Assembly of nuclear DNA-encoded subunits into mitochondrial complex IV, and their preferential integration into supercomplex forms in patient mitochondria Michael Lazarou 1 , Stacey M. Smith 1,2 , David R. Thorburn 2 , Michael T. Ryan 1 and Matthew McKenzie 1 1 Department of Biochemistry, La Trobe University, Melbourne, Australia 2 Murdoch Children’s Research Institute and Genetic Health Services Victoria, Royal Children’s Hospital, and Department of Pediatrics, University of Melbourne, Australia Introduction The mitochondrial respiratory chain consists of four multi-subunit complexes (I–IV) that, through electron- transfer reactions, generate a proton gradient for the F 1 F o -ATPase (complex V) to synthesize ATP. Cyto- chrome c oxidase (complex IV) catalyzes the final step of the electron transfer chain, in which electrons are transferred from reduced cytochrome c to molecular oxygen. Mammalian complex IV comprises 13 differ- ent subunits, and the crystal structure of the bovine complex was solved over 10 years ago [1]. The three largest and most hydrophobic subunits (CO1–3) form the catalytic core, and all are encoded by mitochon- Keywords complex IV; cytochrome c oxidase; membrane proteins; mitochondria; oxidative phosphorylation Correspondence M. T. Ryan, Department of Biochemistry, La Trobe University, 3086 Melbourne, Australia Fax: +61 3 94792467 Tel: +61 3 94792156 E-mail: m.ryan@latrobe.edu.au (Received 4 June 2009, revised 18 August 2009, accepted 16 September 2009) doi:10.1111/j.1742-4658.2009.07384.x Complex IV is the terminal enzyme of the mitochondrial respiratory chain. In humans, biogenesis of complex IV involves the coordinated assembly of 13 subunits encoded by both mitochondrial and nuclear genomes. The early stages of complex IV assembly involving mitochondrial DNA- encoded subunits CO1 and CO2 have been well studied. However, the latter stages, during which many of the nuclear DNA-encoded subunits are incorporated, are less well understood. Using in vitro import and assembly assays, we found that subunits Cox6a, Cox6b and Cox7a assembled into pre-existing complex IV, while Cox4-1 and Cox6c subunits assembled into subcomplexes that may represent rate-limiting intermediates. We also found that Cox6a and Cox7a are incorporated into a novel intermediate complex of approximately 250 kDa, and that transition of subunits from this complex to the mature holoenzyme had stalled in the mitochondria of patients with isolated complex IV deficiency. A number of complex IV subunits were also found to integrate into supercomplexes containing combinations of complex I, dimeric complex III and complex IV. Subunit assembly into these supercomplexes was also observed in mitochondria of patients in whom monomeric complex IV was selectively reduced. We con- clude that newly imported nuclear DNA-encoded subunits can integrate into the complex IV holoenzyme and supercomplex forms by associating with pre-existing subunits and intermediate assembly complexes. Abbreviations BN-PAGE, blue-native polyacrylamide gel electrophoresis; CAP, chloramphenicol; DDM, n-dodecyl-b- D-maltoside; Dw m, membrane potential, LSI, late-stage intermediate. FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS 6701 drial DNA (mtDNA). The remaining 10 subunits are encoded by the nuclear genome, and, like other pro- teins, must be synthesized on cytosolic ribosomes before being imported and subsequently assembled at the mitochondrial inner membrane [2]. The subunits of complex IV encoded by nuclear DNA (nDNA) do not harbor enzymatic activity but function in the structural integrity, regulation and dimerization of the enzyme. For example, subunits Cox6a and Cox5 play roles in the regulation of enzymatic activity [3,4], while Cox6b, along with Cox6a, provides contacts sites for dimeriza- tion [1,5]. Mammalian complex IV forms a complex of approx- imately 200 kDa on blue native (BN) PAGE, but is also found in supercomplexes together with complex I and dimeric complex III [6]. More recently, it has been suggested that this supercomplex can associate with complex II, cytochrome c and complex V in a func- tional respirasome [7]. It has been suggested that supercomplexes enhance respiration due to coordinated channeling of the electron carriers ubiquinol and cyto- chrome c [6,8,9]. Isolated complex IV deficiency is one of the most common respiratory chain defects in humans, and is associated with various clinical phenotypes such as mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), Leigh disease and lactic acidosis [10,11]. Enzymatic deficiencies of complex IV that cause disease are often due to defects in the assembly and ⁄ or stability of the enzyme. To assemble complex IV, subunits translated from both genomes must come together in a coordinated and regulated manner. Studies carried out in the model organism Saccharomyces cerevisiae have provided insights into the biogenesis of complex IV, and led to the identifica- tion of over 20 assembly factors [12]. These nDNA- encoded assembly factors are not associated with assembled complex IV but instead act at various func- tional levels, ranging from subunit insertion and co-factor attachment to regulation of transcription ⁄ translation. Although yeast COX is a close model of its human counterpart, significant differences exist. For example, almost half of the yeast assembly factors do not appear to have human orthologs, and two subunits (Cox7b and Cox8) are found in the human assembly but not in yeast. In addition, as yeast lacks complex I, the supercomplex assemblies differ substantially from those seen in mammalian mitochondria. By tracking the subunit composition of subcomplexes using trans- lational inhibitors and metabolic labeling of mtDNA- encoded proteins, a model for the assembly of human complex IV was proposed [13]. This model was later refined through studies analyzing the composition of sub-assemblies in the mitochondria of patients deficient in complex IV [14,15]. The model proposes that com- plex IV is assembled via pre-formed intermediates in a stepwise process. Of the 10 nDNA-encoded subunits, only Cox4 and Cox5a are integrated at the early stages of assembly that involve formation of the catalytic core. The remaining nDNA-encoded subunits are thought to be added during the last steps of assembly; however, this part of the assembly pathway is the least studied and requires clarification. Furthermore, with the model focusing on the de novo synthesis of com- plex IV, it is unclear how newly imported nDNA- encoded subunits are incorporated in the presence of pre-existing holo-complex IV. In this study, we address the assembly of newly imported complex IV subunits in mitochondria from control cells and from cells of patients with defects in complex IV biogenesis. We found that, in the presence of pre-existing complex IV, newly imported nDNA- encoded subunits can integrate into the holoenzyme as well as into its supercomplex forms. Furthermore, sub- units Cox6a and Cox7a integrate into a novel late- stage intermediate complex of approximately 250 kDa. Assembly into, and progression from, this intermediate was defective in the mitochondria of patients deficient in complex IV, suggesting that it represents an impor- tant step in assembly of the holoenzyme. Results In vitro import and assembly of nDNA-encoded complex IV subunits The assembly of a number of nDNA-encoded com- plex IV subunits was investigated by importing them into isolated mitochondria and monitoring their assem- bly using BN-PAGE. As isolated mitochondria are used and protein synthesis does not take place under the conditions used (data not shown), the integration of a select newly imported subunit into a complex occurs through its association with pre-existing sub- units within the organelle [16,17]. Representative sub- units with cleavable (Cox4-1, Cox6a, Cox7a) and non-cleavable (Cox6b and Cox6c) presequences were selected for investigation. With the exception of Cox4-1, these nDNA-encoded complex IV subunits are postulated to integrate late in the assembly pathway [13,15]. Based on the crystal structure of complex IV [1], the selected subunits are positioned peripherally within the complex (Fig. 1A), and, apart from Cox6b, all contain a single transmembrane-spanning domain. 35 S-labeled complex IV subunit precursor proteins were generated in vitro using rabbit reticulocyte lysate, Mitochondrial complex IV assembly M. Lazarou et al. 6702 FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS and incubated with mitochondria isolated from cul- tured human fibroblasts for 10 or 60 min in the pres- ence or absence of a membrane potential (Dw m ). After import, external proteinase K was added to half of each sample to degrade non-imported protein. Samples were then subjected to SDS–PAGE, and radiolabeled subunits were detected using phosphorimage analysis (Fig. 1B). 35 S-labeled complex IV subunits bound to mitochondria, with the signal increasing over time (Fig. 1B, lanes 2 and 3). For the presequence-contain- ing subunits Cox4-1, Cox6a and Cox7a, an additional faster-migrating species accumulated, representing the mature form of the protein (Fig. 1B, lanes 2 and 3). An additional band seen after proteinase K treatment of Cox7a samples most likely represents a protease- resistant domain of the precursor, as it is also present in the absence of import (lane 7). Successful import of all subunits was determined by their protection from externally added proteinase K (Fig. 1B, lanes 5 and 6), their dependence on the membrane potential (Dw m ) for this protection (Fig. 1B, lanes 4 and 7), and, in the case of Cox4-1, Cox6a and Cox7a, processing of their presequences. We next tested the assembly of newly imported radiolabeled complex IV subunits using BN-PAGE. Radiolabeled subunits were imported into isolated mitochondria and all samples were treated with pro- teinase K before solubilization in n-dodecyl-b-d-malto- side (DDM) and BN-PAGE analysis (Fig. 2A). Use of this detergent results in partial dissociation of respira- tory chain supercomplexes, liberating monomeric (holo-) complex IV and a complex III 2 ⁄ complex IV supercomplex [18]. The migration of complex IV, dimeric complex III, complex I and their super- complexes (CIII 2 ⁄ CIV and CI ⁄ CIII 2 ) was determined by western blot analysis (Fig. 2A, right panels). Imported Cox6a and Cox7a were incorporated into both monomeric and supercomplex (CIII 2 ⁄ CIV) forms of complex IV, with the additional presence of an approximately 250 kDa complex (marked with an asterisk) that resolved after 10 min of import (Fig. 2A, lanes 3, 4, 9 and 10). Radiolabeled Cox6b also appeared to be incorporated into the holoenzyme, albeit weakly (Fig. 2A, lanes 5 and 6), while 35 S-labeled Cox4-1 was not incorporated into any distinct complexes, although some high-molecular- weight smearing was evident (lanes 1 and 2). Two dis- tinct complexes ranging between approximately 100 and 150 kDa were seen with newly imported Cox6c (Fig. 2A, lanes 7 and 8), although neither of these A B Fig. 1. Import of nDNA-encoded complex IV subunits. (A) Structural position of subunits Cox4 (magenta), Cox6b (red), Cox6c (green), Cox7a (blue) and Cox6a (orange) within the crystal structure of bovine complex IV [1]. (B) SDS–PAGE analysis of imported radiola- beled complex IV subunits. Precursor (p) and mature (m) forms of the subunits are identified. Samples were imported into mito- chondria in the presence or absence of a membrane potential (Dw m ), and treated with or without externally added proteinase K (Prot. K). A sample of lysate (representing 20% of added protein ⁄ import) is also shown (lane 1). Radiolabeled proteins were detected by phosphorimage analysis. M. Lazarou et al. Mitochondrial complex IV assembly FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS 6703 complexes co-migrated with holo-complex IV. Based on the BN-PAGE analysis of nDNA-encoded com- plex IV subunits, it can be concluded that some newly imported subunits can assemble with pre-existing subunits into holo-complex IV. Other subunits do not integrate into complex IV, perhaps because there is impaired progression of intermediates along the assem- bly pathway due to the use of isolated mitochondria. Our results are consistent with those of a previous study that analyzed the assembly of yeast complex IV subunits Cox4p, Cox5ap and Cox10p (equivalent to human Cox5b, Cox4-1 and Cox6a, respectively) [19]. In order to characterize the assembly of nDNA- encoded complex IV subunits into the various super- complex forms of complex IV, mitochondria were sol- ubilized in digitonin after subunit import. In this case, the respiratory chain components are also found in their supercomplex forms [18]. The relative positions of supercomplexes comprising complex I, complex III and complex IV (CI ⁄ CIII 2 ⁄ CIV), complex III and complex IV (CIII 2 ⁄ CIV), as well as dimeric com- plex III (CIII 2 ) and monomeric complex IV (CIV), were shown by western blot analysis (Fig 2B, right panels). Radiolabeled Cox6c was incorporated into a complex of approximately 200 kDa that resolved at a slightly lower position than the holo-complex IV, and also into a larger complex at approximately 1 MDa. Likewise, 35 S-labeled Cox4-1 was found in large A CIV * CI 669 440 kDa Cox4-1 Cox6b Cox6c Cox7aCox6a BN-PAGE 0.65% DDM Time (min)10 60 10 60 10 60 10 60 10 60 12345678910 α-complex I α-complex III α-complex IV 134 67 B 669 440 kDa Cox4-1 Cox6b Cox6c Cox7aCox6a CIV BN-PAGE 1% Digitonin Time (min)10 60 10 60 10 60 10 60 10 60 12345678910 CIII α-complex I α-complex III α -complex IV 134 67 * Fig. 2. BN-PAGE analysis of imported radio- labeled complex IV subunits. 35 S-labeled complex IV subunits were individually incu- bated with isolated fibroblast mitochondria for increasing times as indicated. Samples were treated with proteinase K, and solubi- lized in either (A) DDM-containing buffer or (B) digitonin-containing buffer. Radiolabeled proteins were detected by phosphorimage analysis. Right panels: complex IV (CIV), complex I (CI), complex III (CIII 2 ), and their supercomplex forms (CI ⁄ CIII 2 ), (CI ⁄ CIII 2 ⁄ CIV) and (CIII 2 CIV) were identified by western blot analysis using antibodies to the complex I subunit NDUFA9 (a-com- plex I), the core I subunit of complex III (a-complex III) and the COI subunit of complex IV (a-complex IV). The asterisk indicates a complex of approximately 250 kDa. Mitochondrial complex IV assembly M. Lazarou et al. 6704 FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS complexes in the range of approximately 700–1000 kDa (lanes 1 and 2), but these did not co-migrate with any of the complex IV-containing supercomplexes. The identity of these complexes is unknown. The complex of approximately 250 kDa observed for newly imported subunits Cox6a and Cox7a after DDM solu- bilization was poorly resolved using digitonin (Fig. 2B, marked with an asterisk). However, Cox6a and Cox7a as well as Cox6b assembled into holo-complex IV and into complexes co-migrating with the supercomplex forms CIII 2 ⁄ CIV and CI ⁄ CIII 2 ⁄ CIV. Of note, the intensity of assembled 35 S-labeled Cox6b was stronger in mitochondria solubilized in digitonin than those sol- ubilized in DDM (compare lane 6 in Fig. 2A and 2B). This is consistent with a previous report indicating that Cox6b can be preferentially stripped from complex IV in the presence of DDM [20] due to the peripheral nat- ure of this subunit. From these results, we conclude that, in isolated mitochondria, some newly imported subunits have the capacity to integrate into the holoen- zyme as well as into supercomplexes containing com- plex IV. Furthermore, given that yeast mitochondria lack complex I, this analysis also characterized nDNA- encoded subunit integration into more complicated supercomplexes that additionally contain pre-existing complex I. Assembly profile of imported Cox6a in human and yeast mitochondria At early stages of import, the nDNA-encoded com- plex IV subunits Cox6a and Cox7a were found also to be incorporated into a complex of approximately 250 kDa, slightly higher than monomeric complex IV. Both subunits are thought to integrate into the assem- bly pathway at a late stage [13,21]. By studying the import and assembly of Cox6a (see below), we have established that the complex of approximately 250 kDa represents a novel intermediate, and have termed it the late-stage intermediate (LSI) complex (Fig. 3). 35 S-labeled Cox6a was imported into isolated mitochondria for various times before solubilization in DDM and BN-PAGE analysis (Fig. 3A). At early time points, Cox6a was predominantly incorporated into the LSI complex, while a minor amount accumulated into holo-complex IV and its supercomplex, CIII 2 ⁄ CIV. Over the time course of the experiment, Cox6a accumulated into holo-complex IV, while the signal for the LSI complex remained relatively constant. Western blot analysis confirmed the relative positions of com- plex IV and CIII 2 ⁄ CIV (Fig. 3A, right-hand panel); however, the LSI complex was not seen. Assembly intermediates generally cannot be resolved with anti- bodies due to their low steady-state levels, but import using small amounts of radiolabeled protein can detect their presence [16,22–24]. Next, an in vitro import and chase experiment was performed. 35 S-labeled Cox6a was imported for 5 min to accumulate the subunit at the LSI complex before mitochondria were re-isolated and then further incubated in the absence of additional radiolabeled precursor (Fig. 3B, lanes 1–4). Over the chase period, the intensity of 35 S-labeled Cox6a in the LSI complex decreased, with a concomitant increase in the intensity of labeling of holo-complex IV. 35 S-labeled Cox6a was also imported into mitochon- dria isolated from cells that had been pre-incubated with chloramphenicol (CAP). Under these conditions, complex IV assembly intermediates containing mtDNA-encoded subunits are unlikely to be present, and a pool of unassembled nDNA-encoded subunits may accumulate. Any 35 S-labeled Cox6a assembly into holo-complex IV is therefore likely to occur via inte- gration into the pre-existing complex as opposed to new assemblies. As can be seen in Fig. 3C, 35 S-labeled Cox6a was incorporated into the LSI complex and holo-complex IV in mitochondria from both control (lanes 1–3) and CAP-treated cells (lanes 4–6). Given that the import of 35 S-labeled Cox6a was unaffected (Fig. 3D), the decreased signal of assembled Cox6a observed in the CAP-treated samples is probably a result of decreased levels of fully assembled com- plex IV as shown by western blot analysis (bottom panels in Fig. 3C). As in organello labeling is inefficient under the conditions used (data not shown) and Cox6a assembly occurs even in mitochondria isolated from CAP-pretreated cells, these results support the possibil- ity that late-assembling subunits such as Cox6a have the capacity to assemble into complex IV by cycling with pre-existing subunits. In order to eliminate the possibility that the precur- sor form is found in the LSI complex, 35 S-labeled Cox6a was imported into mitochondria for various times (without proteinase K treatment) and subjected to BN-PAGE followed by SDS–PAGE in the second dimension (Fig. 3E). At early time points, the precur- sor form of 35 S-labeled Cox6a (as judged by its co-migration with the lysate control sample) was found in a high-molecular-weight smear, presumably bound to molecular chaperones and ⁄ or the translocase of the outer mitochondrial membrane (TOM) machinery [25]. The mature form of 35 S-labeled Cox6a was initially found in the more slowly migrating LSI complex, and over time assembled into mature complex IV. The position of mature complex IV was confirmed by immunostaining (bottom panel). Based on these results, we conclude that the LSI complex represents a M. Lazarou et al. Mitochondrial complex IV assembly FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS 6705 novel intermediate for the biogenesis of Cox6a, and hence Cox7a, into human complex IV. Coomassie staining of mitochondria did not reveal the presence of the LSI complex even though complex IV could be detected (data not shown), consistent with the LSI complex being a short-lived intermediate. As an ortholog of Cox6a is found in yeast [termed Cox10p (COX13)], we determined whether the LSI complex is evolutionarily conserved. In vitro import of Cox10p has previously been used to analyze com- plex IV assembly in yeast, but digitonin-solubilized mitochondria were employed and hence only super- complexes were observed [19]. Furthermore, a time course of the import was not performed. Radiolabeled Cox10p was imported into isolated yeast mitochondria, and all samples were treated with proteinase K before solubilization in DDM and BN-PAGE analysis (Fig. 4A). The time-course analysis revealed that the assembly pathway of Cox10p resembles that of its human counterpart. The intensity of the LSI complex remained consistent over time, and the holo-com- plex IV of approximately 200 kDa accumulated. Import and chase analysis of 35 S-labeled Cox10p (Fig. 4B, lanes 1–4), revealed that, like its human Cox6a counterpart, Cox10p initially accumulated in the LSI complex and was chased over time into holo- complex IV. Evolutionary conservation of the LSI complex containing Cox6a ⁄ Cox10p suggests that it is an important step in the biogenesis of these subunits and subsequently the holoenzyme. Assembly analysis of 35 S-labeled Cox6a in patient mitochondria The assembly of subunit Cox6a was investigated fur- ther using fibroblast mitochondria from two patients LSIC CIV 669 440 kDa α-complex IV 669 440 kDa LSIC CIII 2 /CIV CIII 2 /CIV CIII 2 /CIV CIV α -complex I V 35 S-Cox6a (Human) Time (min)10 30 60 12 3 Chase (min) 0 15 30 60 1 2 3 4 AB 134 67 134 67 35 S-Cox6a (Human) 669 440 kDa 134 67 LSIC CIV Control + CAP 12 345 6 Time (min) 10 30 60 10 30 60 123 4 56 α-complex IV α-complex II CIV CII Control + CAP 1 23456 Time (min) 10 30 60 10 30 60 p m p m - Prot. K + Prot. K CIV BN-PAGE SDS-PAGE Lysate control 5 min 10 min 60 min 30 min α-complex IV p LSIC CD E Fig. 3. Import and assembly of Cox6a into pre-existing complex IV. (A) 35 S-labeled Cox6a was incubated for various times with mito- chondria isolated from human fibroblasts. Samples were treated with proteinase K and subjected to DDM solubilization, BN-PAGE and phosphorimaging. Right lane: the migration of CIV and CIII 2 ⁄ CIV supercomplexes was identified by western blot analysis using antibodies to the complex IV subunit COI (a-complex IV). (B) 35 S-labeled Cox6a was incubated for 5 min with mitochondria after removal of free 35 S-labeled Cox6a and chase of assembly for vari- ous times as indicated. Mitochondria were treated as in (A). (C) 35 S-labeled Cox6a was incubated for 10–60 min with mitochondria isolated from control fibroblasts that had been pre-treated with or without chloramphenicol (CAP) for 12 h. Samples were treated with proteinase K before being solubilized in DDM-containing buffer and subjected to BN-PAGE, western transfer and phosphorimage analy- sis (top panel), followed by immunodecoration using antibodies to COI (a-complex IV) and 70 kDa subunit (a-complex II) (bottom panel). (D) 35 S-labeled Cox6a was imported into mitochondria as described in (C), with and without proteinase K treatment, before SDS–PAGE and phosphorimage analysis. (E) 35 S-labeled Cox6a was imported into control mitochondria for increasing times as indi- cated. Samples were solubilized in DDM-containing buffer, and sub- jected to BN-PAGE in the first dimension followed by SDS–PAGE in the second dimension. Gels were subjected to phosphorimaging. The position of complex IV was confirmed based on immunoblot analysis using antibodies against COI (bottom panel). The lysate control shows the position of the 35 S-labeled Cox6a precursor spe- cies after one-dimensional SDS–PAGE. p, 35 S-labeled Cox6a precur- sor form; m, 35 S-labeled Cox6a mature form; CIV, complex IV; LSIC, late-stage intermediate complex; CIII 2 ⁄ CIV, complex III 2 ⁄ com- plex IV supercomplex. Mitochondrial complex IV assembly M. Lazarou et al. 6706 FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS with Leigh syndrome involving isolated complex IV deficiency [18]. Patient 1 was homozygous for a patho- genic frameshift mutation in the gene encoding the complex IV assembly factor Surf1. Patient 2 had iso- lated complex IV deficiency and an as yet unknown nuclear gene mutation, although mutations in com- plex IV subunits and known assembly factors have been excluded (data not shown). This analysis served to test whether defects in formation of the LSI com- plex may be involved in altered complex IV assembly and human disease, and also to assess the possible involvement of Surf1 in the formation and assembly of the LSI complex. BN-PAGE and western blot analysis of mitochondria solubilized in DDM indicated that the fibroblasts of patients 1 and 2 had very low levels of mature complex IV (Fig. 5A, right panels). In addi- tion, the CIII 2 ⁄ CIV supercomplex was not detected in patient mitochondria, although the levels of complex I, complex III and the CI ⁄ CIII 2 supercomplex were simi- lar to control. The amount of assembled 35 S-labeled Cox6a in the mitochondria of patient 1 (Fig. 5A, lanes 5–7) was lower relative to control (lanes 1–3), with reduced levels of both the LSI complex and holo-com- plex IV. This is most likely due to the substantially decreased levels of complex IV in these mitochondria. In the mitochondria of patient 2, 35 S-labeled Cox6a was initially incorporated into the LSI complex (Fig. 5A, lane 13) where it accumulated over time (lanes 14 and 15). The radioactivity below the LSI complex and at the position of the CIII 2 ⁄ CIV complex may represent low levels of assembled complex IV (lane 15). Thus, it appears that progression from the LSI complex to holo-complex IV is defective in the mitochondria of patient 2 compared to the assembly profile for the control (compare lanes 9–11 and 13–15). As expected, disruption of the membrane potential (Dw m ) abolished assembly in all samples due to inhibi- tion of import. SDS–PAGE was also performed in order to eliminate the possibility that the assembly defects for Cox6a in patient mitochondria were a result of impaired import. As shown in Fig. 5B, 35 S-labeled Cox6a was efficiently imported into both patient and control mitochondria in a Dw m -dependent manner. Time (min) 10 30 60 10 30 60 Patient 1 Control 6060 ++ +– – – Δ m + ++ CIII 2 /CIV CIV LSIC 669 440 kDa A B 10 30 60 10 30 60 Patient 2 Control 6060 ++ ++ ++– 123 56784 91011 1314151612 α-complex IV α-complex III α-complex I CIII 2 CI/CIII 2 Time (min)10 30 60 10 30 60 6060 +++– – Δ +++ SDS-PAGE Control Patient 1 p m p m –Prot. K +Prot. K Lysate Patient 2 p m 123 56784 9 BN-PAGE 0.65% DDM Control Patient 1 Patient 2 Control Patient 1 Pati ent 2 Control Patient 1 Patient 2 134 67 Fig. 5. Import and assembly of Cox6a in control and patient mitochondria. Mitochondria from control or patient cells were incubated with 35 S-labeled Cox6a for increasing times in the presence or absence of a membrane potential (Dw m ). Half of each sample was treated with proteinase K before being split in two and (A) solubilized in DDM-containing buffer and subjected to BN-PAGE (protease-treated samples only), or (B) SDS–PAGE and phosphorimaging. The right panels in (A) show western blot analysis of complex IV, complex III and complex I in mitochondrial preparations. CI, complex I; CIII 2 , complex III dimer; CIV, complex IV; LSIC, late-stage intermediate complex; CIII 2 ⁄ CIV, complex III 2 ⁄ complex IV supercomplex; CI ⁄ CIII 2 , complex I ⁄ complex III supercomplex. 669 440 kDa AB Time (min) 10 30 60 LSIC CIV 123 134 67 35 S-Cox10p (Yeast) 669 440 kDa Chase (min) 0153060 35 S-Cox10p (Yeast) LSI C CIV 12 3 4 134 67 Fig. 4. Import and assembly of the yeast Cox6a ortholog Cox10p in yeast mitochondria. (A) 35 S-labeled Cox6a was incubated for vari- ous times with mitochondria isolated from yeast cells. Samples were treated with proteinase K and subjected to DDM solubiliza- tion, BN-PAGE and phosphorimaging. (B) 35 S-labeled Cox6a was incubated for 5 min with mitochondria after removal of free 35 S-labeled Cox6a and chase of assembly for various times. Mito- chondria were treated as in (A). CIV, complex IV; LSIC, late-stage intermediate complex. M. Lazarou et al. Mitochondrial complex IV assembly FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS 6707 These results suggest that incorporation of Cox6a into the LSI complex and its progression to complex IV is not dependent on Surf1, and the slowed rate of assem- bly into complex IV is most likely a result of reduced levels of holo-complex IV. In the mitochondria of patient 2, the progression of Cox6a into complex IV is effectively stalled, suggesting that the underlying defect may be related to a late stage in complex IV biogenesis. Assembly of nDNA-encoded complex IV subunits into supercomplexes in control and patient mitochondria When we analyzed respiratory complexes in patient mitochondria after digitonin solubilization and BN- PAGE, we found that the residual complex IV was predominantly found in supercomplexes. Using anti- bodies against the subunits of complexes I, III and IV (Fig. 6A, right panels), it appeared that only the super- complex form (CI⁄ CIII 2 ⁄ CIV) was present in patient mitochondria. An additional faster-migrating species was also detected in patient mitochondria, and is likely to represent the CI ⁄ CIII 2 supercomplex. In control mitochondria, complex IV was also found in its super- complex forms, but its most predominant form was as a monomer. As complex IV was only detected in the CI ⁄ CIII 2 ⁄ CIV supercomplex in patient mitochondria, this suggests that this form is particularly stable and ⁄ or crucial for respiratory function. However, we cannot exclude the possibility that complex IV found Cox4-1Cox6b Cox6c Cox7a Cox6a Time (min) 10 60 10 60 10 60 10 60 10 60 12345678910 669 440 kDa CI/CIII 2 /CIV CIV CIII 2 BN-PAGE 1% Digitonin α -complex I α -complex III α -complex IV CI/CIII 2 669 440 kDa A B BN-PAGE 1% Digitonin α-complex IV α-complex III α-complex I Control Patient 1 Patient 2 CIII 2 /CIV CIV CIII 2 CI/CIII 2 /CIV CI/CIII 2 CI/CIII 2 /CIV Control Patient 1 Patient 2 Control Patient 1 Patient 2 Time (min) 10 30 60 10 30 60 Patient 1 Control 6060 + m + + Δ + ++ 10 30 60 10 30 60 Patient 2 Control 6060 ++ + + ++ 123 56784 9 10 11 13 14 15 1612 134 67 134 67 * LSIC Fig. 6. nDNA-encoded subunits assemble into supercomplexes of control and patient mitochondria. (A) Mitochondria from control or patient cells were incubated with 35 S-labeled Cox6a for increasing times in the presence or absence of a membrane potential (Dw m ). Each sample was treated with proteinase K before being solubilized in digitonin-containing buffer and subjected to BN-PAGE and phosphorimaging. (B) 35 S-labeled complex IV subunits were incubated with mitochondria from patient 1 for 10 and 60 min and treated as described in (A). The right panels in (A) and (B) show the migration of complex IV (CIV), dimeric complex III (CIII 2 ), CIII 2 ⁄ CIV supercomplex, complex I (CI) ⁄ CIII 2 ⁄ CIV supercomplex and CI ⁄ CIII 2 supercomplex by western blot analysis. The asterisk indicates the complex of approximately 100 kDa. Mitochondrial complex IV assembly M. Lazarou et al. 6708 FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS in supercomplexes of patient mitochondria is not all fully assembled. We determined whether the integration of newly imported Cox6a into supercomplexes was defective in patient mitochondria. Radiolabeled Cox6a was imported into mitochondria isolated from patient 1, patient 2 and control fibroblasts, solubilized in digito- nin and analyzed using BN-PAGE (Fig. 6A). As observed above (Fig. 5), the assembly of newly imported Cox6a into monomeric complex IV was reduced in mitochondria from cells of both patient 1 (Fig. 6A, lanes 5–7) and patient 2 (lanes 13–15). Although the LSI complex was not clearly resolved after solubilization using digitonin, mitochondria from cells of patient 2 showed smearing above complex IV (lanes 13–15), and this may represent the impaired pro- gression of 35 S-labeled Cox6a from the LSI complex to holo-complex IV. Incorporation of 35 S-labeled Cox6a into the CIII 2 ⁄ CIV supercomplex was reduced in mito- chondria from both patient 1 and patient 2; however, its incorporation into the CI ⁄ CIII 2 ⁄ CIV supercomplex was not impaired, with an overall signal comparable to that in controls (Fig. 6A). The relatively efficient assembly of newly imported Cox6a into the CI ⁄ CIII 2 ⁄ CIV supercomplex in patient mitochondria led us to investigate the assembly of additional nDNA-encoded subunits. Mitochondria iso- lated from fibroblasts from patient 1 were chosen for this study because of the better growth rate of these cells in culture. As shown in Fig. 6B, newly imported Cox4-1, Cox6b, Cox6c, Cox7a and Cox6a all effi- ciently assembled into the CI ⁄ CIII 2 ⁄ CIV supercomplex in the mitochondria of patient 1. The subunit assembly profile also differed to that observed in control mito- chondria, in that Cox4-1 and Cox6c did not assemble into any complex IV-containing supercomplexes (see Fig. 2B, lanes 1–2 and lanes 5–6). However, in addi- tion to its assembly into supercomplexes in the mito- chondria of patient 1, Cox4-1 also assembled into a complex of approximately 100 kDa (Fig. 6B, lanes 1 and 2) that was not observed in control mitochondria. Cox4-1 is believed to form an early assembly interme- diate with CO1 [15], and the species of approximately 100 kDa may represent such an intermediate; however, further characterization is required. Additional low- molecular-weight complexes were also seen for Cox6c import in the mitochondria of patient 1 that were not seen in control mitochondria (see Fig. 2B, lanes 5 and 6). These smaller complexes may represent rate-limit- ing intermediates due to assembly defects in the mito- chondria of patient 1. It is possible that, in the absence of monomeric complex IV, a portion of these inter- mediates can combine with complex I and complex IV to form the supercomplexes that are seen under these conditions. Discussion The current model of complex IV assembly follows a sequential pathway that begins with the mitochondrial translation of CO1 followed by integration of addi- tional subunits and co-factors through a set of defined intermediates (for reviews on complex IV assembly, see [12,21,26,27]). A number of assembly factors have been identified that assist in the process and act at the levels of regulation [28], co-factor biosynthesis and insertion [29,30] and chaperoning of assembly intermediates [31]. Much of our current knowledge regarding complex IV biogenesis has been provided by studies using the model organism S. cerevisiae. Most studies have focused on the early stages of assembly that involve formation of the catalytic core consisting of the mtDNA-encoded subunits CO1, CO2 and CO3. Thus details of the latter stages in which the majority of nDNA-encoded subunits that surround the core are assembled remain largely unknown. In particular, it is not clear how a newly imported nDNA-encoded subunit assembles in the presence of pre-existing complex IV. Furthermore, there are a number of dif- ferences between mammalian and yeast mitochondria that affect complex IV biogenesis, and these may be relevant to disease. These include the presence of struc- tural subunits and assembly factors in yeast that do not appear to have homologs in mammals, and differences in supercomplex forms. The consequence of these differences is that further analysis of the biogene- sis of the mammalian enzyme is required. Assembly of nDNA-encoded complex IV subunits Of the five nDNA-encoded subunits investigated in this study, Cox6a, Cox7a and Cox6b were found to assemble into both monomeric and supercomplex forms of complex IV (Fig. 1). According to the current model of complex IV assembly, subunit Cox6b assem- bles into the S3 subcomplex together with a host of other nDNA-encoded subunits as well as CO3 [13,15]. However, it has been suggested more recently that Cox6b is incorporated at the very last step of com- plex IV assembly [32], possibly after addition of the late-assembling subunits Cox6a and Cox7a. Based on the results presented here, it appears that subunits that are incorporated late in the assembly pathway have a greater propensity to assemble into pre-existing com- plex IV in isolated mitochondria. Previously, we have shown that individual, newly imported complex I M. Lazarou et al. Mitochondrial complex IV assembly FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS 6709 subunits can dynamically exchange with their pre-exist- ing counterparts to assemble into the complex [16]. Similar findings have also been reported for other complexes [24,33–36]. It is therefore possible that these newly imported nDNA-encoded complex IV subunits are assembling into the pre-existing complex via a similar mechanism. The remaining two subunits, Cox4-1 and Cox6c, were not found to assemble into holo-complex IV; however, they were found to integrate into additional complexes. Subunit Cox6c resolved into two distinct complexes in the range of approximately 100–150 kDa when the detergent DDM was used. The assembly pro- file of Cox6c differed in digitonin-solubilized samples, where it was found to assemble into a complex of approximately 160 kDa and also a large species of approximately 1 MDa. Import of subunit Cox4-1 revealed that it also assembled into large complexes ranging from approximately 700–1000 kDa, although it was not found in any distinct complexes when DDM was used. This subunit is considered to be one of the first nDNA-encoded subunits to integrate into sub- assemblies of complex IV [13,14]. In a previous study, import analysis of the yeast ortholog of human Cox4-1 revealed that it assembles into a number of complexes ranging from 250 to 450 kDa in size [19]. These com- plexes were found to contain assembly factors such as Cox14p, Coa1p and Shy1p as well as complex IV su- bunits. Of these assembly factors, only Shy1p has a human homolog, termed Surf1 [37,38], and this could account for the different complexes observed in yeast and mammalian mitochondria. Nevertheless, the com- plexes identified here may represent rate-limiting inter- mediates that require additional factors not found in yeast mitochondria for Cox4-1 and Cox6c subunit maturation and ⁄ or assembly. A novel late-stage intermediate complex for Cox6a and Cox7a assembly into complex IV In mitochondria solubilized with DDM, newly imported subunits Cox7a and Cox6a were found to integrate into the LSI complex of approximately 250 kDa prior to their incorporation into holo-com- plex IV and its supercomplex forms. Similar results were observed in yeast mitochondria when the assem- bly of the Cox6a ortholog, Cox10p was analyzed, thus indicating that the assembly profile for this subunit is evolutionarily conserved. In comparison to com- plex IV, the LSI complex is not visible on Coomassie- stained 2D gels (data not shown), supporting the conclusion that it is a low-level intermediate complex that is only detected with radiolabeled proteins. Previous import and assembly analysis of Cox10p using digitonin solubilization did not reveal the pres- ence of a complex of approximately 250 kDa [19], con- sistent with our findings that this complex is not well resolved in digitonin. Analysis of mitochondria from a patient harboring an isolated complex IV deficiency of unknown etiology (patient 2) revealed that Cox6a assembly into the LSI complex had stalled. Although a complex of similar size has previously been described in yeast mitochondria [19,39], this complex is likely to differ as yeast contains assembly factors that are not present in humans. Furthermore, the studies in yeast used digitonin for solubilization, and the LSI complex is not clearly resolved in this detergent. Therefore, we conclude that the LSI complex represents a novel com- plex for maturation of at least Cox6a and Cox7a into complex IV, and that this assembly process is per- turbed in human disease. A possible explanation for the larger size of the LSI complex relative to the holo- enzyme is that a specific accessory factor is associated with this late-stage intermediate that is displaced after integration of the subunits into the final complex. Alternatively, Cox6a and Cox7a may integrate into the LSI complex and then be transferred into the maturing complex IV. Integration of nDNA-encoded subunits into supercomplexes in patient mitochondria Using digitonin solubilization of mitochondria to visu- alize supercomplexes, it was found that subunit Cox6a assembled into the CI ⁄ CIII 2 ⁄ CIV supercomplex in the mitochondria of patients 1 and 2. Immunoblot analysis of patient mitochondria revealed that all detectable complex IV was present in a supercomplex with com- plexes I and III. This is in contrast to control mito- chondria in which the majority of complex IV is not associated with supercomplexes and instead resolves in its monomeric form [18,40,41]. As that complex IV may be important for the assembly ⁄ stability of com- plex I [42,43] and functions within respirasomes [7], it follows that limiting amounts of complex IV (in partial or fully assembled forms) could be sequestered within supercomplexes, as observed in patient mitochondria. All subunits tested here (Cox4-1, Cox6a, Cox6b, Cox6c and Cox7a) were found to efficiently assemble into the CI ⁄ CIII 2 ⁄ CIV supercomplex. Of particular interest were the newly imported subunits Cox4-1 and Cox6c, as they did not assemble into complex IV or its supercomplex forms in control mitochondria, but had the ability to integrate into the CI ⁄ CIII 2 ⁄ CIV super- complex in patient mitochondria. As the subunits Cox4-1, Cox6b, Cox6c, Cox7a, and Cox6a all Mitochondrial complex IV assembly M. Lazarou et al. 6710 FEBS Journal 276 (2009) 6701–6713 ª 2009 The Authors Journal compilation ª 2009 FEBS [...]... [19] for the biogenesis of yeast complex IV In summary, the work presented here shows that newly imported complex IV subunits can integrate into complex IV and its supercomplexes by associating with pre-existing subunits and integrating into intermediate complexes A novel late-stage intermediate complex was identified that is evolutionarily conserved The exact composition of this complex awaits further... [14] A small pool of remaining subunits was found predominantly in stalled assembly intermediates Therefore, it is possible that newly imported subunits are channeled more efficiently into assembly intermediates in patient mitochondria as they do not have to compete with a large pool of preexisting subunits In addition, these assembly intermediates may already be in supercomplex forms, as recently proposed... assembled into the supercomplex, this suggests that none of these subunits are rate-limiting for assembly Although it is not clear why these subunits assemble into the supercomplex in patient but not control mitochondria, it has been shown previously that the steady-state levels of various complex IV subunits are reduced in the mitochondria of patients with a mutation in the SURF1 gene [14] A small pool of. .. cytochrome c oxidase assembly defects due to mutations in SCO2 and SURF1 Biochem J 392, 625–632 16 Lazarou M, McKenzie M, Ohtake A, Thorburn DR & Ryan MT (2007) Analysis of the assembly profiles for mitochondrial- and nuclear- DNA-encoded subunits into complex I Mol Cell Biol 27, 4228–4237 17 Lazarou M, Thorburn DR, Ryan MT & McKenzie M (2009) Assembly of mitochondrial complex I and defects in disease Biochim... DNA library and cloned into pGEM-4Z Mitochondrial complex IV assembly sucrose, 1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, and centrifuged at 800 g at 4 °C for 10 min to obtain a postnuclear supernatant Mitochondria were pelleted by centrifugation at 10 000 g at 4 °C for 20 min In vitro import and assembly assays Generation of radiolabeled nDNA-encoded precursor proteins was performed by in vitro... DM, Thorburn DR & Ryan MT (2007) Human CIA30 is involved in the early assembly of mitochondrial complex I and mutations in its gene cause disease EMBO J 26, 3227– 3237 23 Johnston AJ, Hoogenraad J, Dougan DA, Truscott KN, Yano M, Mori M, Hoogenraad NJ & Ryan MT (2002) Insertion and assembly of human tom7 into the preprotein translocase complex of the outer mitochondrial membrane J Biol Chem 277, 42197–42204... turnover in single, functioning membrane protein complexes Nature 443, 355–358 35 Stricker J, Maddox P, Salmon ED & Erickson HP (2002) Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching Proc Natl Acad Sci USA 99, 3171– 3175 Mitochondrial complex IV assembly 36 Daley DO (2008) The assembly of membrane proteins into complexes Curr Opin Struct... O, Guerin B & Rigoulet M (1999) ATP-regulation of cytochrome oxidase in yeast mitochondria: role of subunit VIa, Eur J Biochem 263, 118–127 4 Allen LA, Zhao XJ, Caughey W & Poyton RO (1995) Isoforms of yeast cytochrome c oxidase subunit V affect the binuclear reaction center and alter the kinetics of interaction with the isoforms of yeast cytochrome c J Biol Chem 270, 110–118 5 Yoshikawa S, Shinzawa-Itoh... in a high molecular weight complex and is required for efficient assembly of cytochrome c oxidase in yeast FEBS Lett 498, 46–51 40 McKenzie M, Lazarou M, Thorburn DR & Ryan MT (2006) Mitochondrial respiratory chain supercomplexes are destabilized in Barth syndrome patients J Mol Biol 361, 462–469 41 Schagger H & Pfeiffer K (2001) The ratio of oxidative phosphorylation complexes I–V in bovine heart mitochondria. .. Import of Cox10p was performed as described by Ryan et al [44] Cell culture and mitochondrial isolation Primary skin fibroblasts grown from patient skin biopsy material (obtained by consent) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) containing 10% v ⁄ v fetal bovine serum at 37 °C under an atmosphere of 5% CO2 ⁄ 95% air supplemented with 50 lgÆmL)1 uridine For . Assembly of nuclear DNA-encoded subunits into mitochondrial complex IV, and their preferential integration into supercomplex forms in patient mitochondria Michael. into the maturing complex IV. Integration of nDNA-encoded subunits into supercomplexes in patient mitochondria Using digitonin solubilization of mitochondria