mr 1s interacts with pet100 and pet117 in module based assembly of human cytochrome c oxidase

Article MR-1S Interacts with PET100 and PET117 in ModuleBased Assembly of Human Cytochrome c Oxidase Graphical Abstract Authors Sara Vidoni, Michael E Harbour, Sergio Guerrero-Castillo, , Susanne Arnold, Erika Fernandez-Vizarra, Massimo Zeviani Correspondence emfvb2@mrc-mbu.cam.ac.uk (E.F.-V.), mdz21@mrc-mbu.cam.ac.uk (M.Z.) In Brief By quantitatively analyzing the COX intermediates accumulated in a MT-CO3 mutant, Vidoni et al modify the existing sequential COX assembly model to a module-based pathway and describe MR-1S as a vertebrate-specific factor involved in COX maturation, cooperating with the highly conserved PET100 and PET117 chaperones Highlights d d d d Human cytochrome c oxidase (COX) assembles in a modular rather than sequential fashion HIGD1A is associated to COX and is assembled in the initial phase MR-1S, the smallest product of PNKD, is a vertebratespecific COX assembly factor MR-1S interacts with the highly conserved COX chaperones PET100 and PET117 Vidoni et al., 2017, Cell Reports 18, 1727–1738 February 14, 2017 ª 2017 Elsevier Inc http://dx.doi.org/10.1016/j.celrep.2017.01.044 Cell Reports Article MR-1S Interacts with PET100 and PET117 in Module-Based Assembly of Human Cytochrome c Oxidase Sara Vidoni,1 Michael E Harbour,1 Sergio Guerrero-Castillo,2 Alba Signes,1 Shujing Ding,1 Ian M Fearnley,1 Robert W Taylor,3 Valeria Tiranti,4 Susanne Arnold,2 Erika Fernandez-Vizarra,1,* and Massimo Zeviani1,5,* 1MRC Mitochondrial Biology Unit, Cambridge CB2 0XY, UK Center for Mitochondrial Medicine, Radboud University Medical Center, 6525 Nijmegen, the Netherlands 3Wellcome Trust Centre for Mitochondrial Research, Institute of Neuroscience, Newcastle University, Newcastle upon Tyne NE1 7RU, UK 4Unit of Molecular Neurogenetics, Fondazione Istituto Neurologico Carlo Besta, 20133 Milan, Italy 5Lead Contact *Correspondence: emfvb2@mrc-mbu.cam.ac.uk (E.F.-V.), mdz21@mrc-mbu.cam.ac.uk (M.Z.) http://dx.doi.org/10.1016/j.celrep.2017.01.044 2Radboud SUMMARY The biogenesis of human cytochrome c oxidase (COX) is an intricate process in which three mitochondrial DNA (mtDNA)-encoded core subunits are assembled in a coordinated way with at least 11 nucleus-encoded subunits Many chaperones shared between yeast and humans are involved in COX assembly Here, we have used a MT-CO3 mutant cybrid cell line to define the composition of assembly intermediates and identify new human COX assembly factors Quantitative mass spectrometry analysis led us to modify the assembly model from a sequential pathway to a module-based process Each module contains one of the three core subunits, together with different ancillary components, including HIGD1A By the same analysis, we identified the short isoform of the myofibrillogenesis regulator (MR-1S) as a new COX assembly factor, which works with the highly conserved PET100 and PET117 chaperones to assist COX biogenesis in higher eukaryotes INTRODUCTION Cytochrome c oxidase (COX) or complex IV (cIV) is the multiheteromeric terminal oxidase of the mitochondrial respiratory chain Of the 14 structural subunits composing mammalian COX, three (MT-CO1, CO2, and CO3) are encoded in the mitochondrial DNA (mtDNA) and translated inside the organelle The remaining 11 subunits and all of the other proteins assisting the assembly process are imported into the organelle from the cytoplasm More than 30 different ancillary proteins are required for the correct assembly of the 11 structural subunits of yeast COX (Soto et al., 2012) Several orthologs have been found in humans, mainly because their mutations determine COX defective mitochondrial disease (Antonicka et al., 2003a, 2003b; Bugiani et al., 2005; Jaksch et al., 2000; Leary et al., 2004; Lim et al., 2014; Ola´hova´ et al., 2015; Szklarczyk et al., 2013; Valnot et al., 2000a, 2000b) The current assembly model for human COX (Nijtmans et al., 1998; Stiburek et al., 2005, 2006) consists of four steps: early (step [S1]), intermediate (S2), late (S3), and last (S4) MT-CO1 is deemed as the first COX component to be inserted in the inner mitochondrial membrane and constitute the ‘‘seed’’ onto which the other subunits are assembled (S1) The incorporation of two early subunits, COX4 and COX5A, with MT-CO1 forms the S2 intermediate This is followed by the incorporation of subunits MT-CO2 and then MT-CO3, which in turn triggers the ‘‘late’’ incorporation of most of the remaining accessory subunits (S3), with the exception of COX6A, COX6B, and COX7A or COX7B, which are incorporated last (S4) (Ferna´ndez-Vizarra et al., 2009) The mechanisms and players involved in this process are relatively well established in yeast (Mick et al., 2011), whereas the process is less clear and possibly different in mammals (Dennerlein and Rehling, 2015; Soto et al., 2012) For instance, specific factors present in mammals but not in yeast are necessary for the incorporation of MT-CO1 (Clemente et al., 2013; Dennerlein et al., 2015; Mick et al., 2012) and for stabilization and metalation of MT-CO2 (Bourens et al., 2014; Leary et al., 2009; Pacheu-Grau et al., 2015; Stroud et al., 2015) In order to further define the key steps in COX assembly and identify new players in human COX biogenesis, we took advantage of a nearly homoplasmic frameshift mutation in MT-CO3 (Tiranti et al., 2000), characterized by the accumulation of assembly intermediates RESULTS Analysis of Assembly Intermediates Recapitulates COX Biogenesis Fragment length polymorphism (RFLP) analysis on a cybrid cell line harboring the m.9536_9537insC (p.Gln111Profs*113) mutation (Tiranti et al., 2000) in MT-CO3 showed that the mutation load was >95% (Figure S1) By western blot (WB) analysis of dodecyl-maltoside (DDM)-treated mitochondrial fractions, Cell Reports 18, 1727–1738, February 14, 2017 ª 2017 Elsevier Inc 1727 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Figure COX Assembly Intermediates in Cybrids Carrying a MT-CO3 Mutation (A) Mitoplasts isolated from WT and MT-CO3 mutant cybrids analyzed by BNGE and WB using a-MT-CO1 Arrows indicate MT-CO1 in mature COX (cIV), subassembly intermediates (Sub-cIV), COX dimer (cIV2), and the cIII2 + cIV supercomplex (B) Bi-directional SILAC-labeled mitoplasts from both cybrid cell lines were isolated, mixed, and subjected to COX immunocapture Each data point represents a specific protein All of the identified COX subunits were located in the bottom left quadrant A group of proteins known to be involved in COX assembly, in which MR-1S was found, is also shown in detail in the top right quadrant The values in the x axis correspond to the log2 heavy-to-light (H/L) ratio of the peptides detected in experiment 1, where the heavy (H)-labeled MT-CO3 mutant and unlabeled (L) WT cells were mixed The values in the y axis correspond to the inverted log2 H/L ratio (Àlog2 H/L) of the peptides detected in experiment 2, where the unlabeled (L) MT-CO3 mutant and the labeled (H) WT cells were mixed (C) Table showing the log2 H/L and the Àlog2 H/L ratios obtained for the proteins plotted in (B) by the two-way SILAC mass-spectrometry analysis run through first-dimension (1D) blue native gel electrophoresis (BNGE), we detected numerous MT-CO1-containing subassembly species (Figure 1A), which were likely to contain chaperones involved in COX assembly (Dennerlein and Rehling, 2015) In order to characterize the composition of the subassembly COX species, we carried out a comparison of COX immunopurified from mitoplasts of wild-type (WT) and cybrid mutant cell lines by quantitative mass spectrometry (MS) analysis using stable isotope-labeled amino acids in cell culture (SILAC) These experiments were performed in duplicate with reciprocal isotopic labeling between mutant and WT cell lines and showed that the COX subunits were clustered in four distinct groups according to their relative abundance (Figure 1B; Table S1), in nearly complete concordance with the current COX assembly model (Ferna´ndez-Vizarra et al., 2009; Fornuskova et al., 2010; Nijtmans et al., 1998; Stiburek et al., 2006) Interestingly, the first group 1728 Cell Reports 18, 1727–1738, February 14, 2017 included COX4I1, COX5A, and HIGD1A, a protein previously proposed to bind MT-CO1 and regulate COX activity in hypoxia (Hayashi et al., 2015) The next group was composed of proteins with $3.5-fold reduction in the mutant cell line and included MT-CO2, COX5B, COX6C, COX7C, COX8A, and, unexpectedly, MT-CO1 The third group, with more than 5-fold difference between mutant and WT complexes, included MT-CO3, COX6A1, COX6B1, and COX7A2 The last group contained COX7A2L (also termed super-complex assembly factor [SCAFI]) (Lapuente-Brun et al., 2013), NDUFA4 (Balsa et al., 2012; Pitceathly et al., 2013), and virtually all of the subunit components of complex III (Figure 1B; Table S1) These results reflect the stalling of COX assembly due to the virtual absence of MT-CO3 in the mutant cell line, which prevents the formation of cIII2 + cIV supercomplexes and causes the accumulation of COX assembly intermediates Figure SILAC and Complexome Profiling Isolated mitochondria from MT-CO3 mutant and control cybrids cells treated with SILAC medium in both orientations were pooled, solubilized with DDM, and separated by BNGE Gel lanes were cut into 64 slices (number at the top; number 64 at bottom) followed by liquid chromatography (LC) MS analysis (A) Complexome heatmap of wild-type (WT, left) and MT-CO3 mutant (right) 143B cybrids Highlighted in red is slice 39, corresponding to fully assembled COX in WT cells (215 kDa) COX subunits found under that point are considered to be in COX subassemblies (Sub-cIV) (B) Profiles of selected proteins in the early, intermediate (intermed.), and last groups (see main text for details) The heatmaps and profiles are shown for the light WT and heavy mutant direction; virtually identical results were obtained by the reverse light mutant and heavy WT direction For each protein, the relative intensity (to a maximum of 1) of a representative peptide along the 64 slices was plotted with respect to the molecular mass These assembly intermediates were also analyzed by ‘‘complexome profiling’’ (Heide et al., 2012) Proteins from mutant and WT cybrid cells were labeled with SILAC, combined and resolved by 1D-BNGE, and excised in 64 slices (Figures 2A and 2B) Figure 2A shows the heatmap of WT mitochondria in comparison with the mutated mitochondria In WT mitochondria, COX subunits are focalized in slice 39 (at a molecular size of 215 kDa), whereas they are much more scattered in the MTCO3 mutated cells The profiles for the individual subunits (Figure 2B) indicate that subunits COX4I1 and COX5A (not shown), as well as HIGD1A, are distributed in the bottom part of the gel (from 30 kDa upward), thus representing early subcomplexes In mutant mitochondria, these subunits are predominantly found in assembly intermediates rather than in (residual) fully assembled COX The ‘‘intermediate’’ group of subunits was detected from higher positions along the gel (from 120 kDa upward), and again, in the mutant cell line, they were predominantly found in subassemblies rather than in residual fully assembled COX Interestingly, the intermediate group included MT-CO1, suggesting that in mammalian cells, the incorporation of this subunit occurs at a stage later than the initial one, which instead corresponds to the formation of the COX4I1, COX5A, and HIGD1A cluster Finally, the last subunits to be incorporated were exclusively present in fully assembled COX, and their relative amount was virtually negligible in the mutant cell line Identification of a Novel COX Assembly Factor In order to identify COX-specific assembly factors associated with assembly intermediates, we sought proteins associated with mutant COX assemblies in greater abundance than with WT cybrid lines In a cluster of proteins with mutant/WT ratios of $1.5–2, four were already known to participate in COX biogenesis, namely, Cell Reports 18, 1727–1738, February 14, 2017 1729 COA3 (CCDC56 or MITRAC12) (Clemente et al., 2013; Mick et al., 2012), PET100 (Lim et al., 2014; Ola´hova´ et al., 2015; Szklarczyk et al., 2012), and the human orthologs of the yeast Pet117 (McEwen et al., 1993; Szklarczyk et al., 2012) and Cmc2 (Horn et al., 2010) In this cluster, we identified a fifth protein, myofibrillary-related protein short isoform (MR-1S; also known as PNKD isoform 2; Uniprot: Q8N490-2), which we investigated further as a potential, new candidate assembly factor (Figures 1B and 1C) All the other entries in the cluster were non-mitochondrial proteins according to Mitocarta (http://archive.broadinstitute.org/ pubs/MitoCarta/index.html) and Mitominer (http://mitominer mrc-mbu.cam.ac.uk/release-4.0/begin.do), two mitochondrialspecific proteome databases, and therefore were not considered relevant for this study A protein of unknown function, TMEM214, was further investigated However, immunofluorescence-based subcellular localization showed a non-mitochondrial pattern, and knockdown expression of the protein did not affect COX assembly or function (data not shown) MR-1S Knockdown Affects COX Assembly and Function MR-1S is encoded by transcript of the paroxysmal non-kinesigenic dyskinesia (PNKD) gene To further define the role of MR1S in COX assembly, we performed lentiviral transduction of three small hairpin RNAs (shRNAs) specific for PNKD-3 (shMR1SRNA1,2,3) From WT cybrids treated with shMR-1SRNA1, we selected two clones (shMR-1SRNA1À7 and shMR-1SRNA1À11), which showed the virtual absence of MR-1S (Figure 3A) Both shMR-1SRNA1À7 and shMR-1SRNA1À11 cell lines displayed 30% reduction in COX activity (Figure 3B) compared with cells transduced with the ‘‘empty’’ vector or with an shMR-1SRNA1À3 clone, which showed MR-1S levels similar to the control (Figure 3A) Likewise, shMR-1SRNA1À7 and shMR-1SRNA1À11 cell lines showed reduced oxygen consumption rate (OCR) measured by Seahorse analysis (Figure 3C) Furthermore, the shRNA cells displayed a reduction in the amount of MT-CO1 and COX6A1 incorporated into mature COX (by $35% and $60%, respectively) (Figures 3D–3F) The amount of MT-CO1-immunoreactive subassembly species was not changed in the shRNA clones, except for a nearly fully assembled species, which was undetectable in the knockdown cells (Figures 3E and 3F) MR-1S Interacts with COX Subunits and Assembly Factors The migration pattern of MR-1S-containing assembly intermediates was determined in reciprocal complexome analyses with SILAC-labeled samples In the MT-CO3 cybrid cell line, and, albeit in a lesser amount, in the WT cell line as well, MR-1S was localized in two main bands in the COX area One was in slices 35-37, corresponding to 300-255 kDa, respectively, thus migrating faster than the COX main band (214 kDa in slice 39), whereas the second was migrating slower ($170 kDa) (Figure 4A) Importantly, in WT mitochondria, many COX structural subunits were also detected in the same slices as MR-1S, although in lower amounts as compared to the main COX band (Figure 2A) In addition, MR-1S was also detected in the lowermolecular-weight area at the bottom of the gel (49 and 30 kDa) in both WT and mutant cells These data strongly suggest a role for MR-1S as a chaperone assisting COX assembly 1730 Cell Reports 18, 1727–1738, February 14, 2017 Next, we transduced WT and MT-CO3 mutant cybrids with a C-terminal hemagglutinin (HA)-tagged recombinant MR-1S cDNA (MR-1SHA) (Figure 4B) As previously reported (Ghezzi et al., 2009), MR-1SHA was targeted to mitochondria (Figure S2) By HA immunovisualization, we detected the same two distinct bands (by WB of 1D-BNGE) or spots (by WB of 2D-BNGE) localized around the band or spot corresponding to fully assembled COX (Figures 4C and 3D), as previously found by complexome and SILAC analysis of naive cell lines This result indicates that the MR-1SHA protein is incorporated in the same supramolecular structures as the endogenous one To test the specific interaction of MR-1SHA with COX subunits, we performed co-immunoprecipitation experiments using an a-HA antibody No COX subunits were detected in the immunoprecipitates in control cells (i.e., WT cybrids transduced with the empty vector), whereas MT-CO1, MT-CO2, COX5A, and COX5B, but not COX6B, were present in the immunopurified material from MR-1SHA-expressing cells (Figure S3A) In mtDNA translation experiments, MR-1SHA was able to bind newly synthesized MT-CO2 and, in smaller proportions, MT-CO1 as well (Figure S3B) To further explore the MR-1S-specific interactome, we examined the protein interactions of HA-tagged MR-1S by MS of SILAC-labeled a-HA immunoprecipitates from both WT and MTCO3 cybrids The specific interactors consistently detected by these analyses included (1) several COX structural subunits belonging to the early or intermediate assembly groups and (2) two COX assembly factors, i.e., PET100 (Church et al., 2005; Lim et al., 2014; Ola´hova´ et al., 2015) and PET117 (McEwen et al., 1993; Soto et al., 2012; Szklarczyk et al., 2012) (Figures 4E and 4F) MR-1S-PET100 Complexes Identified by Complexome Profiling To gain further insight on the physiological interactions of MR1S, we performed complexome profiling of mitochondria isolated from 143B cells and C-124 human skin fibroblasts (Figures 5A and 5B) In both cell types, HIGD1A was detected in all blue native (BN) gel slices where COX subunits were present (i.e., in the monomeric COX, cIII2 + cIV supercomplex and in the larger COX-containing supercomplexes), qualifying HIGD1A as a constitutive subunit of the COX complex (Figure 5B) Both MR-1S and PET100 were predominantly detected in the region around the COX holocomplex (214 kDa) Again, both their migration profiles showed two clear peaks at $160 kDa and 270 kDa, which were distinctly different from that of the COX monomer (Figure 5B), indicating that they were bound to COX assembly intermediates The migration profiles also revealed that the putative assembly factor PET117 was present in a peak overlapping the COX holocomplex but showing a ‘‘shoulder’’ coinciding with the higher-molecular-weight peak containing MR-1S and PET100 (Figure 5B) This suggests an interaction between PET117 and MR-1S plus PET100 during COX biogenesis MR-1S Interacts with PET100 and PET117 The absence of human PET100 is associated with severe, isolated COX deficiency and impaired COX assembly, leading to Figure COX Functional and Assembly Defects after MR-1S Knockdown (A) SDS-PAGE and WB analysis showing MR-1S steady-state levels in three different clones (clones shMR-1SRNA1À3, 7, and 11), isolated after lentiviral transduction of WT cybrids with pLKO.1 containing a shRNA sequence specifically targeting the MR-1S isoform, in comparison with the mock transduced cells (pLKO.1 is an empty vector [E.V.]) Tubulin (TUB) was used as the loading control (B) COX enzyme activity normalized to citrate synthase (CS) activity Plotted data are mean ± SD (n = 4) (C) Respiration measured in the knock-down clones, in comparison with the control (E.V.), as oxygen consumption rate (OCR) in basal conditions (BASAL) and after addition of the uncoupler FCCP (maximum respiration rate, MRR) Histograms represent the mean ± SD values from three independent experiments, each with five or six replicates (D) 1D-BNGE and WB using a-MT-CO1 The densitometric quantification of the MT-CO1 cIV signal, normalized to the CS signal and expressed as the percentage of the normalized control signals, of three independent experiments is shown below (mean ± SD) (E) 1D-BNGE + 2D-SDS-PAGE and WB analysis of the distribution of MT-CO1, MT-CO2, and COX6A1 signals in shMR-1S clones RNA1À7 and RNA1À11 in comparison with the E.V control (F) Graphs showing the densitometric profiles MT-CO1 and COX6A1 signals The tables show the quantification of the area below the curve (subtracting the background), expressed as the percentage of the control signal normalized by the corresponding cII-SDHA signal (not depicted) Red arrows indicate an MT-CO1- and MT-CO2-containing sub-complex present in the control (empty vector [E.V.]) cells but absent in knockdown clones RNA1À7 and RNA1À11 a fatal early-onset mitochondrial encephalopathy (Lim et al., 2014; Ola´hova´ et al., 2015) The role of human PET117 in COX biogenesis is deemed to be similar to that shown in yeast, although no experimental confirmation has thus far been provided in mammalian systems (Szklarczyk et al., 2012) In fibroblasts carrying a truncating homozygous p.Gly48* mutation in PET100 (Ola´hova´ et al., 2015), the MR-1S steady-state levels were markedly reduced (Figure 6A) and the typical MR-1S-containing supramolecular structures were absent (Figures 6B and S5), suggesting that the stability and interaction of MR-1S with COX assembly intermediates depend on the presence of PET100 Next, we transduced WT cybrids, control, and PET100G48* immortalized fibroblasts transduced with FLAG-tagged versions of either PET100 or PET117 All cell lines showed very high expression of recombinant PET117FLAG, whereas the levels of PET100FLAG were much lower (Figure 6C) but sufficient to rescue the COX assembly and activity (Figure S4) and (partially) Cell Reports 18, 1727–1738, February 14, 2017 1731 Figure MR-1S Is Found in Two Distinct Supra-molecular Complexes and Binds COX Structural Subunits and Assembly Factors (A) SILAC-complexomic profiles for MR-1S and MT-CO2 in WT and MT-CO3 mutant cybrids (B) SDS-PAGE and WB analyses showing overexpression of HA-tagged MR-1S (MR-1SHA) in the transduced WT and MT-CO3 mutated cybrids (C and D) Native first-dimension (1D-BNGE) (C) and denaturing second-dimension (1D-BNGE + 2D-SDS-PAGE) (D) and WB analyses showing migration of MR1SHA inside the two distinct complexes with respect to the MT-CO2 and COX5A signals (E and F) Scatterplots (E) and tables (F) showing the log2 and Àlog2 H/L ratios obtained after mass spectrometry analysis of a-HA co-immunoprecipitation fractions of bi-directional SILAC labeling of WT (left graph) or MT-CO3 mutant (right) cybrid cells expressing MR-1SHA, combined with material from empty vector control cells Only the upper right quadrants of the plots, showing the statistically significant interactions (based on significance B, Perseus analysis platform; Cox and Mann, 2008; Tyanova et al., 2016), are displayed pWPXLd-ires-HygroR, empty vector [E.V.] See also Figure S3 complement the molecular phenotype of PET100G48* fibroblasts (Figure 6A) In SILAC experiments using naive WT cybrids and cybrids transduced with PET117FLAG, MR-1S was co-immunoprecipitated by an a-FLAG monoclonal antibody, together with early (COX4I1 and COX5A) and intermediate (MT-CO1, MT-CO2, COX5B, and COX6C) COX structural subunits and COX11, another COX assembly factor (Figure 6D) More work is necessary to establish whether COX11 and MR-1S co-operate in COX assembly Likewise, in PET100G48* immortalized fibroblasts transduced with PET100FLAG, the a-FLAG antibody coimmunoprecipitated MR-1S, together with MT-CO1, MT-CO2, COX5A, and COX5B (Figure 6E) However, no co-immunoprecip1732 Cell Reports 18, 1727–1738, February 14, 2017 itation of any of these proteins was obtained in the PET100G48* fibroblast cell line transduced with PET117FLAG (Figure 6E) Taken together, these results clearly demonstrate that PET117 physically interacts with MR-1S and with some COX subunits and that this interaction requires the presence of PET100 DISCUSSION Revisiting the COX Assembly Pathway Both immunocapture/SILAC and complexome/SILAC analyses converge in defining four groups of subunits (early, intermediate, late, and last) differentially clustering in subassembly intermediates during COX biogenesis Figure MR-1S and PET100 Are Found in the Same Complexes in Control Cells (A) Complexome heatmap of 143B cells (left) and control fibroblasts (right) (B) Profiles of selected proteins (HIGD1A, PET117, PET100, and MR-1S) The profile of COX holocomplex is in black (see main text for details) The first group includes COX4I1 and COX5A subunits, which were proposed to be the first to join MT-CO1 and determine the transition from assembly step (S1, corresponding to monomeric MT-CO1) to S2 (Figure 7A) Accordingly, these two subunits were found in several early, low-molecular-weight subcomplexes In immunoprecipitation and SILAC experiments, HIGD1A, which is proposed to mainly interact with MT-CO1 (Hayashi et al., 2015), appeared with the same relative abundance in the MT-CO3 mutant versus WT cells and in lowmolecular-weight subcomplexes, like the subunits COX4I1 and COX5A However, in complexome experiments, HIGD1A also co-migrates with higher-order COX structures (from 300 to 500 kDa) in a non-stoichiometric proportion compared to the other COX subunits (Figure 2B) HIGD1A is a mammalian ortholog of Rcf1, a COX-interacting factor that helps stabilize the cIII2 + cIV(1-2) supercomplexes in yeast (Chen et al., 2012; Strogolova et al., 2012; Vukotic et al., 2012), and when bound to COX, it increases its activity (Hayashi et al., 2015) (Rydstroăm Lundin et al., 2016) Thus, HIGD1A may be a regulatory component of COX, which is incorporated in the early stages of the enzyme assembly Unexpectedly, both the immunoprecipitation/SILAC and complexome analyses showed MT-CO1 to be part of the intermediate assembly group of COX subunits rather than the early-assembly group including COX4I1 and COX5A Previous studies have shown that the knockdown of COX4I1 expression results in a similar decrease in the COX5A steadystate levels and vice versa In addition, the levels of fully assembled COX were the same as the residual levels of either subunit (Fornuskova et al., 2010) This is consistent with the idea that these subunits interact and play a seminal role in the first stages of COX assembly Importantly, the total amount of MT-CO1 was unchanged, but there was an accumulation of MT-CO1-containing assembly intermediates These findings support the idea that shortage of the COX4I1 + COX5A early subassembly impairs the incorporation of MT-CO1, leading to accumulation of MT-CO1 intermediates, concordant with the idea of a separate MT-CO1 module This later incorporation of MT-CO1 was also previously suggested in S cerevisiae (Church et al., 2005), and is in agreement with kinetic experiments of mitochondrial translation after doxycycline treatment (Moreno-Lastres et al., 2012) Our complexome profiling confirmed that the intermediate assembly group includes, in addition to MT-CO1, the subunits MT-CO2, COX5B, COX6C, COX7C, and COX8A In the current model, the incorporation of MT-CO3 occurs just before or concomitantly with the intermediate group of subunits (Figure 7A) However, our data indicate that the incorporation of the nucleus-encoded accessory subunits (COX5B, COX6C, COX7C, and COX8A) parallels that of MT-CO2 and MT-CO1 and occurs before, rather than after, the incorporation of MTCO3 (Figures 1, 2, and 7B) In fact, our data indicate that the late assembly group includes subunit MT-CO3, together with COX6A1, COX6B1, and COX7A2 The ‘‘last’’ group includes two proteins: NDUFA4, Cell Reports 18, 1727–1738, February 14, 2017 1733 Figure PET117 Interaction with MR-1S and COX Subunits Is Mediated by PET100 (A) Steady-state levels of proteins visualized by SDS-PAGE, WB, and immunodetection in the PET100G48* patient and in control (WT) immortalized fibroblasts transduced with PET100FLAG The proteins were detected with the indicated antibodies The signal intensities expressed as percentage of the control (WT_E.V.) of MT-CO2, MT-CO1, and MR-1S normalized to tubulin are plotted in the top graph (mean ± SD; n = 2) The signal intensities expressed as percentage of the control (WT_E.V.) of MT-CO2 and MT-CO1 normalized to the MR-1S signal are plotted in the bottom graph (mean ± SD; n = 2) See also Figure S4 (B) 1D-BNGE, WB, and immunodetection analysis showing the absence of fully assembled COX, detected with a-MT-CO2, and of the MR-1S supramolecular complexes in the PET100G48* primary fibroblasts See also Figure S5 (C) SDS-PAGE, WB, and immunodetection using a-FLAG Actin and tubulin (TUB) are loading standards Notice that a long exposure was necessary to visualize the PET100FLAG band (D) Scatterplots and heavy-to-light (H/L) ratio table obtained after mass spectrometry analysis of a-FLAG co-immunoprecipitation fractions of bi-directional SILAC labeling of cybrid cells expressing recombinant PET117FLAG, combined with material from empty vector cybrid cells Only the top right quadrant of the plot and the statistically significant interactions are shown (significance B, Perseus analysis platform; Cox and Mann, 2008; Tyanova et al., 2016) (E) SDS-PAGE, WB analysis, and immunodetection of the co-immunoprecipitation fractions using a-FLAG in PET100G48* immortalized fibroblasts overexpressing PET100FLAG or PET117FLAG TOT, total mitoplast lysate before co-immunoprecipitation FT, flow-through fraction with the unbound proteins WASH1 and WASH9, fractions obtained after the first and ninth washings of the a-FLAG-M2-agarose resin ELUTE, eluted fractions of the material bound to the resin after treatment with the specific FLAG peptide which is peripherally associated with COX (Balsa et al., 2012; €ttemann, 2015; Pitceathly et al., 2013), and Kadenbach and Hu COX7A2L, which is necessary for the formation of cIII2 + cIV (Cogliati et al., 2016; Pe´rez-Pe´rez et al., 2016; Williams et al., 2016) The incorporation of COX6B1 and COX6A1 in the late assembly group is in agreement with data obtained from the analysis of COX6B1 mutant human fibroblasts (Massa et al., 2008) and knockdown expression of COX6A1 in human cells (Fornuskova et al., 2010) In addition, our results confirm that COX7A2 (the 1734 Cell Reports 18, 1727–1738, February 14, 2017 non-muscular isoform of COX7A), rather than COX7B (Nijtmans et al., 1998; Stiburek et al., 2006), belongs to the late assembly group (Fornuskova et al., 2010) (Figure 7A) We have evidence that COX7B could in fact be incorporated with the intermediate assembly group (data not shown) The current human COX assembly model is based on the sequential incorporation of individual subunits into a progressively bigger supramolecular structure However, a modular assembly model has recently been suggested for the yeast Figure New Complex IV Assembly Model versus the Reference Assembly Model (A) Reference assembly model based on Nijtmans et al (1998); Stiburek et al (2005), (2006) (B) New modular assembly model Each of the modules corresponds to one of the mtDNA-encoded subunits (MT-CO1, MT-CO2, and MT-CO3) The MT-CO1 module is labeled as the MITRAC complex (Mick et al., 2012) Proteins whose genes have been found mutated in mitochondrial disorders are displayed in red enzyme (McStay et al., 2013a, 2013b, 2013c; Su et al., 2014) Likewise, in human cultured cells, MT-CO1 was found to participate in an assembly module termed MITRAC (for mitochondrial translation/regulation of assembly intermediates of COX) (Dennerlein et al., 2015; Mick et al., 2012), together with COX structural subunits and the assembly factors COA3 (MITRAC12), COA1 (C7orf44 or MITRAC15), COX14 (C12orf62), CMC1, and SURF1 In yeast, a second module contains Cox3 together with Cox4 (ortholog of human COX5B), Cox7 (COX7A), and Cox13 (COX6A) A third module consists of Cox2 together with other yet-to-be-determined subunits (Su et al., 2014) These three modules then join together, release the chaperones, and give rise to mature COX The heavy/light ratios obtained in our SILAC analyses support the existence of assembly modules rather than sequential incorporation, where MT-CO2 would form a module with at least COX6C, COX7C, COX8A, and COX5B, while MTCO3 would go together with COX6A1, COX6B1, and COX7A2 (Figure 7B) MR-1S Role in COX Assembly and Interaction with PET117 and PET100 We found MR-1S to interact with COX-related proteins Accordingly, MR-1SHA binds to a subassembly species composed of the structural subunits defining the ‘‘S3’’ intermediate in the COX assembly model, except for MT-CO3 Drastic knockdown of MR-1S expression had functional consequences on COX activity and respiratory function, confirming its involvement in COX biogenesis A role in the intermediate assembly steps of COX is also supported by the fact that the advanced intermediates and fully assembled COX are less abundant in the MR-1S knockdown cells Cell Reports 18, 1727–1738, February 14, 2017 1735 Mutations in exon of the PNKD gene, encoding the mitochondrial targeting sequence of MR-1S (S for short) and MR1L (L for long), have been associated with the autosomaldominant neurological disease paroxysmal non-kinesigenic dyskinesia (PNKD) (Ghezzi et al., 2009) In contrast with the ubiquitous distribution of MR-1S, MR-1L is only expressed in the brain Interestingly, low OCR was found by Seahorse analysis in patient fibroblasts carrying the A9V mutation (Ghezzi et al., 2015), but the molecular pathogenic mechanisms of PNKD remain elusive, since the mutation does not affect the mature forms of both MR-1S and MR-1L The function of both MR-1L, which localizes to mitochondria, and MR-1M, which localizes to the Golgi apparatus and endoplasmic reticulum (ER), remains unknown but is likely not related to the COX-specific chaperone role that we have demonstrated for MR-1S Our data show that MR-1S co-immunoprecipitates with PET100, a known COX assembly factor (Church et al., 2005; Lim et al., 2014; Ola´hova´ et al., 2015), and vice versa Yeast Pet100 is necessary for COX assembly and forms a subcomplex with Cox7 (COX7A), Cox7a (COX6C), and Cox8 (COX7C) (Church et al., 1996, 2005) Interestingly, two of the human ortholog subunits (COX6C and COX7C) were present in the same fractions as PET100 when MR-1SHA was immunoprecipitated Human skin fibroblasts deficient in PET100 show profoundly reduced COX levels (Lim et al., 2014; Ola´hova´ et al., 2015) In PET100G48* mutant cells, MT-CO2 is drastically reduced, whereas MT-CO1 is also low, but to a lesser extent (Ola´hova´ et al., 2015; Figure 6A) In PET100M1? mutant fibroblasts (Lim et al., 2014), the turnover of MT-CO2 and MT-CO3 was much higher than in control cells This is totally consistent with the idea that PET100 is important for the formation and/or stabilization of the intermediate lacking the MT-CO3 module, which would be generated when the COX4I1 + COX5A and the MTCO1 + MT-CO2 modules get together, preceding the incorporation of MT-CO3 and its partners This is the same step in which we propose that MR-1S binds the COX assembly intermediates In addition to PET100, MR-1S interacts with PET117, whose involvement in COX assembly in human cells was never described before By using PET100-defective cells (PET100G48*), we were able to determine that the interactions among MR-1S, PET117, and COX structural subunits require PET100 In contrast with PET100, PET117, and many other COX assembly factors, MR1S is present only in vertebrates, suggesting for this protein a peculiar function that is probably specific to the higher complexity and plasticity of COX in these organisms compared to that in yeast or non-vertebrate pluricellular eukaryotes EXPERIMENTAL PROCEDURES Cell Culture Human 143B osteosarcoma cells and primary and immortalized skin fibroblasts were grown at 37 C and 5% CO2 in high-glucose plus Glutamax and sodium pyruvate DMEM (Life Technologies) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin, and 50 mg/mL uridine For puromycin and hygromycin, concentrations were mg/mL and 100 mg/mL, respectively Cell Line Generation Primary skin fibroblasts were immortalized by lentiviral transduction of pLOXTtag-iresTK (Addgene #12246, Tronolab) 1736 Cell Reports 18, 1727–1738, February 14, 2017 C-terminal HA or FLAG tags were added by PCR amplification PCR fragments were cloned into pWPXLd-ires-PuroR and pWPXLd-ires-HygroR lentiviral expression vectors, modified versions of pWPXLd (Addgene #12258, Tronolab) Lentiviral particles were generated in HEK293T packaging cells, and target cells were transduced as described previously (Perales-Clemente et al., 2008) 24 hr after transduction, cells were selected for puromycin or hygromycin resistance RNAi was performed by lentiviral transduction of shRNA sequences targeting specifically the MR-1S isoform (target 1, 50 -GTGGTATCCTCCTCTTCA TCC-30 ; target 2, 50 -GCCAGCCAAAGGCAAATAAAG-30 ; and target 3, 50 -CTA GGCTATTGACTGTTAAGT-30 ), cloned into the pLKO.1-TRC vector (Addgene #10878) according to the manufacturer’s instructions (https://www.addgene org/tools/protocols/plko/) Native and Denaturing Protein Electrophoresis and WB Samples for BNGE were prepared as described previously (Nijtmans et al., 2002; Wittig et al., 2006) Native samples were run through pre-cast NativePAGE 3%– 12% Bis-Tris gels, while Novex NuPAGE 4%–12% Bis-Tris Gels (Life Technologies) were used for denaturing conditions For WB, samples were electroblotted to PVDF membranes and immunodetected using commercial specific antibodies A detailed list can be found in Supplemental Experimental Procedures Immunodetection signal intensities were quantified using ImageJ Quantitative SILAC Mass Spectrometry The two cell lines to be compared by SILAC (Ong et al., 2002) were grown in ‘‘heavy’’ DMEM containing 15N- and 13C-labeled Arg and Lys and in ‘‘light’’ DMEM containing 14N and 12C Arg and Lys (Sigma-Aldrich) Equal portions of the differentially labeled cell lines were mixed and solubilized; affinity purifications were performed using anti-HA-agarose (Cell Signaling), antiFLAG-agarose (Sigma-Aldrich), or cIV immunocapture kit (Abcam) beads and analyzed as described previously (Andrews et al., 2013) See Supplemental Experimental Procedures for a detailed explanation Complexome Profiling Mitochondrial samples from 143B cells and human skin fibroblasts (cell line C-124 from the Radboud Center for Mitochondrial Medicine) or from differentially labeled MT-CO3 mutated and WT cybrids (1:1 mixture of the cells grown in heavy and light SILAC media) were used for ‘‘complexome profiling,’’ which was performed and analyzed as previously described (Heide et al., 2012), with some modifications for SILAC sample analysis For a detailed explanation, see Supplemental Experimental Procedures Functional Assays The activities of COX and citrate synthase (CS) were measured in digitoninsolubilized cell samples (Tiranti et al., 1995) by spectrophotometry as described previously (Kirby et al., 2007) The respiratory activity was measured in whole cells in an XF24 extracellular flux analyzer (Seahorse Biosciences) (Invernizzi et al., 2012) Statistical analysis was by unpaired Student’s t test using GraphPad Prism 5.0a software SUPPLEMENTAL INFORMATION Supplemental Information includes Supplemental Experimental Procedures, five figures, and one table and can be found with this article online at http:// dx.doi.org/10.1016/j.celrep.2017.01.044 AUTHOR CONTRIBUTIONS Conceptualization, E.F.-V and M.Z.; Methodology, E.F.-V., S.V., M.E.H., I.M.F., and S.A.; Investigation, S.V., M.E.H., S.G.-C., A.S., S.D., and E.F.-V.; Formal Analysis, S.V., M.E.H., S.D., S.G.-C., S.A., and E.F.-V.; Resources, V.T., R.W.T., E.F.-V., and M.Z.; Writing – Original Draft, E.F.-V.; Writing – Review & Editing, M.Z., I.M.F., S.A., S.V., and E.F.-V.; Visualization, S.V., M.E.H., A.S., S.A., and E.F.-V.; Supervision, E.F.-V and M.Z.; Funding Acquisition, M.Z ACKNOWLEDGMENTS We are very grateful to Joe Carroll (MRC-MBU, Cambridge, UK) for excellent advice, Daniele Ghezzi (Istituto Besta, Milan, Italy) for providing the MR1SHA cDNA clone, and Ester Perales-Clemente and Pilar Bayona-Bafaluy (University of Zaragoza, Spain) for providing the PuroR and HygroR lentiviral expression vectors We would like to thank Ulrich Brandt for his support and helpful discussions This work was supported by MRC-QQR grant 20152020, ERC advanced grant ERC FP7-322424 (to M.Z.), Excellence Initiative of the German Federal and State Governments grant EXC 115 (to S.A.), a Humboldt Foundation fellowship (to S.G.-C.), Wellcome Trust strategic award 096919/Z/11/Z (to R.W.T.), the MRC Centre for Neuromuscular Diseases (G0601943), and the UK NHS Highly Specialised ‘‘Rare Mitochondrial Disorders of Adults and Children’’ Service Received: September 16, 2016 Revised: December 6, 2016 Accepted: January 19, 2017 Published: February 14, 2017 Cox, J., and Mann, M (2008) MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Nat Biotechnol 26, 1367–1372 Dennerlein, S., and Rehling, P (2015) Human mitochondrial COX1 assembly into cytochrome c oxidase at a glance J Cell Sci 128, 833–837 Dennerlein, S., Oeljeklaus, S., Jans, D., Hellwig, C., Bareth, B., Jakobs, S., Deckers, M., Warscheid, B., and Rehling, P (2015) MITRAC7 acts as a COX1-specific chaperone and reveals a checkpoint during cytochrome c oxidase assembly Cell Rep 12, 1644–1655 Ferna´ndez-Vizarra, E., Tiranti, V., and Zeviani, M (2009) Assembly of the oxidative phosphorylation system in humans: what we have learned by studying its defects Biochim Biophys Acta 1793, 200–211 Fornuskova, D., Stiburek, L., Wenchich, L., Vinsova, K., Hansikova, H., and Zeman, J (2010) Novel insights into the assembly and function of human nuclear-encoded cytochrome c oxidase subunits 4, 5a, 6a, 7a and 7b Biochem J 428, 363–374 REFERENCES Ghezzi, D., Viscomi, C., Ferlini, A., Gualandi, F., Mereghetti, P., DeGrandis, D., and Zeviani, M (2009) Paroxysmal non-kinesigenic dyskinesia is caused by mutations of the MR-1 mitochondrial targeting sequence Hum Mol Genet 18, 1058–1064 Andrews, B., Carroll, J., Ding, S., Fearnley, I.M., and Walker, J.E (2013) Assembly factors for the membrane arm of human complex I Proc Natl Acad Sci USA 110, 18934–18939 Ghezzi, D., Canavese, C., Kovacevic, G., Zamurovic, D., Barzaghi, C., Giorgi, C., Zorzi, G., Zeviani, M., Pinton, P., Garavaglia, B., and Nardocci, N (2015) A family with paroxysmal nonkinesigenic dyskinesias (PNKD): evidence of mitochondrial dysfunction Eur J Paediatr Neurol 19, 64–68 Antonicka, H., Leary, S.C., Guercin, G.H., Agar, J.N., Horvath, R., Kennaway, N.G., Harding, C.O., Jaksch, M., and Shoubridge, E.A (2003a) Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency Hum Mol Genet 12, 2693–2702 Antonicka, H., Mattman, A., Carlson, C.G., Glerum, D.M., Hoffbuhr, K.C., Leary, S.C., Kennaway, N.G., and Shoubridge, E.A (2003b) Mutations in COX15 produce a defect in the mitochondrial heme biosynthetic pathway, causing early-onset fatal hypertrophic cardiomyopathy Am J Hum Genet 72, 101–114 Balsa, E., Marco, R., Perales-Clemente, E., Szklarczyk, R., Calvo, E., Landa´zuri, M.O., and Enrı´quez, J.A (2012) NDUFA4 is a subunit of complex IV of the mammalian electron transport chain Cell Metab 16, 378–386 Bourens, M., Boulet, A., Leary, S.C., and Barrientos, A (2014) Human COX20 cooperates with SCO1 and SCO2 to mature COX2 and promote the assembly of cytochrome c oxidase Hum Mol Genet 23, 2901–2913 Bugiani, M., Tiranti, V., Farina, L., Uziel, G., and Zeviani, M (2005) Novel mutations in COX15 in a long surviving Leigh syndrome patient with cytochrome c oxidase deficiency J Med Genet 42, e28 Chen, Y.C., Taylor, E.B., Dephoure, N., Heo, J.M., Tonhato, A., Papandreou, I., Nath, N., Denko, N.C., Gygi, S.P., and Rutter, J (2012) Identification of a protein mediating respiratory supercomplex stability Cell Metab 15, 348–360 Church, C., Chapon, C., and Poyton, R.O (1996) Cloning and characterization of PET100, a gene required for the assembly of yeast cytochrome c oxidase J Biol Chem 271, 18499–18507 Church, C., Goehring, B., Forsha, D., Wazny, P., and Poyton, R.O (2005) A role for Pet100p in the assembly of yeast cytochrome c oxidase: interaction with a subassembly that accumulates in a pet100 mutant J Biol Chem 280, 1854–1863 Clemente, P., Peralta, S., Cruz-Bermudez, A., Echevarrı´a, L., Fontanesi, F., Barrientos, A., Fernandez-Moreno, M.A., and Garesse, R (2013) hCOA3 stabilizes cytochrome c oxidase (COX1) and promotes cytochrome c oxidase assembly in human mitochondria J Biol Chem 288, 8321–8331 Cogliati, S., Calvo, E., Loureiro, M., Guaras, A.M., Nieto-Arellano, R., GarciaPoyatos, C., Ezkurdia, I., Mercader, N., Va´zquez, J., and Enriquez, J.A (2016) Mechanism of super-assembly of respiratory complexes III and IV Nature 539, 579–582 Hayashi, T., Asano, Y., Shintani, Y., Aoyama, H., Kioka, H., Tsukamoto, O., Hikita, M., Shinzawa-Itoh, K., Takafuji, K., Higo, S., et al (2015) Higd1a is a positive regulator of cytochrome c oxidase Proc Natl Acad Sci USA 112, 1553–1558 Heide, H., Bleier, L., Steger, M., Ackermann, J., Droăse, S., Schwamb, B., Zoărnig, M., Reichert, A.S., Koch, I., Wittig, I., and Brandt, U (2012) Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex Cell Metab 16, 538–549 Horn, D., Zhou, W., Trevisson, E., Al-Ali, H., Harris, T.K., Salviati, L., and Barrientos, A (2010) The conserved mitochondrial twin Cx9C protein Cmc2 Is a Cmc1 homologue essential for cytochrome c oxidase biogenesis J Biol Chem 285, 15088–15099 Invernizzi, F., D’Amato, I., Jensen, P.B., Ravaglia, S., Zeviani, M., and Tiranti, V (2012) Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells Mitochondrion 12, 328–335 Jaksch, M., Ogilvie, I., Yao, J., Kortenhaus, G., Bresser, H.G., Gerbitz, K.D., and Shoubridge, E.A (2000) Mutations in SCO2 are associated with a distinct form of hypertrophic cardiomyopathy and cytochrome c oxidase deficiency Hum Mol Genet 9, 795–801 €ttemann, M (2015) The subunit composition and funcKadenbach, B., and Hu tion of mammalian cytochrome c oxidase Mitochondrion 24, 64–76 Kirby, D.M., Thorburn, D.R., Turnbull, D.M., and Taylor, R.W (2007) Biochemical assays of respiratory chain complex activity Methods Cell Biol 80, 93–119 Lapuente-Brun, E., Moreno-Loshuertos, R., Acı´n-Pe´rez, R., Latorre-Pellicer, A., Cola´s, C., Balsa, E., Perales-Clemente, E., Quiro´s, P.M., Calvo, E., Rodrı´guez-Herna´ndez, M.A., et al (2013) Supercomplex assembly determines electron flux in the mitochondrial electron transport chain Science 340, 1567–1570 Leary, S.C., Kaufman, B.A., Pellecchia, G., Guercin, G.H., Mattman, A., Jaksch, M., and Shoubridge, E.A (2004) Human SCO1 and SCO2 have independent, cooperative functions in copper delivery to cytochrome c oxidase Hum Mol Genet 13, 1839–1848 Leary, S.C., Sasarman, F., Nishimura, T., and Shoubridge, E.A (2009) Human SCO2 is required for the synthesis of CO II and as a thiol-disulphide oxidoreductase for SCO1 Hum Mol Genet 18, 2230–2240 Lim, S.C., Smith, K.R., Stroud, D.A., Compton, A.G., Tucker, E.J., Dasvarma, A., Gandolfo, L.C., Marum, J.E., McKenzie, M., Peters, H.L., et al (2014) A Cell Reports 18, 1727–1738, February 14, 2017 1737 founder mutation in PET100 causes isolated complex IV deficiency in Lebanese individuals with Leigh syndrome Am J Hum Genet 94, 209–222 Soto, I.C., Fontanesi, F., Liu, J., and Barrientos, A (2012) Biogenesis and assembly of eukaryotic cytochrome c oxidase catalytic core Biochim Biophys Acta 1817, 883–897 Massa, V., Fernandez-Vizarra, E., Alshahwan, S., Bakhsh, E., Goffrini, P., Ferrero, I., Mereghetti, P., D’Adamo, P., Gasparini, P., and Zeviani, M (2008) Severe infantile encephalomyopathy caused by a mutation in COX6B1, a nucleus-encoded subunit of cytochrome c oxidase Am J Hum Genet 82, 1281–1289 Stiburek, L., Vesela, K., Hansikova, H., Pecina, P., Tesarova, M., Cerna, L., Houstek, J., and Zeman, J (2005) Tissue-specific cytochrome c oxidase assembly defects due to mutations in SCO2 and SURF1 Biochem J 392, 625–632 McEwen, J.E., Hong, K.H., Park, S., and Preciado, G.T (1993) Sequence and chromosomal localization of two PET genes required for cytochrome c oxidase assembly in Saccharomyces cerevisiae Curr Genet 23, 9–14 Stiburek, L., Hansikova, H., Tesarova, M., Cerna, L., and Zeman, J (2006) Biogenesis of eukaryotic cytochrome c oxidase Physiol Res 55 (Suppl 2), S27–S41 McStay, G.P., Su, C.H., Thomas, S.M., Xu, J.T., and Tzagoloff, A (2013a) Characterization of assembly intermediates containing subunit of yeast cytochrome oxidase J Biol Chem 288, 26546–26556 McStay, G.P., Su, C.H., and Tzagoloff, A (2013b) Modular assembly of yeast cytochrome oxidase Mol Biol Cell 24, 440–452 McStay, G.P., Su, C.H., and Tzagoloff, A (2013c) Stabilization of Cox1p intermediates by the Cox14p-Coa3p complex FEBS Lett 587, 943–949 Mick, D.U., Fox, T.D., and Rehling, P (2011) Inventory control: cytochrome c oxidase assembly regulates mitochondrial translation Nat Rev Mol Cell Biol 12, 14–20 Mick, D.U., Dennerlein, S., Wiese, H., Reinhold, R., Pacheu-Grau, D., Lorenzi, I., Sasarman, F., Weraarpachai, W., Shoubridge, E.A., Warscheid, B., and Rehling, P (2012) MITRAC links mitochondrial protein translocation to respiratory-chain assembly and translational regulation Cell 151, 1528–1541 Moreno-Lastres, D., Fontanesi, F., Garcı´a-Consuegra, I., Martı´n, M.A., Arenas, J., Barrientos, A., and Ugalde, C (2012) Mitochondrial complex I plays an essential role in human respirasome assembly Cell Metab 15, 324–335 Nijtmans, L.G., Taanman, J.W., Muijsers, A.O., Speijer, D., and Van den Bogert, C (1998) Assembly of cytochrome-c oxidase in cultured human cells Eur J Biochem 254, 389–394 Nijtmans, L.G., Henderson, N.S., and Holt, I.J (2002) Blue Native electrophoresis to study mitochondrial and other protein complexes Methods 26, 327–334 Ola´hova´, M., Haack, T.B., Alston, C.L., Houghton, J.A., He, L., Morris, A.A., Brown, G.K., McFarland, R., Chrzanowska-Lightowlers, Z.M., Lightowlers, R.N., et al (2015) A truncating PET100 variant causing fatal infantile lactic acidosis and isolated cytochrome c oxidase deficiency Eur J Hum Genet 23, 935–939 Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., Steen, H., Pandey, A., and Mann, M (2002) Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics Mol Cell Proteomics 1, 376–386 Pacheu-Grau, D., Bareth, B., Dudek, J., Juris, L., Voăgtle, F.N., Wissel, M., Leary, S.C., Dennerlein, S., Rehling, P., and Deckers, M (2015) Cooperation between COA6 and SCO2 in COX2 maturation during cytochrome c oxidase assembly links two mitochondrial cardiomyopathies Cell Metab 21, 823–833 Perales-Clemente, E., Bayona-Bafaluy, M.P., Pe´rez-Martos, A., Barrientos, A., Ferna´ndez-Silva, P., and Enriquez, J.A (2008) Restoration of electron transport without proton pumping in mammalian mitochondria Proc Natl Acad Sci USA 105, 18735–18739 Pe´rez-Pe´rez, R., Lobo-Jarne, T., Milenkovic, D., Mourier, A., Bratic, A., Garcı´aBartolome´, A., Ferna´ndez-Vizarra, E., Cadenas, S., Delmiro, A., Garcı´a-Consuegra, I., et al (2016) COX7A2L Is a Mitochondrial Complex III Binding Protein that Stabilizes the III2+IV Supercomplex without Affecting Respirasome Formation Cell Rep 16, 2387–2398 Pitceathly, R.D., Rahman, S., Wedatilake, Y., Polke, J.M., Cirak, S., Foley, A.R., Sailer, A., Hurles, M.E., Stalker, J., Hargreaves, I., et al.; UK10K Consortium (2013) NDUFA4 mutations underlie dysfunction of a cytochrome c oxidase subunit linked to human neurological disease Cell Rep 3, 17951805 Rydstroăm Lundin, C., von Ballmoos, C., Ott, M., Aădelroth, P., and Brzezinski, P (2016) Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae Proc Natl Acad Sci USA 113, E4476–E4485 1738 Cell Reports 18, 1727–1738, February 14, 2017 Strogolova, V., Furness, A., Robb-McGrath, M., Garlich, J., and Stuart, R.A (2012) Rcf1 and Rcf2, members of the hypoxia-induced gene protein family, are critical components of the mitochondrial cytochrome bc1-cytochrome c oxidase supercomplex Mol Cell Biol 32, 1363–1373 Stroud, D.A., Maher, M.J., Lindau, C., Voăgtle, F.N., Frazier, A.E., Surgenor, E., Mountford, H., Singh, A.P., Bonas, M., Oeljeklaus, S., et al (2015) COA6 is a mitochondrial complex IV assembly factor critical for biogenesis of mtDNA-encoded COX2 Hum Mol Genet 24, 5404–5415 Su, C.H., McStay, G.P., and Tzagoloff, A (2014) The Cox3p assembly module of yeast cytochrome oxidase Mol Biol Cell 25, 965–976 Szklarczyk, R., Wanschers, B.F., Cuypers, T.D., Esseling, J.J., Riemersma, M., van den Brand, M.A., Gloerich, J., Lasonder, E., van den Heuvel, L.P., Nijtmans, L.G., and Huynen, M.A (2012) Iterative orthology prediction uncovers new mitochondrial proteins and identifies C12orf62 as the human ortholog of COX14, a protein involved in the assembly of cytochrome c oxidase Genome Biol 13, R12 Szklarczyk, R., Wanschers, B.F., Nijtmans, L.G., Rodenburg, R.J., Zschocke, J., Dikow, N., van den Brand, M.A., Hendriks-Franssen, M.G., Gilissen, C., Veltman, J.A., et al (2013) A mutation in the FAM36A gene, the human ortholog of COX20, impairs cytochrome c oxidase assembly and is associated with ataxia and muscle hypotonia Hum Mol Genet 22, 656–667 Tiranti, V., Munaro, M., Sandona`, D., Lamantea, E., Rimoldi, M., DiDonato, S., Bisson, R., and Zeviani, M (1995) Nuclear DNA origin of cytochrome c oxidase deficiency in Leigh’s syndrome: genetic evidence based on patient’sderived rho degrees transformants Hum Mol Genet 4, 2017–2023 Tiranti, V., Corona, P., Greco, M., Taanman, J.W., Carrara, F., Lamantea, E., Nijtmans, L., Uziel, G., and Zeviani, M (2000) A novel frameshift mutation of the mtDNA COIII gene leads to impaired assembly of cytochrome c oxidase in a patient affected by Leigh-like syndrome Hum Mol Genet 9, 2733–2742 Tyanova, S., Temu, T., Sinitcyn, P., Carlson, A., Hein, M.Y., Geiger, T., Mann, M., and Cox, J (2016) The Perseus computational platform for comprehensive analysis of (prote)omics data Nat Methods 13, 731–740 Valnot, I., Osmond, S., Gigarel, N., Mehaye, B., Amiel, J., Cormier-Daire, V., Munnich, A., Bonnefont, J.P., Rustin, P., and Roătig, A (2000a) Mutations of the SCO1 gene in mitochondrial cytochrome c oxidase deficiency with neonatal-onset hepatic failure and encephalopathy Am J Hum Genet 67, 1104–1109 Valnot, I., von Kleist-Retzow, J.C., Barrientos, A., Gorbatyuk, M., Taanman, J.W., Mehaye, B., Rustin, P., Tzagoloff, A., Munnich, A., and Roătig, A (2000b) A mutation in the human heme A:farnesyltransferase gene (COX10 ) causes cytochrome c oxidase deficiency Hum Mol Genet 9, 1245–1249 Vukotic, M., Oeljeklaus, S., Wiese, S., Voăgtle, F.N., Meisinger, C., Meyer, H.E., Zieseniss, A., Katschinski, D.M., Jans, D.C., Jakobs, S., et al (2012) Rcf1 mediates cytochrome oxidase assembly and respirasome formation, revealing heterogeneity of the enzyme complex Cell Metab 15, 336–347 Williams, E.G., Wu, Y., Jha, P., Dubuis, S., Blattmann, P., Argmann, C.A., Houten, S.M., Amariuta, T., Wolski, W., Zamboni, N., et al (2016) Systems proteomics of liver mitochondria function Science 352, aad0189 Wittig, I., Braun, H.P., and Schaăgger, H (2006) Blue native PAGE Nat Protoc 1, 418–428  ... an interaction between PET117 and MR- 1S plus PET100 during COX biogenesis MR- 1S Interacts with PET100 and PET117 The absence of human PET100 is associated with severe, isolated COX deficiency and. .. Assembly and Interaction with PET117 and PET100 We found MR- 1S to interact with COX-related proteins Accordingly, MR- 1SHA binds to a subassembly species composed of the structural subunits defining... Figure MR- 1S Is Found in Two Distinct Supra-molecular Complexes and Binds COX Structural Subunits and Assembly Factors (A) SILAC-complexomic profiles for MR- 1S and MT-CO2 in WT and MT-CO3 mutant cybrids