino acid substitutions in the hydrophobic domain, with the exception of the negative glutamate at position 58 in higher primates. The most notable was the difference in the spacing of the two positive charges in this segment, which are separated by one amino acid in the hamster and by three amino acids in primates. The mouse MWFE protein is missing a positive side chain in this region, but mouse cDNA could complement the hamster mutation at a slightly lower efficiency than hamster cDNA, indicating both the charge spacing and the identity of amino acid residue in this region are important for function of MWFE. The relevance of these sites for explaining the observed incompatibility was verified experimentally by site-directed mutagenesis [61]. When the hamster MWFE was modified at positions 41–42 (mutation L41F, R42G), the complemented mutant cells (B2-A41/42) showed a ~50% loss in activity. The mutant allele A41/42 resembles the mouse MWFE with a positive charge removed at position 41, and a comparable reduction had been observed with the mouse MWFE. When the human MFWE was modified at the same positions (mutation F41L, G42R), activity was partially restored (50%) in complemented cells (B2-U41/42); the addition of a positive charge at position 41 converted a completely inactive human protein into a partially functional one. Thus, this relatively small region of six residues can contain one (mouse, A41/42 mutant), two (wt hamster), or three charges (U41/42 mutant) in a still functioning protein. A more detailed analysis must await the publication of a crystal structure. We believe that this example represents the most specific illustration to date of the incompatibility of mitochondrial

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proteins from different species when a multi-subunit complex is formed. These alleles yielding partial complex I activity may also be suitable starting points for constructing mouse models for mitochondrial diseases. In the extreme cases, it is clear that when mutant MWFE proteins are undetectable there are also no intermediate or large (~900 kDa) complexes detectable on BN-gels [by Western blot analysis with antibodies against other subunits, or NADH-nitroblue tetrazolium (NBT) oxidoreductase activity]. A detectable, steady state level of MWFE on a Western blot may therefore be taken as an indication of complex I assembly, and it may be a useful diagnostic parameter. On the other hand, it has been experimentally challenging to make a precise distinction between a reduced specific activity of complex I due to a MWFE mutation and a reduced level of active complex I. The MWFE mutation could cause a decreased rate of assembly or lower the stability of the complex. The original mutant selection also yielded a mutant NDUFA1 allele with a point mutation causing an R50K substitution. The substitution is conservative and the highly deleterious consequence (zero activity) is surprising, since the positive charge is preserved. A similar result was observed with an R50I mutation. Another highly conserved residue, D51, cannot be altered; a D51A substitution yields an unstable, inactive complex I (Yadava, unpublished). Here also a crystal structure will be needed for a full explanation/interpretation. A third mutant allele (V79-G14) selected in cells in tissue culture created an MWFE protein with a small alteration and deletion at the C-terminus (positions N65). This protein was also completely inactive. The mutation destroyed the antigenic determinant against which the anti-MWFE antiserum had been produced and hence the fate of this protein could not be followed. When mutant proteins or the human protein was completely inactive in hamster cells (complex I activity measured by polarography, complex I assembly determined on BN-gels), the mutated MWFE protein was also undetectable on Western blots from SDS-PAGE of whole mitochondria. The construction of a di-cistronic vector has permitted us to express wild-type or partially active MWFE proteins with an HA epitope tag in the presence of the endogenous MWFE; stable cell lines can be selected with the drug-resistance marker in the second cistron. The two proteins can be co-expressed in the complex I if the mutated protein is still compatible. Thus, one can also test these alleles in normal human cells (HT1080): human MWFE-HA is accumulated in human cells, but hamster MWFE-HA and the mutated proteins A41/42 (L41F, R42G) and U41/42 (F41L, G42R) were not detectable [61]. It appears that the human complex is less tolerant of changes in this region compared to the hamster complex. In the future, our experimental model system will allow us to test other mutations in the MWFE protein. A rational selection of mutations to be tested will be possible when a

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crystal structure for the mammalian complex becomes available.

3. The ESSS subunit 3.1. ESSS, an essential component of complex I Several other Chinese hamster cell mutants with a severe complex I deficiency described by our laboratory were shown to have X-linked mutations, but they were complemented in somatic cell hybrids with the CCL16-B2 (NDUFA1) mutants [51]. Until recently, the NDUFA1 gene was the only known X-linked gene for the list of subunits determined from highly purified bovine complex I. When the ESSS subunit was added to the list [11], its gene was also localized on the X chromosome, and thus it became an obvious candidate for a second complementation group of mutants in our collection [40]. The hamster ESSS cDNA was expressed from a dicistronic expression vector and shown to complement the group of mutants including CCL16-B11, V79-G18 and V79-G35. The hamster ESSS protein is 122 amino acids in length, with one predicted transmembrane domain (residues 60–80), and therefore with hydrophilic domains of approximately equal size (50 aa) on either side of the membrane (Fig. 3). 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 differences in the N-terminal domain (located on the matrix side). In each of our mutants a chain-terminating codon had been introduced by the mutation. In CCL16-B11 and V79-G35 cells, the truncated protein is terminated at the C-terminal end of

the mitochondrial targeting sequence; they are effectively null mutants. The truncated protein in the V79-G18 mutant is missing most of the C-terminal domain predicted to extend into the intermembrane space [40]. The ESSS protein is an integral membrane protein that has been purified in the h subcomplex (Fig. 1) in the scheme developed by Walker’s laboratory [10,27]. It belongs to the group of 32 proteins outside of the core group present in prokaryotes and eukaryotes, and therefore its initial categorization was as a bsupernumeraryQ protein. Our findings with the three mutant cell lines/alleles prove that ESSS is an essential protein for complex I assembly and activity. In its absence, no ~900-kDa complex is detectable by BN-PAGE, and no rotenone-sensitive respiration can be measured by polarography. The C-terminal of ESSS subunit can be tagged with haemagglutinin (HA) or hexahistidine (HIS6) epitope monomers, and these tags allowed us to monitor the expression of this protein and its incorporation into the ~900-kDa complex seen on BN gels. As seen with the MWFE protein, a successful complementation required a hamster ESSS cDNA/protein, again suggesting that highly species-specific interactions take place between ESSS and its neighbors in the h subcomplex [40]. 3.2. Accumulation of hamster ESSS-HA in other mutants and heterologous cells Mutated and heterologous ESSS proteins tagged with HA or HIS6 at the C-terminus can be expressed in stably transfected mutant or wild-type cells from a di-cistronic vector. Like MWFE, it can be co-expressed with the endogenous wild-type protein. Hamster ESSS-HA (or HIS6) expressed in wild-type hamster cells appears to lower total complex I activity slightly relative to the wild-

Fig. 3. Sequence homologies in the ESSS protein from several mammalian species. The protein shown is the mature subunit found in the complex I after removal of the mitochondrial targeting sequence. The segment between L60 and L80 is the transmembrane domain. Acidic side chains are represented in red, basic side chains are represented in blue, and hydrophobic side chains are represented in green.

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type parent, and we speculate that the epitope on the relatively short C-terminal domain may slow down assembly or interfere with activity. Hamster ESSS-HA expressed in human cells is not incorporated into complex I, but it is accumulated to a significant level in human mitochondria. In contrast to MWFE, it is therefore not so much dependent on assembly into a final complex for its stabilization. That is not to say that it exists in the inner mitochondrial membrane in complete isolation (see below). It was of interest to investigate the expression and accumulation of transgenic hamster ESSS-HA in other respiration-deficient mutants in our collection. The ESSSHA protein is accumulated in the mitochondria of the CCL16-B2 mutants (lacking MWFE), in the V79-G7 mutants (defective in mitochondrial protein synthesis and hence missing all ND subunits), and in the V79-G8 mutants defective in complex I due to a mutation in an as yet unidentified X-linked gene. Since ESSS is part of the h membrane subcomplex, and MWFE is associated with the g membrane subcomplex, these two integral membrane proteins may be incorporated into distinct pre-complexes and hence stabilized independently of each other. It may be more significant that the ESSS protein can be stabilized and accumulated in the absence of any of the mitochondrially encoded subunits, suggesting a major interaction with one or more nuclear-encoded subunits [40]. 3.3. Cross-linking studies Studies have been initiated with mitochondria from various cell lines expressing HA-tagged ESSS after exposure to the cross-linking agent MBS (maleimidobenzoyl N-hydroxysuccinimide ester). In such experiments, approximately 40% of the HA antigen can be shifted to a second distinct band on SDS gels, suggesting crosslinking to an unidentified protein of ~18 kDa (Potluri, unpublished). Such an estimate is highly approximate for such cross-linked proteins. The most striking observation is that the same cross-linked species can be detected at comparable abundance in all cells expressing this transgenic hamster ESSS-HA. In V79-G18 cells, it is found in the large ~900-kDa complex and thus in its proper physiological context. In V79-G7 cells lacking all ND subunits, the cross-linked partner must therefore represent a nuclear-encoded subunit. In CCL16-B2 cells the finding of the cross-linked species is consistent with an association in a pre-complex that is formed independently of the MWFE protein. Finally, a cross-linked species with the same electrophoretic mobility is found in human mitochondria expressing hamster ESSS-HA. One can hypothesize that the heterologous hamster protein can form the same pre-complex in human cells, but further assembly into a mature complex is prevented due to protein incompatibilities further along the assembly pathway. Experiments to identify the cross-linked protein by MALDI-TOF are under way.

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4. Phosphorylation of complex I subunits There have been several reports in the literature suggesting that certain subunits of complex I can be phosphorylated in vitro [12,29,35–38,41] and in vivo [39]. The initial focus was on the 18-kDa subunit (AQDQ, NDUFS4) [35,38], but a recent publication has challenged the identification of this subunit as a target of protein kinases. In the most thorough analysis, Chen et al. [12] have identified the MWFE and ESSS subunits as the subunits (from beef heart) that are phosphorylated in vitro by a cAMP-dependent protein kinase. Furthermore, the sites of phosphorylation of these subunits were determined. Another intriguing recent paper has identified mutations in the protein kinase PINK1 as the cause of a rare familial form of Parkinson’s disease [55]. This kinase has been localized in mitochondria. The authors speculate that bwild-type PINK1 may protect neurons from stress-induced mitochondrial dysfunctionQ. The inhibition of complex I has been implicated in a number of models attempting to explain the selective death of neurons of the substantia nigra, and it is tempting to make complex I a target of this kinase, although there is no proof at this time. One problem in most of these studies is that phosphorylation was observed only in vitro, and no in vivo data are available. There is uncertainty about the nature of the kinase, about the signal that might control the kinase (or a phosphatase), and about the physiological conditions that would trigger a signaling cascade leading to either phosphorylation or dephosphorylation of a specific target in complex I (or any other subunit in the ETC). A possible in vivo role for phosphorylation of the NDUFS4 subunit was suggested by the studies of Papa et al. [38] in a patient with a fatal neurological syndrome. A homozygous 5-bp duplication in the cDNA of the NDUFS4 18 kDa subunit of complex I abolishes cAMP-dependent phosphorylation site of this protein. The authors claim that failure to phosphorylate also causes failure of the activation of the complex. However, the altered amino acid sequence of the protein could also be responsible for a significant structural perturbation that influences complex I assembly or activity. A definite conclusion must await a test of the role (if any) of this phosphorylation site by site-directed mutagenesis. Since the MWFE and ESSS subunits have gained prominence in this story from the results of the Cambridge group, our characterization of null mutants and the ability to complement them with MWFE or ESSS cDNAs provide an opportunity to investigate the potential significance of phosphorylation of these proteins. Accordingly, we have made Ser/Thr to Ala and Ser/Thr to glutamate mutations at most of the potential sites in these two proteins, and stable transfected cell lines have been established by using an independent selection with a dicistronic vector [61]. The consequences of these mutations are currently being evaluated. It should be noted

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that we can estimate complex I activity and assembly, but we do not know yet how to stimulate one or more potential mitochondrial kinases in vivo. Furthermore, such regulatory mechanisms may also operate in a tissuespecific manner, and fibroblasts may not be the ideal cell type for such an investigation. In the long term it may require the production of transgenic mice (knock-in mutations) for a complete physiological evaluation of such mutations.

5. Model for mammalian complex I assembly Results may be too preliminary to attempt to make an explicit model for complex I assembly in mammalian mitochondria. The formulation of such models could be guided or aided by several theoretical and experimental observations. It is highly unlikely that the complex is assembled one subunit at a time, and an initial assembly of modules (pre-complexes) is strongly suggested from several considerations. A modular nature of the complex has been proposed from evolutionary analyses and comparisons, suggesting that it is the result of a combination of an NADH dehydrogenase, hydrogenases, and ion transporters [19,20]. Thus, assembly of complex I could re-capitulate the evolutionary history in which subcomplexes or pre-complexes represent the functional units. Much of that model is based on homologies of the bacterial subunits with other known proteins, and it excludes the role of 32 additional subunits of the mammalian complex. A priori, it might have been plausible to imagine the assembly of the core of the mammalian complex, consisting of 14 subunits shared with the prokaryotes, followed by the addition of the other eukaryotic subunits. At this time there is no evidence for such a pathway. The ability to fractionate the complex into at least four distinct subcomplexes by a judicious choice of detergents (see Ref. [27] for a summary) could serve as another guide. In particular, one could assume, as in the proposed model for the Neurospora complex I [57,58], that the peripheral

membrane subcomplex and the integral membrane subcomplex are assembled independently and joined together in a final step. Evidence for such a model is missing in mammalian systems. Studies of complex I from human patients with isolated complex I deficiencies by BN gel electrophoresis have suggested the existence of assembly intermediates [1,53]. These intermediates are presumably detectable because in the presence of specific subunits with missense mutations the assembly is slow. However, as pointed out earlier, the observed pre-complexes could also be the result of a weakened complex that is dissociated under the conditions of solubilization required for BN-PAGE. In these human mutants complex I deficiency is partial and all the mutations were in nuclear genes encoding subunits of the peripheral subcomplex. Studies described here investigated the role of two integral membrane subunits, MWFE and ESSS, found in different subdomains (Ig and Ih, respectively) of the integral membrane domain of complex I (Fig. 1). The results are summarized in Table 1, together with results from another complex I mutant and from a mutant cell line defective in mitochondrial protein synthesis. The ESSS subunit (in Ih) can be imported and accumulated in mitochondria in the absence of any of the mitochondrially encoded subunits, and it is also stable in the absence of MWFE, or as a hamster protein in human mitochondria where it is not found in the complete complex I [40]. The simplest interpretation that it is stable as an isolated subunit is unlikely, since it can be specifically cross-linked with another complex I subunit in all these conditions (Potluri, unpublished). The MWFE and PSST subunits are not accumulated/stable in the absence of ESSS. Similarly, stabilization of MWFE requires at least the ND4 and ND6 subunits, while PSST is dependent on ND5 and ND6 [60]. A very speculative model might suggest that the incorporation of some (or all) of the ND subunits depends on a pre-complex containing ESSS and other nuclear-encoded subunits, followed by the incorporation of MWFE. At the same time, independent of the presence of MWFE, a large proportion of the subunits of the peripheral complex are assembled, and this assembly may start in

Table 1 Western blot analysis of mitochondria from respiration-deficient Chinese hamster mutants with available antisera against complex I proteins Ia (peripheral)

Ia (integral)

Peripheral subcomplex Wild type V79-G7a CCL16-B2b V79-G8b V79-G18b V79-G11 V79-G29 a b

Ih

Integral membrane subcomplex

51 kDa

30 kDa

TYKY

PSST

B8

39 kDa

MWFE

ESSS

B17

+ + + + +

+ + +/ + +/ + +

+ + + + +

+

+ + + + +

+ + +/ + +/ + +/

+

+ + + +

+ + + + +

+

The V79-G7 mutant is completely defective in mitochondrial protein synthesis. These complex I mutants represent three complementation groups of X-linked genes.

+

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solution (in the matrix), but is completed with some integral membrane subunits, since the peripheral membrane proteins are associated with the mitochondrial membrane in the absence of MWFE. As shown with the inducible MWFE model system, the synthesis of MWFE is not the ratelimiting step in complex I assembly. It should be mentioned that we have been unable to find any definitive evidence for the accumulation of assembly intermediates that can be identified by BN-PAGE. In all the mutants investigated under our conditions, no signal representing such an intermediate has been detected in the lower regions of the gels. The ~900-kDa complex is clearly present and identifiable by various means in wild-type and complemented mutant cells, and completely absent in the mutants. Probing with the available antisera failed to detect anything other than the occasional faint band or smear on BN-PAGE, even though the same antisera could detect strong bands for individual subunits on gels from SDSPAGE. Milder conditions for solubilization may be required in future experiments.

6. Summary and conclusions The isolation and characterization of Chinese hamster cell mutants with complete defects in complex I function due to the absence of either the MWFE or the ESSS subunits have provided us with valuable model systems for addressing a number of unsolved problems related to the assembly and function of this complex. The most striking results are that these small subunits are absolutely essential for assembly and function. Complementation of the null mutants has allowed the investigation of altered proteins, and in the future additional amino acid substitutions can be tested for their role in the catalytic activity or in the regulation of the complex. In the absence of either subunit, partially assembled subcomplexes appear to be assembled, but their complete composition remains to be elucidated.

Acknowledgements Research described in this review was supported by a grant from the United States Public Health Service (GM59909), by the Muscular Dystrophy Association, and by the United Mitochondrial Disease Foundation.

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