Accessory subunits are integral for assembly and function of mitochondrial complex I

David A. Stroud1*, Elliot E. Surgenor1, Luke E. Formosa1,2, Boris Reljic2†, Ann E. Frazier3,4,

Marris G. Dibley1, Laura D. Osellame1, Tegan Stait3, Traude H. Beilharz1, David R.

Thorburn3-5, Agus Salim6, Michael T. Ryan1*

1Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery

Institute, Monash University, 3800, Melbourne, Australia.

2Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La

Trobe University 3086, Melbourne, Australia.

3Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne 3052,

Australia

4Department of Pediatrics, University of Melbourne, Melbourne 3052, Australia.

5Victorian Clinical Genetics Services, Royal Children’s Hospital 3052, Melbourne, Australia.

6Department of Mathematics and Statistics, La Trobe University 3086, Melbourne Australia.

*Correspondence to: [email protected] and [email protected]

†Current address: Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne,

Victoria 3052, Australia

Complex I (NADH:ubiquinone ) is the first of the mitochondrial respiratory chain (RC) and is composed of 44 different subunits in , making it one of the largest known multi-subunit membrane complexes1. Complex I exists in supercomplex forms with RC complexes III and IV, which are together required for the generation of a transmembrane proton gradient used for the synthesis of ATP2.

Complex I is also a major source of damaging reactive oxygen species and its dysfunction is associated with , Parkinson’s disease and aging3-5.

Bacterial and human complex I share 14 core subunits essential for enzymatic function, however the role and requirement of the remaining 31 human accessory subunits is unclear1,6. The incorporation of accessory subunits into the complex increases the cellular energetic cost and has necessitated the involvement of numerous assembly factors for complex I biogenesis. We used -editing to generate human knockout cell lines for each accessory subunit. We found that 25 subunits are strictly required for assembly of a functional complex and one subunit is essential for cell viability.

Quantitative proteomic analysis of cell lines revealed that loss of each subunit affects the stability of other subunits residing in the same structural module. Analysis of proteomic changes following loss of specific modules revealed ATP5SL and DMAC1 as novel factors required for assembly of the distal portion of the complex I membrane arm. Our results demonstrate the broad importance of accessory subunits in the structure and function of human complex I. Coupling gene-editing technology with proteomics represents a powerful tool for dissecting large multi-subunit complexes and enabling the study of complex dysfunction at a cellular level.

Mitochondrial complex I is a boot-shaped structure of ~1MDa with a hydrophilic matrix arm and a hydrophobic membrane arm7-9. These arms are assembled via intermediate modules through transient association with assembly factors10. The N-module at the tip of the matrix arm is involved in oxidation of NADH while the Q-module bridges the matrix and membrane arms and is involved in transfer of electrons along Fe-S clusters to ubiquinone. With the reduction of ubiquinone, four protons are pumped across the inner-membrane (IM) into the intermembrane space (IMS). The core structure of the membrane arm is defined by 7 subunits encoded by mitochondrial DNA (mtDNA); ND1 at the base of the Q-module, followed by

ND3, 6, and 4L, and the antiporter-like subunits ND2, ND4 and ND58,9. The mechanisms of

NADH oxidation and proton pumping are conserved from bacteria to humans, with 14 core

(including the 7 mtDNA-encoded) subunits performing these roles11.

To investigate the importance of the 31 accessory subunits, we used TALEN and

CRISPR/Cas9 gene-editing tools to disrupt their in human HEK293T cells

(Supplementary Table 1). Of the knockout (KO) lines generated, 24 were unable to grow on galactose-containing media indicating mitochondrial respiration defects (Fig. 1). Blue-native

(BN)-PAGE and immunoblot analysis for subunits NDUFA9, NDUFA13 and NDUFB11,

(located in different regions of the complex) revealed that loss of an individual accessory subunit often disrupted assembly of complex I (Fig. 1). Analysis of the supercomplex was also disrupted in the same cell lines (Extended Data Fig. 1) while assembly of complexes III and IV was not affected (Extended Data Fig. 2). For cell lines still capable of growth on galactose, a complex was present that did not markedly differ from the migration of mature complex I (Fig. 1, lanes 2-6). Other cell lines showed different subcomplexes including one that migrated slightly faster than complex I, consistent with loss of the N-module12 (Fig. 1, lanes 5-8, marked with ‡).

In contrast to all other subunits, we found NDUFAB1 to be essential for cell viability

(Extended Data Fig. 3a-c). NDUFAB1 is unique as it is the only subunit with a 2:1 stoichiometry within the complex, where it binds LYR motifs present in NDUFA6 and

NDUFB97. NDUFAB1 is also the mitochondrial acyl carrier protein13 and associates with involved in fatty-acid synthesis (LIPT2) and other proteins (Extended Data Fig. 3d;

Supplementary Table 2) including LYRM7 that promotes biogenesis of the complex III

Rieske subunit (UQCRFS1). An NDUFAB1KO cell line was generated by complementing cells with the yeast mitochondrial acyl carrier protein (yACP1) (Extended Data Fig. 3e, Supplementary Table 1). Since NDUFAB1KO lacks assembled complex I and dies in galactose media (Fig. 1; Extended Data Fig. 3c, e), the essential role of NDUFAB1 is independent of complex I.

We selected a subset of representative KO lines for further analysis (Fig. 2a). Rescue of each line restored complex I assembly in all cases (Fig. 2b). Cell lines lacking NDUFV3,

NDUFA12 or NDUFA7 that still grew on galactose, had negligible to moderate reductions in complex I activity and mitochondrial respiratory capacity (Fig. 2c). Knockout of NDUFS6 led to most of the N-module dissociating from complex I, however this did not severely impact complex I activity or respiratory capacity (Fig. 2c). This is consistent with previous patient cell studies12 and suggests that the complex is less stable during BN-PAGE (Extended

Data Fig. 4a). In NDUFA12KO cells, the complex I assembly factor NDUFAF2, substituted for its paralog NDUFA12 leading to complex I appearing fully assembled (Extended Data

Fig. 4b). In NDUFA2KO cells, no N-module was present (Extended Data Fig. 4a) and these cells showed severe defects in complex I activity and respiration (Fig. 2c). We hypothesize that NDUFV3 may be the terminally assembled subunit of complex I due to its location9 and lack of defects upon its loss. In vitro imported NDUFV3 also readily exchanged with the endogenous assembled protein (Extended Data Fig. 4c), while only bona fide subunits were enriched without assembly factors when complex I was isolated using NDUFV3 as bait

(Extended Data Fig. 3d, Supplementary Tables 3-4). In contrast to most N-module subunits, knockout of membrane arm subunits resulted in severe mitochondrial respiration defects (Fig.

2c) and loss of assembled complex I with the concomitant accumulation of subcomplexes

(Fig. 1).

The severity of knockouts observed for each accessory subunit on complex I assembly appeared to largely predict the impact of in patients with mitochondrial disease

(Extended Data Table 1)14. Almost all patients reported with mutations in genes encoding three of the subunits that exhibit mild assembly defects (NDUFA12, NDUFS4, NDUFS6), have two nonsense mutations that block subunit expression. In contrast, patients having defects in 8 accessory subunits showing severe assembly defects carry missense mutations, suggesting that the complete loss of any of these subunits may be incompatible with human life (Extended Data Table 1).

Next we employed stable isotope labelling with amino acids in cell culture (SILAC) and quantitative mass-spectrometry to determine changes in levels of cellular proteins in the representative KO lines (Supplementary Table 5). Most of the >6,000 cellular proteins detected did not significantly differ from control except for complex I subunits themselves, which were consistently downregulated (Fig. 2e, Extended Data Fig. 5a). Of the other 20 mitochondrial proteins whose levels were changed >2-fold, 9 were similarly responsive in a cell line lacking functional complex IV15 (Extended Data Fig. 5b; Supplementary Table 6) pointing to these gene products being related to general defects in oxidative .

Besides affected gene sets related to complex I and oxidative phosphorylation, other affected pathways related to metabolism, transporter activity, and DNA replication (Fig.

2e; Extended Data Fig. 5d).

Hierarchical clustering of protein ratios in the representative KO cell lines (Extended Data

Fig. 5a) identified clusters of complex I subunits that are similarly located in the structure

(e.g. NDUFS4, NDUFA7 and NDUFA12). To increase the resolution of our clustering analysis, we measured the levels of mitochondrial proteins in the remaining 20 KO cell lines

(Fig. 3a; Supplementary Table 5). Using the colour scheme from our heat maps, we mapped the levels of individual subunits in each KO onto the recently solved structure of bovine complex I9 (Fig. 3b; Extended Data Fig. 6a). We uncovered clear structural correlations including the loss of subunits around the N-module upon KO of subunit NDUFA2 as well as loss of subunits from the distal membrane module in NDUFB11KO cells (Fig. 3b). Transcriptomic analysis of the representative KO cell lines revealed that the only genes with more than a two-fold difference in expression are those that were gene-edited (Extended Data

Fig. 7). We conclude that the mutated target genes may be subject to nonsense-mediated mRNA decay while the other complex I subunits whose levels decrease are most likely proteolytically degraded16.

Hierarchical clustering analysis of complex I subunits (Supplementary Table 7) identified five clusters containing subunits with similar stabilities across knockouts (Fig. 3a). Mapping of these clusters to the structure of bovine complex I9 revealed distinct modules (Fig. 3c;

Extended Data 6b). One cluster contains subunits encompassing the N-module while each other cluster partitions with mtDNA-encoded “ND” subunits. NDUFAB1 could not be assigned to any cluster with its level being almost unchanged, consistent with its separate functions. Subunits NDUFA9, NDUFB4, NDUFB6 and NDUFA11 were not clearly mapped to an individual module and may reside at module interfaces.

Complex I is assembled via a series of intermediate assembly modules and requires the involvement of >10 known assembly factors17,18. We generated KO cell lines of assembly factors known to function at different steps – NDUFAF1, NDUFAF2, NDUFAF4,

NDUFAF6 and TIMMDC1 (Extended Data Fig. 8a). BN-PAGE analysis showed a reduction or loss of complex I assembly (Fig. 4a). Proteomic analysis indicated that complex I subunits belonging to different modules were affected to varying degrees (Extended Data Fig. 8b, c).

The profile of changes in complex I subunits in assembly factor KO lines correlated with groups of complex I KO lines whose subunits belong to distinct modules (Fig. 4b) consistent with assembly models18. Since little is known about the assembly of the distal membrane module, we searched our proteomic dataset for proteins altered in KOs belonging to the ND4 and ND5 module relative to those belonging to ND1 and ND2 modules (Supplementary

Table 8). ATP5SL, recently identified in a complex I subassembly17, accumulated in ND4- and ND5-module KO lines (Fig. 4c). In a separate analysis, the uncharacterized DMAC1

(TMEM261) was at elevated levels in membrane arm subunit KO lines when compared against matrix arm subunit KO lines (Fig. 4c; Supplementary Table 9). KO of either ATP5SL or DMAC1 led to specific and severe complex I assembly defects (Fig. 4d, Extended Data

Fig. 9a, b) and turnover of N-module and distal membrane arm subunits (Extended Data Fig.

9c, d). Integration of the proteomic profiles in DMAC1KO and ATP5SLKO lines with those originating from our accessory subunit KO lines, indicated a strong correlation with the ND5 module (Fig. 4b).

While DMAC1 is absent from the MitoCarta2.0 database19, we found it to be a mitochondrial inner-membrane protein (Extended Data Fig. 9e, f). Pulse-chase analysis revealed that mtDNA-encoded subunits formed a 600 kDa intermediate complex20 in DMAC1KO cells but then dissociated (Extended Data Fig. 9g) indicating a late-stage assembly defect similar to that seen upon loss of complex I assembly factor FOXRED121. Proteins highly enriched with

ATP5SL included complex I subunits of the ND4 module and FOXRED1 (Fig. 4e) while proteins enriched with DMAC1 included subunits ND4 and ND5, plus ATP5SL and

FOXRED1 along with OXA1L, the membrane insertase for mtDNA-encoded subunits (Fig.

4e; Supplementary Table 10-11). ATP5SL and DMAC1 also interacted with newly translated

ND5 (Fig. 4f). Since other complex I subunits, assembly factors and subunits of complexes

III and IV were enriched in DMAC1 pull-downs, the integration of the ND4 and ND5 modules in the assembly pathway may intersect with supercomplex formation and occur concurrently with addition of the N-module, the final step in complex I assembly22. Due to the association of DMAC1 with the biogenesis of the distal region of complex I, we termed the protein Distal Membrane-arm Assembly Component 1.

In summary, we demonstrate that accessory subunits are integrally associated in modules, defined by the core structural and functional subunits of human complex I, assembly of which require the concerted action of assembly factors. By defining the impact of individual subunit KOs, our data will facilitate validation of putative pathogenic variants found in complex I genes in patients while DMAC1 and ATP5SL also represent new pathological gene targets. Our approach also serves as a powerful example of how coupling gene-editing and quantitative proteomics allows rapid insights into previously inaccessible aspects of human cellular function.

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Figure 1. Analysis of complex I assembly in KO cell lines. Mitochondria were solubilized in triton X-100 and analysed by BN-PAGE and immunoblotting (IB). CI, complex I; SDHA

(complex II subunit), loading control; ‡, loss of N-module; #, subcomplexes. HEK293T*, control for NDUFAB1KO. Galactose growth and subunit positions indicated.

Figure 2. Metabolic and proteomic analysis of representative complex I accessory subunit KO lines. (a) Positions of subunits in complex I9. (b) Cell lines complemented with cDNA encoding the targeted gene. Analysis per Fig. 1. CI+III, supercomplex; ‡, loss of N- module; #, subcomplexes. (c) Upper panel, CI activity relative to (CS). n = 3 or n = 4 biological replicates (HEK293T, NDUFA7, NDUFV3). P values are from an unpaired t-test; *, P<0.05; **, P<0.01. Middle, mitochondrial basal and maximal oxygen consumption rates (OCR). Lower panel, glycolytic capacity. ECAR, extracellular acidification rate. n = 3 or n = 4 biological replicates (NDUFA7, NDUFA12, NDUFV3).

Data are mean ± S.E.M. (d) Volcano plots showing relative levels of proteins in KO cells. P values are from an unpaired t-test; n = 3 biological replicates; red dots, CI subunits; black dots, P<0.05, >1.5-fold change; light grey dots, n.s. (e) (GO) enrichment map of pathways and functions altered in respiration deficient knockouts. Example GO terms are grouped according to general role.

Figure 3. Subunit stability correlates with structural modules. (a) Levels of CI subunits in KO lines. n.d., not detected. (b) Subunit levels for KOs mapped to the CI structure9. Grey, no data; yellow and arrow, KO subunit. Scale per (a). (c) Clusters defined in (a) mapped to the CI structure. Italics, core subunits; subunits not clustered removed for clarity.

Figure 4. Analysis of complex I assembly factors including DMAC1 and ATP5SL. (a)

Complex I in assembly factor KO lines as per Fig.1. (b) KO lines compared via Pearson correlation and hierarchical clustering. (c) Volcano plots showing proteins with altered levels in KOs of subunits in specific modules. Light grey dots (n.s.), >5% False Discovery Rate

(FDR), <1.5-fold change; module N/A, module not assigned. P values from an unpaired t-test employing permutation based FDR statistics; n = 27 biological replicates (ND4/ND5,

ND1/ND2 modules), n = 56 (membrane arm), n = 23 (matrix arm). (d) Complementation of

KOs per Fig. 2b. (e) Affinity enrichment of proteins from DMAC1FLAG or ATP5SLFLAG cells.

P values are from an unpaired single-sided t-test. n = 3 biological replicates; light grey dots, n.s (P-value>0.05). (f) Radiolabelled mtDNA-encoded subunits in ATP5SLFLAG or

DMAC1FLAG lines were immunoprecipitated with FLAG beads, analysed by SDS-PAGE and autoradiography.

Methods

Cell lines, gene-editing and screening

HEK293T cells23, commonly used in complex I assembly studies15,20,21,24,25, interactome26 and mitochondrial complexome studies24, were originally purchased from the ATCC and a clonal cell line was obtained after single cell sorting20 and used as the parental line for all gene editing and proteomic work. Knockout cell lines were validated by sequencing of targeted for insertions and deletions (indels), immunoblotting and subsequent proteomic analysis. Cell lines regularly undergo testing for mycoplasma contamination using

PlasmaTest (InvivoGen). Gene-editing was performed using TALEN27 pairs as described15,28, or the pSpCas9(BB)-2A-GFP (PX458) CRISPR/Cas9 construct (a gift from Feng Zhang;

Addgene plasmid # 4813829). Briefly, in the first round, TALEN constructs were designed using the ZiFiT Targeter30. For genes unsuccessfully targeted in the first round,

CRISPR/Cas9 guide RNAs were designed for a second round of gene-disruption using

CHOPCHOP31. Successful targeting strategies and constructs can be found in Supplementary

Table 1. Gene edited and control HEK293T cells15 were cultured in DMEM (ThermoFisher) supplemented with 10% (v/v) FBS and 50 µg/mL uridine. Transfection reagents used were:

Lipofectamine 2000 and Lipofectamine LTX (ThermoFisher). During screening, glucose-free

DMEM supplemented with 5mM galactose, 1mM sodium pyruvate, 10% (v/v) dialyzed FBS

(ThermoFisher) and 50 µg/mL uridine was used to identify respiratory incompetent knockout clones. Respiratory competent knockout clones were identified by sequencing of a mixed

PCR product covering the target region, where a loss of sequencing fidelity at the target indicates a candidate clone28. With the exception of the NDUFA9KO and COA6KO cell lines which were described previously15,20, indels for individual alleles are summarized in

Supplementary Table 1. To generate NDUFAB1 knockout cells, clonal HEK293T cells were transduced with lentiviruses pLVX-TetOne-Puro-NDUFAB1*FLAG or pLVX-TetOne-Puro-yACP1FLAG

(Clontech). NDUFAB1*FLAG represents the C-terminally FLAG tagged human NDUFAB1 protein encoded by cDNA having undergone silent mutagenesis to remove the CRISPR/Cas9 target site. yACP1FLAG indicates cDNA encoding the C-terminally FLAG tagged yeast (S. cerevisiae) ACP1. Transduced cells were grown in the presence of 2 µg/mL puromycin for

72 h, and expression of NDUFAB1*FLAG or yACP1FLAG was confirmed after a further 72 h of treatment with 1 µg/mL doxycycline (DOX; Sigma-Aldrich) followed by SDS-PAGE and immunoblotting with NDUFAB1 (Abcam) and FLAG (Sigma-Aldrich) antibodies. For subsequent gene-editing, cells cultured in the presence of 50 ng/mL DOX were transfected with pSpCas9(BB)-2A-GFP-NDUFAB1 and screened as described above.

For complementation, cDNAs encoding NDUFV3FLAG, NDUFS6FLAG, NDUFA8FLAG,

ATP5SLFLAG and DMAC1FLAG (TMEM261FLAG) were cloned into pBABE-puro (Addgene

#1764;32), whereas NDUFA1, NDUFA2, NDUFB7, NDUFB10, NDUFB11 and NDUFC1 cDNAs were cloned into pBMN-Z (Addgene #1734) in place of the LacZ insert. Retroviral constructs were used to transduce the corresponding main clone (Supplementary Table 1), following which expression was selected for through growth in galactose DMEM with the exception of NDUFS6KO and NDUFV3KO which were selected using 2 µg/mL puromycin.

Transduction was verified by BN-PAGE or SDS-PAGE followed by immunoblotting with

NDUFA9 or FLAG antibodies respectively.

Mitochondrial isolation, gel electrophoresis, immunoblotting and antibodies

Mitochondria were isolated as previously described33. Protein concentration was estimated by bicinchoninic acid assay (BCA; Pierce), and aliquots of crude mitochondria stored at -80˚C until use. SDS-PAGE was performed using samples solubilized in LDS sample buffer and separated on NuPAGE Novex Bis-Tris protein gels according to manufacturer’s instructions (ThermoFisher). Tris-Tricine SDS-PAGE, BN-PAGE and 2D-PAGE were performed as described previously34-36. Carbonate and swelling experiments were performed as described37. Immunoblotting onto PVDF membranes was performed using a Novex Semi-

Dry Blotter (ThermoFisher) according to manufacturer’s instructions. Horseradish peroxidase coupled secondary antibodies and ECL chemiluminescent substrate (BioRad) were used for detection on a BioRad ChemiDoc XRS+ imaging system. The following primary antibodies were used in this study: COX2 (ThermoFisher #A-6404), COX4 (Abcam, #ab110261),

FLAG (Sigma-Aldrich, M2 clone), MIC10 (Aviva Systems Biology, #ARP44801_P050),

NDUFA13 (Mitosciences #MS103-SP), NDUFAB1 (Abcam, #ab96230), NDUFB11

(Abcam, #ab183716), NDUFV1(Proteintech #11238-1-AP), NDUFS2 (Mitosciences,

#MS114), anti-RC (Abcam, #ab110413 which contains antibodies against ATP5A,

UQCRC2, COX1, SDHB and NDUFB8), SDHA (Abcam, #ab14715), TIMMDC1 (Sigma, #

HPA053214), TOMM20 (Santa Cruz, #Sc11415) and UQCRC1 (ThermoFisher,

#16D10AD9AH5), while rabbit polyclonal antibodies against NDUFA912, NDUFAF1

(CIA30)38, NDUFAF221, NDUFAF421, NDUFB638 and HSP7020 were raised in-house. mRNA expression level analysis

For analysis of mRNA expression levels, total RNA was harvested from each cell-line in replicate with TRIzol™ (Thermo scientific). Total RNA was purified using Direct-zol™ columns according to the manufacturers specifications (Zymo Research). For cDNA synthesis 1 µg of total RNA was processed as the T12VN-PAT assay39 adapted for multiplexing on the Illumina MiSeq instrument. We refer to this assay as mPAT for multiplexed PAT. The approach is based on a nested-PCR that sequentially incorporates the

Illumina platform’s flow-cell specific terminal extensions onto 3’ RACE PCR amplicons.

First, cDNA was generated using the anchor primer mPAT Reverse, next this primer and a pool of 50 gene-specific primers were used in 5 cycles of amplification. Each gene-specific primer had a universal 5’ extension (see Supplementary Table 12) for sequential addition of the 5’ (P5) Illumina elements. These amplicons were then purified using NucleoSpin columns

(Macherey-Nagel), and entered into second round of amplification using the universal

Illumina Rd1 sequencing Primer and TruSeq indexed reverse primers from Illumina. Second round amplification was for 14 cycles. Note, that each experimental condition was amplified separately in the first round with identical primers. In the second round, a different indexing primer was used for each experimental condition. All PCR reactions were pooled and run using the MiSeq Reagent Kit v2 with 300 cycles (i.e. 300 bases of sequencing) according to the manufacturer’s specifications. Data were analysed using established bioinformatics pipelines40. Figures were generated using the R framework.

Oxygen consumption and enzymatic activity measurements

Oxygen consumption (OCR) and extracellular acidification (ECAR) rates were measured in live cells using a Seahorse Bioscience XF24-3 Analyzer as described15. Briefly, 50,000 cells were plated per well in Seahorse Bioscience culture plates treated with 50 µg/mL poly-D-

Lysine and grown overnight in standard culture conditions. The cellular OCR and ECAR were analyzed in non-buffered DMEM (Seahorse Biosciences) containing 5 mM glucose, 1 mM sodium pyruvate and 50 µg/mL uridine with the following inhibitors: 2 µM Oligomycin;

0.5 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP); 0.5 µM Rotenone; and 0.3 µM Antimycin A. For each assay cycle, four measurement time points of 2 min mix,

2 min wait and 5 min measure were collected. For each cell line, 3-4 replicate wells were measured in multiple plates and CyQuant (ThermoFisher Scientific) was used to normalize measurements to cell number. Basal OCR and non-mitochondrial respiration (following rotenone and antimycin A injections) were calculated as a mean of the measurement points.

Basal ECAR was calculated from the initial basal measurement cycle. To calculate proton leak and maximal respiration, the initial measurement following addition of oligomycin or FCCP was used. Enzymatic activity measurements were performed as previously described41 in three separate subcultures of each cell line. To accommodate unequal variance, statistical significance was determined through an unpaired two-sample, two-sided t-test using Welch’s correction.

Radiolabeling of mtDNA-encoded translation products and protein import

Radiolabelling of mtDNA-encoded proteins was performed as previously described15,34.

Isolated mitochondria were subjected to BN-PAGE or 2D-PAGE as described above, following which proteins were transferred to PVDF membranes and analysed by phosphorimager digital autoradiography (GE Healthcare Life Sciences). For immunoprecipitation of newly translated proteins, mitochondria were isolated from cells pulsed for 2 h and solubilized in 1% (w/v) digitonin, 20 mM Bis-Tris (pH 7.0), 50 mM NaCl,

0.1 mM EDTA, 10% (v/v) glycerol. After a brief clarification spin, complexes were incubated with anti-FLAG affinity gel (SigmaAldrich), the gel washed with 0.2 % (w/v) digitonin, 20 mM Bis-Tris (pH 7.0), 60 mM NaCl, 0.5 mM EDTA, 10% (v/v) glycerol, and enriched proteins eluted with the addition of 150 µg/mL FLAG peptide (SigmaAldrich).

Samples were TCA precipitated to remove detergent and analysed by SDS-PAGE and phosphorimaging as above.

For protein import, NDUFA12, NDUFA7 and NDUFV3 cDNA were cloned into the pGEM-

4Z plasmid (Promega). mRNA was transcribed using the mMESSAGE mMACHINE SP6 transcription kit (ThermoFisher Scientific) according to the manufacturer’s instructions.

Radiolabelled proteins were translated in the presence of [35S]-methionine/ using a

Rabbit Reticulocyte Lysate System (Promega). Translated proteins were incubated with isolated mitochondria at 37˚C as previously described12, following which mitochondria were analysed by SDS-PAGE or BN-PAGE as described above. Quantitative mass-spectrometry using mitochondrial and whole-cell starting material, and data analysis

For NDUFV3KO, NDUFS6KO, NDUFA2KO, NDUFA8KO, NDUFA1KO, NDUFS5KO,

NDUFC1KO, NDUFB4KO, NDUFB7KO, NDUFB10KO, NDUFB11KO mass-spectrometry was performed from SILAC labelled whole-cell starting material as described by Kulak et al.42

13 15 13 15 with modifications. Briefly, cells cultured in “heavy” C6 N4-, C6 N2-lysine- containing or “light” SILAC DMEM15 were harvested, washed in PBS and protein content determined by BCA assay. Measurements were performed in batches of 3-4 knockout cell lines in triplicate with a label switch. Each batch utilized a single pool of clonal HEK293T cells (1 sample grown in “heavy” DMEM, and 2 independent samples grown in “light”

DMEM) and knockout cell lines were grown with the complementary label orientation (1 in

“light” DMEM, and 2 in “heavy” DMEM). Equal amounts of “heavy” and “light” (typically

250 µg) control HEK293T and knockout cells were mixed, and cells were solubilized in 1% w/v sodium deoxycholate, 100 mM Tris-HCl (pH 8.1). Lysates were sonicated for 30 min at

60˚C in a sonicator waterbath, followed by denaturation and alkylation through the addition of 5 mM Tris(2-carboxyethy)phosphine (TCEP), 20 mM chloroacetamide and incubation for

5 min at 99˚C with vortexing. Samples were digested with trypsin overnight at 37˚C.

Detergent was removed by ethyl acetate extraction in the presence of 2% formic acid (FA), following which the aqueous phase was concentrated by vacuum centrifugation. Peptides were reconstituted in 0.5% FA and loaded onto pre-equilibrated small cation exchange

(Empore Cation Exchange-SR, Supelco Analytical) stage-tips made in-house. Tips were washed with 6 load volumes of 20% acetonitrile (ACN), 0.5% FA and eluted in 5 sequential fractions of increasing amounts (45-300 mM) of ammonium acetate, 20% ACN, 0.5% FA. A sixth elution was collected using 5% ammonium hydroxide, 80% ACN following which fractions were concentrated, desalted and reconstituted as previously described15. Peptides were reconstituted in 0.1% trifluoroacetic acid (TFA) and 2% ACN and fractions analysed sequentially by online nano-HPLC/electrospray ionization-MS/MS on a Q Exactive

Plus connected to an Ultimate 3000 HPLC (Thermo-Fisher Scientific). Peptides were first loaded onto a trap column (Acclaim C18 PepMap nano Trap x 2 cm, 100 µm I.D, 5 µm particle size and 300 Å pore size; ThermoFisher Scientific) at 15 µL/min for 3 min before switching the pre-column in line with the analytical column (Acclaim RSLC C18 PepMap

Acclaim RSLC nanocolumn 75 µm x 50 cm, PepMap100 C18, 3 µm particle size 100 Å pore size; ThermoFisher Scientific). The separation of peptides was performed at 250 nL/min using a non-linear ACN gradient of buffer A (0.1% FA, 2% ACN) and buffer B (0.1% FA,

80% ACN), starting at 2.5% buffer B to 35.4% followed by ramp to 99% over 120 minutes

(runs had a total acquisition time of 155 min to accommodate void and equilibration volumes). Data were collected in positive mode using Data Dependent Acquisition using m/z

375 - 1800 as MS scan range, HCD for MS/MS of the 12 most intense ions with z ≥ 2. Other instrument parameters were: MS1 scan at 70,000 resolution (at 200 m/z), MS maximum injection time 50 ms, AGC target 3E6, Normalized collision energy was at 27% energy,

Isolation window of 1.8 Da, MS/MS resolution 17,500, MS/MS AGC target of 1E5, MS/MS maximum injection time 100 ms, minimum intensity was set at 1E3 and dynamic exclusion was set to 15 sec.

For the remaining knockouts we utilized isolated mitochondria as starting material. Cells were cultured in SILAC DMEM as above and mitochondrial isolations performed in batches of 1-6 knockout cell lines in triplicate. Each batch contained a single set of clonal HEK293T mitochondria (2 independent isolations from “heavy” and 1 from “light” cells), with knockout mitochondria having the complementary label orientation (2 independent isolations from “light” DMEM and 1 from “heavy” cells). Mitochondria were isolated from cell pellets stored at -80˚C as previously described 43, but with modifications. Cells were resuspended in 20 mM HEPES-KOH (pH 7.6), 220 mM mannitol, 60 mM sucrose, 1 mM EDTA, 1 mM

PMSF and homogenized as described above. The homogenate was centrifuged at 800 x g for

5 minutes at 4˚C, and the supernatant again centrifuged at 10,000 x g for 10 minutes at 4˚C.

Crude mitochondria were resuspended in the above buffer and the two differential centrifugation steps repeated. The resuspended pellet was then layered onto a sucrose cushion consisting of 10 mM HEPES-KOH (pH 7.6), 500 mM sucrose, 1 mM EDTA. Samples were centrifuged at 10,000 x g for 10 minutes at 4˚C, following which the protein concentration was estimated by BCA assay. Equal amounts of “heavy” and “light” (typically 20 µg) control

HEK293T and knockout mitochondria were mixed as described above, collected by centrifugation at 18,000 x g and solubilized in 8 M urea, 50 mM ammonium bicarbonate.

Proteins were acetone precipitated, reduced and alkylated and desalted as previously described15. Peptides reconstituted in 0.1% TFA and 2% ACN were analysed on a Q Exactive

Plus, or a LTQ-Orbitrap Elite Instrument. Instrument and method parameters for Q Exactive

Plus were as described above however utilized a shorter gradient (90 min separation, 120 min total acquisition). For the Orbitrap Elite, instrument and method parameters were as previously described15. A single technical re-injection was collected for all mitochondrial samples.

All raw file names included identifiers for the batch, instrument and gradient used, knockout cell line being studied, and applicable label orientation. Raw files were analysed using the

MaxQuant platform44 version 1.5.4.1 searching against the Uniprot human database containing reviewed, canonical and isoform variants in FASTA format (June 2015) and a database containing common contaminants by the Andromeda search engine45. Default search parameters for a Arg10 and Lys8 labelled experiment were used with modifications.

Briefly, cysteine carbamidomethylation was used as a fixed modification, and N-terminal acetylation and methionine oxidation were used as variable modifications. False discovery rates of 1% for proteins and peptides were applied by searching a reverse database, and “Re- quantify” and “Match from and to”, “Match between runs” options were enabled with a match time window of 2 minutes. Experimental groups based on data gathered using different instrumentation and/or acquisition parameters were given odd numbered fractions to avoid falsely matched identifications, whereas fractionated whole-cell samples were given sequential fraction numbers. Unique and razor peptides with a minimum ratio count of 2 were used for quantification.

Using the Perseus platform (version 1.5.4.1), identifications were matched to the

MitoCarta2.0 database19 using Ensembl ENSG id and gene name identifiers. Identifications labeled by MaxQuant as “Only identified by site”, “Reverse” and “Potential Contaminant” were removed. Proteins having < 3 valid values in a single experimental group were removed. For mitochondrial samples, we found the correlation of log2-ratio data from biological replicates in the same experimental group to be moderate at best and as low as 0.3 in some cases. We surmised the main cause of this to be batch and labelling effect, the former due to differences in mitochondrial isolations between batches and latter due to one (of three) replicates within each experimental group always being subjected to a label switch. To account for these and potentially other factors, we adopted an approach that borrows principles from RUV-246 and SVA47 methods for removing unwanted variations, with modifications in the algorithm for choosing the control proteins (i.e. those not found in

MitoCarta 2.019) and moderating the amount of adjustment for genes with small sample size due to missing values. Adjustments were performed in the R framework, following which the adjusted ratios were imported back into Perseus. The log2 ratio values for proteins in replicates were normally distributed and had equal variances. The mean log2-transformed ratios for each experimental group were calculated along with their standard deviation and P- value based on single sample two-sided t-test15. This statistical approach was consistent with published quantitative SILAC analyses employing similar instrumentation and methods15,48,49. Groups having < 2 valid values were converted to “NaN” (not a number). A quality matrix was generated based on the standard deviation, and corresponding values having a standard deviation greater than 1 converted to “NaN”. This threshold was determined empirically to remove outliers from the main distribution of standard deviations across all samples. These data can be found in Supplementary Data Table 5.

Figs 3b and Extended Data Fig. 6a, 8c and 9d were generated from a matrix containing log2- transformed median SILAC ratios having a standard deviation <1 for complex I subunits

(Supplementary Table 7) and data were mapped to homologous subunits in PDB XXXX9.

For Fig. 3a, hierarchical clustering on rows (proteins) was performed using Pearson distance and average linkage. Data were pre-processed using k-means (clusters = 300). Images were generated using the PyMOL Molecular Graphics System, Version 1.7.2.1 (Schrödinger,

LLC). Log2 SILAC ratios for some proteins in their corresponding knockout cell line had very low (generally >4-fold reduction) ratios, whereas others were reported “NaN”. This could be either due to the “Re-quantify” option being turned on for the MaxQuant search, which results in translation of peak shapes from an identified isotope pattern being translated to its unidentified label partner, or indels in some lines generating a non-functional (but still translated) protein as we have seen previously15.

For the identification of proteins dysregulated between knockouts of discrete modules (Fig.

4c; Supplementary Tables 8 and 9), triplicate log2–transformed SILAC ratios from

Supplementary Table 5 were assigned to one of two groups based on the knockout being associated with the indicated module. Groups tested had comparable variance, and a modified

Welch’s two-sample t-test with permutation based FDR statistics50,51 was used to determine significance. Parameters for the test were: 70% minimum valid values, 250 permutations and significance being an FDR of <0.05. For the gene ontology enrichment analysis in Fig. 2c, proteins with a P-value <0.05 and with

>1.5 fold change up or down were submitted to the DAVID online tool

(david.abcc.ncifcrf.gov/home.jsp) for enriched biological processes (GOTERM_BP_FAT) and molecular function (GOTERM_MF_FAT). Functional annotation charts were exported and visualised using Cytoscape (version 3.4.0) and the Enrichment Map app52 (version 2.1.0;

P<0.005). Contents of enriched terms indicated in Fig. 2c are detailed in Extended Data Fig.

5d.

Affinity enrichment mass-spectrometry and data analysis

Affinity-enrichment experiments in Fig. 4e, Extended Data Figs 3d and 4d, and

Supplementary Tables 2-4 and 10-11 were performed from HEK293T and knockout cells complemented with the FLAG-tagged protein cultured in “heavy” or “light” SILAC DMEM as previously described15. Mass-spectrometry was performed on a Q Exactive Plus as above but using a shorter gradient (25 min separation, 60 min total acquisition). For data analysis, raw files were analyzed using the MaxQuant platform as above using default search parameters for a Arg10 and Lys8 labelled experiment, with modifications. Briefly, cysteine carbamidomethylation was used as a fixed modification, and N-terminal acetylation and methionine oxidation were used as variable modifications. False discovery rates of 1% for proteins and peptides were applied by searching a reverse database, and “Re-quantify” and

“Match from and to”, “Match between runs” options were enabled with a match time window of 2 minutes. Unique and razor peptides with a minimum ratio count of 1 were used for quantification. Data analysis was performed using the Perseus framework. Identifications were matched to MitoCarta2.0 dataset19 as above. Only proteins with a sequence coverage of

2 or more unique peptides were included in further analysis. Normalized SILAC ratios were inverted to achieve the orientation FLAG-tagged/HEK293T and proteins not present in > 2/3 replicates were removed. Log10-transformed transformed values had a normal distribution and comparable variance. For affinity-enrichment experiments, statistical method, sample size and analysis approaches were chosen based on published quantitative affinity- enrichment analyses employing similar instrumentation and methods15,21,53,54. P-values were calculated by a single (FLAG-tagged cell line enriched) sided t-test and the negative logarithmic P-value plotted against the mean of the three replicates.

Miscellaneous molecular biology cDNA inserts were obtained from an in-house cDNA library generated from our clonal

HEK293T line. Briefly, RNA was isolated using TRIzol Reagent (ThermoFisher) according to manufacturer’s instructions. The Superscript III first strand synthesis kit (ThermoFisher

Scientific) was used to generate cDNA primed with either Oligo(dT) or random hexamers.

Inserts were amplified from the library using Q5 High Fidelity DNA Polymerase (NEB) and

Gibson assembled into the relevant plasmid (see above) using the NEBuilder HiFi DNA

Assembly Master Mix (NEB) according to manufacturer’s instructions. Sanger sequencing was performed from PCR product or plasmid template DNA. DNA sequence assembly and alignment to sequencing reads was performed using SnapGene (GSL Biotech) and Geneious

(Biomatters).

Immunofluorescence microscopy was performed as previously described55 using primary antibodies (FLAG or TOMM20) at 1:500 dilutions. Primary antibodies were labelled with anti-mouse conjugated Alexa Fluor 488 and anti-rabbit conjugated Alexa Fluor 568 secondary antibodies (Molecular Probes). Hoechst (1µg/mL) was used to stain nuclei. Cells were visualized using either a Leica TCS SP8 equipped with HyD detectors. Images were processed using Image J56. All figures were prepared using Adobe Photoshop and Illustrator

(CC2015.5).

Data Reporting No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment.

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Acknowledgments

We thank M. Curtis, P. Faou, M. Lazarou, B. Porebski, L. Twigg, R. Schittenhelm (Monash

Biomedical Proteomics Platform), A. Barugahare and P. Harrison (Monash Bioinformatics

Platform), Monash Micro Imaging and the Micromon NGS Facility for assistance. We acknowledge funding from NHMRC Project Grants (1068056, 1107094) and fellowships

(1070916 to D.A.S., 541920 to A.E.F., 1022896 to D.R.T.), the Australian Mitochondrial

Disease Foundation and the Victorian Government’s Operational Infrastructure Support

Program.

Contributions D.A.S and M.T.R conceived the project and wrote the manuscript; D.A.S., D.R.T., and

M.T.R. designed the experiments; D.A.S., E.E.S., L.E.F., B.R., M.G.D., L.D.O. and M.T.R. generated and analysed KO lines; D.A.S. performed proteomic experiments; A.E.F. and T.S. performed enzymology; T.H.B. undertook transcript analysis; A.S. developed normalization algorithms.

Author information

Data available via ProteomeXchange (PXD004666) and NCBI's Gene Expression Omnibus

(GSE84913). The authors declare no competing financial interests. Correspondence to D.A.S.

([email protected]) or M.T.R ([email protected]).

Extended Data Figures

Extended Data Figure 1. Assembly analysis of the complex I/III/IV supercomplex (SC) in knockout cell lines. Mitochondria were solubilized in digitonin and complexes separated by BN-PAGE followed by immunoblotting (IB) using the indicated antibodies. An antibody against complex V (CV) subunit ATP5A was used as loading control. #, subcomplexes; *, non-specific.

Extended Data Figure 2. Steady state levels of respiratory chain complexes I-IV and supercomplex forms in the 28 complex I accessory subunit knockout lines generated in this study. NDUFA9KO has been analysed previously20, whereas the NDUFAB1KO is described in Extended Data Fig. 3. Mitochondria were solubilized in triton X-100 (TX100) or digitonin (DIG) and analysed by BN-PAGE and immunoblotting with antibodies against

NDUFA9 (complex I), SDHA (complex II), UQCRC1 (complex III) and COX4 (complex

IV). In TX100 samples, some Complex III-IV supercomplex is retained. #, secondary clone later identified be an incomplete knockout.

Extended Data Figure 3. Generation and analysis of NDUFAB1KO cell lines. (a) Scheme detailing knockout strategy of genomic NDUFAB1 using doxycycline (DOX) -inducible expression of CRISPR/Cas9-resistant NDUFAB1 (NDUFAB1*FLAG) or yACP1FLAG. (b)

NDUFAB1 knockouts complemented with NDUFAB1FLAG (NDUFAB1*-2) cells were cultured in media lacking DOX for the indicated times. Isolated mitochondria were analysed by BN-PAGE (triton X-100) or SDS-PAGE and immunoblotting (IB) with the indicated antibodies. (c) Brightfield images of cells grown +/- DOX, or + DOX in glucose or galactose cell culture medium. Scale bar, 25 µm. (d) SILAC labelled mitochondria from DOX-treated

HEK293T or NDUFAB1FLAG (NDUFAB1*-2) cells were solubilized in triton X-100 and incubated with anti-FLAG affinity gel. Elutions were mixed and analysed by LC-MS as described in the methods. Proteins enriched with NDUFAB1 include complex I subunits and

LYRM proteins. P values are from an unpaired single-sided t-test. n = 3 biological replicates; light grey dots, P-value>0.05. (e) Mitochondria isolated from NDUFAB1 knockouts complemented with yACP1FLAG or NDUFAB1FLAG were solubilized in triton X-100 and analysed by BN-PAGE and immunoblotting with the indicated antibodies.

Extended Data Figure 4. Analysis of N-module accessory subunits. (a) Mitochondria were isolated from cell lines, solubilized in triton X-100 and analysed by BN-PAGE and immunoblotting for N-module subunit NDUFV1 or non N-module subunit NDUFA9. ‡, complex lacking N-module; N*, subcomplex containing N-module. SDHA was used as a loading control. (b) Mitochondria were solubilized in digitonin and analysed by BN-PAGE and immunoblotting for NDUFAF2. †, NDUFAF2 associated complex I. (c) [35S]-methionine labelled proteins were imported into the indicated mitochondria, solubilised in digitonin and analysed by BN-PAGE and autoradiography. 10% of the input lysate was analysed by SDS-

PAGE and autoradiography. CISC, complex I supercomplex *, non-specific band. (d)

Mitochondria isolated from NDUFV3KO cells complemented with NDUFV3FLAG were solubilized in triton X-100 or digitonin and complexes bound to anti-FLAG affinity gel.

Eluted proteins were analysed by LC-MS as described in the supplementary materials. P values are from an unpaired single-sided t-test. n = 3 biological replicates; light grey dots, n.s.

P-value>0.05.

Extended Data Figure 5. Proteomic analysis of KO cell lines. (a) Relative levels of proteins in representative accessory subunit KO cell lines, clustered according to Euclidean distance. Column order is as per Fig. 2b. The inset shows complex I subunit specific clusters.

(b) Volcano plot depicting proteins regulated in representative accessory subunit KO cell lines harbouring respiration defects (NDUFA2KO, NDUFA8KO, NDUFS5KO, NDUFC1KO,

NDUFB10KO, NDUFB11KO, NDUFB7KO). Proteins found to be regulated in a cell line with a severe complex IV defect15 are shaded light blue (down) and green (up), suggesting their response is due to general defects in respiration. Inset, volcano plot depicting the relative level of proteins in a complex IV knockout cell line. P values are from an unpaired t-test; n =

8 independent means comprised each of 3 biological replicates (main panel), n = 3 (inset) biological replicates; light grey dots, P-value>0.05, <1.5-fold change. Data is reproduced in

Supplementary Table 6. (c) Proteins affected >2-fold in levels in respiration-deficient subunit

KO cell lines. Colour key according to (b). Bold, proteins listed in MitoCarta2.0. (d) Proteins associated with GO terms and groups outlined in Fig. 2d.

Extended Data Figure 6. Mapping of complex I subunit levels onto the structure. (a)

Subunit levels in complex I accessory subunit KO lines were mapped to homologous subunits in the bovine single-particle electron cryo-microscopy structure of complex I9 as in Fig. 3b.

Both sides of complex I are shown. Median ratio data used in the preparation of this figure can be found in Supplementary Table 7. (b) Opposite side view of Fig. 3c. n.d., dark grey shading on the structures, subunits not quantified. Subunits not clustered to modules removed for clarity.

Extended Data Figure 7. mRNA expression levels in selected accessory subunit knockout lines. Transcripts were measured for nuclear-encoded complex I subunit genes along with control genes from complex II (SDHA), complex III (UQCRC1, UQCRFS1), complex IV (COX4L1, NDUFA4), complex V (ATP5B, ATP5H) and mt-ribosome (MRPS2,

MRPL46) in KO lines (performed in duplicate).

Extended Data Figure 8. Analysis of assembly factor knockout lines. (a) Mitochondrial proteins from the indicated cell lines were separated by SDS-PAGE and subjected to western blot analysis. (b) Volcano plots showing fold changes vs P-values for the mitochondrial proteins in assembly factor knockout cell lines. P values are from an unpaired t-test; n = 3 biological replicates; coloured dots are according to the key at bottom right; n.s., P=>0.05.

(c) Subunit levels mapped to homologous subunits in the bovine single-particle electron cryo- microscopy structure as for Fig. 3b. n.d., dark grey shading on the structures, subunits not quantified. Both sides of complex I are shown.

Extended Data Figure 9. Characterization of DMAC1 and ATP5SL. (a) ATP5SLKO mitochondria were solubilized in triton X-100 or digitonin and analysed by BN-PAGE and immunoblotting with the indicated antibodies. (b) As for (a) using DMAC1KO mitochondria.

(c) Volcano plots showing fold changes vs P-values for the mitochondrial proteins in

ATP5SL and DMAC1 KO cell lines. P values are from an unpaired t-test; n = 3 biological replicates; coloured dots represent complex I subunits depicted in the key; n.s., P>0.05. (d)

Subunit levels mapped to homologous subunits in the bovine single-particle electron cryo- microscopy structure as for Fig. 3b. n.d., dark grey shading on the structures, not quantified.

Both sides of complex I are shown. (e) Mitochondria isolated from DMAC1 cells complemented with DMAC1FLAG were resuspended in isotonic buffer, hypoosmotic swelling buffer, or triton X-100 (TX-100) followed by proteinase K (PK) incubation where indicated.

Alternately, mitochondria were treated with 100 mM Na2CO3 and membrane-integral (pellet) and soluble or peripherally attached (supernatant, SN) proteins were separated by ultracentrifugation. Samples were analysed by SDS-PAGE and immunoblotting for

TOMM20 (outer membrane); MIC10 (integral inner membrane protein exposed to intermembrane space); NDUFAF1 (matrix, soluble); NDUFS2 (matrix, peripheral). (f)

DMAC1KO cells complemented with DMAC1FLAG were analysed by immunofluorescence microscopy with the indicated antibodies. TOMM20, mitochondrial protein. Scale bar, 20

µm. Representative result from 3 independent experiments. (g) Cells were pulsed with [35S]- methionine for 1 h and chased for the indicated times as described in the supplementary materials. Isolated mitochondria were solubilized in triton X-100 and analysed by 2D-PAGE and autoradiography. à, 600 kDa complex; #, subcomplex containing ND1 and ND2.

Extended Data Table 1: Pathogenic mutations in complex I accessory subunit genes in patients with mitochondrial disease

aThree accessory subunits in which knockouts cause mild complex I assembly defects have had patients reported with pathogenic mutations; in almost all cases the mutations are expected to cause two null alleles, suggesting that almost complete loss of function of these subunits may be required to cause human disease. Eight accessory subunits in which knockouts cause severe complex I assembly defects have had patients reported with pathogenic mutations; in almost all cases the patients have at least one missense mutation or some evidence that some residual subunit protein is present. This suggests that complete loss of function of these subunits may not be compatible with human life. Interpretation of the data for the NDUFB11 and NDUFA1 subunits is complicated by their being encoded on the X . Males thus have only one copy of these genes while females have 2 copies with some cells expressing the wildtype and some expressing the mutant . All reported NDUFB11 patients are female and all had stop codon or frameshift mutations expected to cause null alleles. Such patients often have skewed X-inactivation, with most cells expressing the wildtype allele, which may compensate partly for the severity of the defect. An additional subunit, NDUFA5, has not had patients with mutations identified but knockout of the mutation in mice results in embryonic lethality. This is consistent with the suggestion that human foetuses may not be viable if they have null-type mutations in both alleles of genes encoding accessory subunits linked to severe assembly defects. #, Online Mendelian Inheritance in Man, OMIM®. McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) 01/20/2016 World Wide Web URL: http://omim.org KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO HEK293T NDUFV3 NDUFA7 NDUFA12 NDUFS6 NDUFS4 NDUFA2 NDUFA6 NDUFA9 NDUFA3 NDUFA8 NDUFA13 NDUFA1 NDUFS5 NDUFA10 NDUFC1 NDUFB10 HEK293T* NDUFC2 NDUFB1 NDUFB5 NDUFB11 NDUFB7 NDUFB3 NDUFB2 NDUFB9 NDUFB8 NDUFA11 NDUFAB1 NDUFB6 NDUFA5 NDUFB4 viability in galactose ++++++−−−−−−−−−−−− −−−−−−−−−−−−−+ kDa 669 CI ‡ 440 Matrix IM # IMS 134 IB: NDUFA9

IB: SDHA CII kDa 669 CI ‡ 440 Matrix IM # IMS 134 IB: NDUFA13 IB: SDHA CII kDa 669 CI 440 ‡ Matrix IM # IMS 134 IB: NDUFB11 IB: SDHA CII 12345678910 11 12 13 14 15 16 17 18 19 20 2122 23 24 25 26 27 28 2930 3132

Stroud et al. Figure 1 KO KO KO KO a b c KO KO KO KO KO KO KO KO NDUFV3 BN-PAGE, IB: NDUFA9

N--mod. KO KO KO KO KO KO KO KO KO NDUFA2 NDUFV3 NDUFA7 NDUFS6 NDUFA2 NDUFA8 NDUFA1 NDUFS5 NDUFC1 NDUFB7 “heel” “toes” HEK293T NDUFA12 NDUFB10 NDUFB11 NDUFA7 3 NDUFS6 NDUFS6 NDUFA2 NDUFA8 NDUFA1 NDUFS5 NDUFC1 NDUFB10 NDUFB11 NDUFB7 HEK293T − +−+−+−+−+−+−+−+−+rescue CI/CS NDUFA12 NDUFB11 2 kDa CI+III activity * x10 1 * NDUFC1 CI * * * * * * * * 0 * * * * * * * * Matrix NDUFA1 NDUFB7 669 ‡ 150 Basal mito. respiration IM % HEK293T 100 Max. mito. respiration # basal mito. 440 50 IMS respiration NDUFA8 NDUFS5 NDUFB10 IB: SDHA CII 0 CI d other 12345678910 11 12 13 14 15 16 17 18 19 100 n.s. / <1.5-fold change % HEK293T

4 4 4 4 4 Oligomycin ECAR/ 50 Basal ECAR e Oxidative 0 phosphorylation p-value p-value p-value p-value p-value 2 2 2 2 2 Down 10 10 10 10 10 GO:0003954 Up NADH Translation

-Log -Log -Log -Log -Log dehydrogenase activity GO enrichment 0 0 0 0 0 GO:000412 Translation Up -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 Down

Log2 ratio Log2 ratio Log2 ratio Log2 ratio Log2 ratio Metabolism NDUFV3KO/HEK293T NDUFS6KO/HEK293T NDUFA2KO/HEK293T NDUFA8KO/HEK293T NDUFA1KO/HEK293T GO:0005544 Calcium 4 4 4 4 4 dependent GO:0006260 phospholipid DNA replication GO:0050662 binding Coenzyme Transporters p-value p-value p-value

2 2 2 2 2 binding p-value p-value 10 10 10 10 10 -Log -Log -Log -Log -Log GO:0055114 Oxidation reduction GO:0030554

0 0 0 0 0 Adenyl nucleotide GO:006091 binding -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 -4 -2 0 2 4 Generation of GO:0015399 precursor Primary active Log2 ratio Log2 ratio Log2 ratio Log2 ratio Log2 ratio metabolites and transmembrane NDUFS5KO/HEK293T NDUFC1KO/HEK293T NDUFB10KO/HEK293T NDUFB11KO/HEK293T NDUFB7KO/HEK293T energy transporter activity

Stroud et al. Figure 2 Log ratio KO lines: a 2 c vs HEK293T N module Q mod. ND1 mod. ND2 mod. -2 -1 0 12 n.d. NDUFA2 NDUFS6 NDUFA5 MT-ND1 MT-ND2

NDUFV3 NDUFA7 NDUFA12 NDUFS6 NDUFS4 NDUFA2 NDUFA6 NDUFA9 NDUFA3 NDUFA5 NDUFA8 NDUFA13 NDUFA1 NDUFS5 NDUFA10 NDUFC1 NDUFC2 NDUFB1 NDUFB5 NDUFB10 NDUFB11 NDUFB4 NDUFB6 NDUFB7 NDUFB3 NDUFB2 NDUFB9 NDUFB8 NDUFA11 NDUFAB1 NDUFA6 NDUFV1 NDUFS2 NDUFA3 MT-ND3

NDUFV3L NDUFA7 NDUFV2 NDUFS3 NDUFA8 NDUFA1 NDUFV3S NDUFA12 NDUFV3 NDUFS7 NDUFA13 NDUFA10 NDUFS6 NDUFA7 NDUFS1 NDUFS8 NDUFC1 NDUFA12 NDUFS4 NDUFC2 NDUFS4 NDUFV1 NDUFS5 NDUFS1 N mod. NDUFA11 NDUFA9 NDUFV2 NDUFA2 NDUFA6 NDUFAB1 ND4 mod. ND5 mod. NDUFA5 NDUFS3 NDUFS2 MT-ND4 MT-ND5 NDUFS7 NDUFB1 NDUFB3 NDUFS8 NDUFB5 NDUFB7 NDUFA3 ND1/Q NDUFA8 ND1/Q NDUFB10 NDUFB8

Subunits: NDUFA13 NDUFB11 NDUFB9 MT-ND1 NDUFA9 NDUFB4 NDUFB6 NDUFA10 MT-ND3 MT-ND2 NDUFC2 ND2 NDUFC1 ND2 NDUFS5 NDUFA1 NDUFB4 NDUFA11 MT-ND4 Matrix NDUFB10 NDUFB11 ND4 NDUFB1 ND4 NDUFB5 NDUFB6 IM MT-ND5 NDUFB9 NDUFB7 ND5 NDUFB8 ND5 NDUFB3 NDUFAB1 IMS

b

Matrix

IM

IMS NDUFA2KO NDUFA8KO NDUFS5KO NDUFB11KO NDUFB7KO

Stroud et al. Figure 3 Pearson a b correlation BN-PAGE 0 10.5 KO KO KO KO KO NDUFB11 NDUFB5 NDUFB10 ND4 mod. NDUFB1 NDUFAF1 NDUFA10

NDUFAF1 NDUFC2 NDUFAF2 NDUFAF6 NDUFAF4 HEK293T TIMMDC1 NDUFS5 ND2 mod. NDUFC1 +NDUFAF1 kDa NDUFA1 NDUFA8 CI TIMMDC1 ND1 mod. 669 NDUFA3 NDUFA13 +TIMMDC1 NDUFAF6 440 NDUFA5 Q mod. NDUFAF4 +NDUFAF6 DMAC1 KO lines: NDUFB3 NDUFAF4 NDUFB9 ND5 mod.

IB: NDUFB11 NDUFB2 134 ATP5SL +ATP5SL NDUFB7 subcomplexes NDUFB8 DMAC1 NDUFA6 NDUFA2 IB: SDHA CII NDUFAF2 N mod. NDUFS6 +NDUFAF2 123456 NDUFS4 c N mod. Complex III d BN-PAGE ND1/Q modules Complex IV

matrix KO ND2 module Mitocarta 2.0 KO ND4 module other 15 arm ND5 module n.s. / <1.5 module N/A fold change Matrix IM 10 ND1/ND2 ND4/5 DMAC1 IMS ATP5SL ATP5SL HEK293T −+ −+rescue

p-value NDUFAF2 kDa CI+III 10

p-value CI

5 DMAC1 669 10

-Log 440 -Log 134 IB: NDUFA9

0 66 0 5 10 -1 01-2 -1 0 21 IB: SDHA CII protein changes in protein changes in 12345 ND4/ND5 vs ND1/ND2 modules membrane vs matrix arm subunits e f [35S]-met. pulse, 2h IP: FLAG, SDS-PAGE DMAC1 ATP5SL ND4 ND5 Total Elution Total Elution FOXRED1 OXA1L 1

1 ATP5SL FOXRED1 FLAG FLAG FLAG FLAG NDUFAF2 NDUFAF4 ND4 0.5 0.5 HEK293T HEK293T HEK293T HEK293T ATP5SL DMAC1 DMAC1 ND5 kDa ATP5SL NDUFAF3 50

ratio enrichment * ratio enrichment TIMMDC1 10 10 ND5 NDUFAF3 37 COXI 0 0 Log NDUFAF4 Log CytB COXII/III n.s. n.s. 25 ATP6 20 0 2 0 2 4 15 -Log10 p-value -Log10 p-value FLAG FLAG ATP5SL /HEK293T DMAC1 /HEK293T 1234 5678

Stroud et al. Figure 4 KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO HEK293T NDUFV3 NDUFA7 NDUFA12 NDUFS6 NDUFS4 NDUFA2 NDUFA6 NDUFA9 NDUFA3 NDUFA5 NDUFA8 NDUFA13 NDUFA1 NDUFS5 NDUFA10 NDUFC1 NDUFB10 HEK293T* NDUFC2 NDUFB1 NDUFB5 NDUFB11 NDUFB7 NDUFB3 NDUFB2 NDUFB9 NDUFB8 NDUFA11 NDUFAB1 NDUFB6 NDUFB4 kDa SC 669 440 # IB: NDUFA9

IB: ATP5A CV kDa SC 669 * 440 # IB: NDUFA13

IB: ATP5A CV kDa SC 669 440 # IB: NDUFB11

IB: ATP5A CV 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 2223 2425 26 2728 29 30 3132

Stroud ED Fig. 1 CRISPR/Cas9 generated: NDUFA3 NDUFA7 NDUFA12 NDUFB7 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA12-6 NDUFA12-23 HEK293T NDUFA7-3 NDUFA7-13 HEK293T NDUFA7-13 HEK293T NDUFA7-3 NDUFA7-13 HEK293T NDUFA7-3 NDUFA7-13 HEK293T NDUFA7-3 NDUFA7-13 HEK293T NDUFA7-3 HEK293T NDUFA7-3 NDUFA7-13 HEK293T NDUFA7-3 NDUFA7-13 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFB7-1 NDUFB7-21 HEK293T NDUFA3-1 NDUFA3-9 HEK293T NDUFA3-1 NDUFA3-9 NDUFA7-3 NDUFA7-13

SC SC SC SC CI CI CI CI CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

TALEN generated: NDUFA1 NDUFA2 NDUFA5 NDUFA6 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG NDUFA1-2 HEK293T NDUFA2-3 NDUFA2-5# NDUFA2-3 NDUFA2-5# HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA6-7 NDUFA6-9 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA1-3 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA1-2 NDUFA1-3 HEK293T NDUFA2-3 NDUFA2-5# HEK293T NDUFA2-3 NDUFA2-5# HEK293T NDUFA2-3 NDUFA2-5# HEK293T HEK293T NDUFA2-3 NDUFA2-5# HEK293T NDUFA2-3 NDUFA2-5# HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 HEK293T NDUFA5-3# NDUFA5-9 NDUFA6-7 NDUFA6-9 HEK293T HEK293T NDUFA2-3 NDUFA2-5# SC SC SC SC CI CI CI CI CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

NDUFA8 NDUFA10 NDUFA11 NDUFA13 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG

NDUFA13-3 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA11-2 NDUFA11-23 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-3 NDUFA13-2 HEK293T NDUFA13-2 NDUFA10-8 NDUFA10-10 NDUFA10-8 NDUFA10-10 NDUFA10-8 NDUFA10-10 NDUFA10-8 NDUFA10-10 NDUFA10-8 NDUFA10-10 NDUFA10-8 NDUFA10-10 HEK293T HEK293T HEK293T HEK293T HEK293T HEK293T NDUFA10-8 NDUFA10-10 HEK293T NDUFA10-8 NDUFA10-10 HEK293T HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 HEK293T NDUFA8-3 NDUFA8-22 SC SC SC SC CI CI CI CI CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

NDUFB1 NDUFB2 NDUFB3 NDUFB4 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB4-3 NDUFB4-7 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB2-1 NDUFB2-2 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB3-2 NDUFB3-8 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 HEK293T NDUFB1-1 NDUFB1-9 SC SC SC SC CI CI CI CI

CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

NDUFB5 NDUFB6 NDUFB8 NDUFB9 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB5-3 NDUFB5-6 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB6-6 NDUFB6-9 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB9-6 NDUFB9-8 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 HEK293T NDUFB8-10 NDUFB8-3 SC SC SC SC CI CI CI CI CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

NDUFB10 NDUFB11 NDUFC1 NDUFC2 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC2-4 NDUFC2-8 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFC1-2 NDUFC1-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB10-3 NDUFB10-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 HEK293T NDUFB11-4 NDUFB11-5 SC SC SC SC CI CI CI CI

CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

NDUFS4 NDUFS5 NDUFS6 NDUFV3 Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV Complex I Complex II Complex III Complex IV TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG TX100 DIG HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFS6-1 NDUFS6-11 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFV3-3 NDUFV3-4 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS5-10 NDUFS5-9 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 HEK293T NDUFS4-3# NDUFS4-8 SC SC SC SC CI CI CI CI CIII CIII CIII CIII CIV CIV CIV CIV CII CII CII CII

Stroud ED Fig. 2 a b c KO KO NDUFAB1*-2 FLAG HEK293T cells SDS-PAGE FLAG + DOX inducible days -DOX 024 NDUFAB1*FLAG or NDUFAB1* in NDUFAB1 (NDUFAB1*-2) yACP1 in NDUFAB1 (ACP1-3) IB: mtHSP70 HEK293T yACP1FLAG IB: FLAG (NDUFAB1) CI Transfect NDUFAB1 669 CRISPR/Cas9 0 days +DOX 440 * screen clones − DOX

KO BN-PAGE, for NDUFAB1 IB: NDUFA9 kDa 8 days 123

e BN-PAGE, IB: glucose NDUFA9 SDHA UQCRC1 COX4 + DOX FLAG FLAG FLAG FLAG galactose FLAG FLAG FLAG FLAG

d 2 NDUFAB1 HEK293T + yACP1 HEK293T + NDUFAB1* HEK293T + yACP1 HEK293T + NDUFAB1* HEK293T + yACP1 HEK293T + NDUFAB1* HEK293T + yACP1 HEK293T + NDUFAB1*

LYRM7 NDUFAF5 LIPT2 LYRM2 1 parental yACP1-1 yACP1-3 parental NDUFAB1*-2 NDUFAB1*-3 parental yACP1-1 yACP1-3 parental NDUFAB1*-2 NDUFAB1*-3 parental yACP1-1 yACP1-3 parental NDUFAB1*-2 NDUFAB1*-3 parental yACP1-1 yACP1-3 parental NDUFAB1*-2 NDUFAB1*-3 kDa ratio enrichment

CI 10 669 UQCRFS1

CIII Log 440 CI 0 CII CIV other n.s. 134 CII 024 -Log p-value 1 23456 78910 11 12 13 14 15 16 17 18 19 20 21 22 23 24 10 NDUFAB1*FLAG/HEK293T

Stroud ED Fig. 3 a b c BN-PAGE BN-PAGE, IB: NDUFV1 IB: NDUFA9 IB: NDUFAF2 KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO S]- NDUFV3 S]- NDUFA7 S]- NDUFA12 35 35 35 [ [ [ SC

HEK293T NDUFA12 NDUFA7 NDUFV3 NDUFS4 CI HEK293T NDUFV3 NDUFA7 NDUFA12 NDUFS6 NDUFS4 NDUFA2 HEK293T NDUFV3 NDUFA7 NDUFA12 NDUFS6 NDUFS4 NDUFA2 HEK293T CI+III kDa † KO SC CI NDUFA12 CI ‡ ‡ 669 kDa SC N* NDUFA7KO CI 440

669 BN-PAGE, SC N mod. in vitro import NDUFV3KO CI 132 440 SDS-PAGE 20 15 IB:SDHA CII input lysate kDa * 1 2 3 4 5 6 7 8 9 10 11 121314 1 2 3 4 5 1 2 3 d NDUFV3 1.5 1.5 NDUFV3 1.0 1.0 0.5 0.5 CI ratio enrichment ratio enrichment CIII 10 10 CIV other Log Log 0 0 n.s. NDUFAF2 NDUFAF2 NDUFAF4 NDUFAF4 TIMMDC1 TIMMDC1

0 2 4 0 2 4 -Log p-value -Log10 p-value 10 FLAG NDUFV3FLAG Triton X-100 NDUFV3 Digitonin

Stroud ED Fig. 4 a b 8 Log2 ratio KO KO KO KO KO KO KO KO KO KO vs HEK293T 4 -4 0 4 6 NDUFV3 NDUFB7 NDUFS6 NDUFA2 NDUFA8 NDUFA1 NDUFS5 NDUFC1 NDUFB10 NDUFB11 2 p-value

NDUFS4 10 NDUFA7

NDUFA12 -Log 4 NDUFS6 0

NDUFB11 p-value MT-ND4 10 -2 0 2 NDUFB10 KO Log2 CIV /HEK293T NDUFB4 -Log

NDUFA10 2 >2 fold >2 fold Complex I NDUFS5 P<0.05 P<0.05 Down >1.5 fold CIVKO MT-ND2 KO NDUFA9 Up >1.5 fold CIV NDUFC2 Mitocarta 2.0 NDUFC1 non-mito. NDUFB7 0 n.s. / <1.5-fold change NDUFB9 -4 -2 0 24 MT-ND5 NDUFB8 Log2 significant (P<0.05) protein ratios NDUFV2 in respiratory deficient KOs/HEK293T NDUFV1 NDUFA2 NDUFS1 c Down >2 fold, P<0.05 Up >2 fold, P<0.05 2 2 2 2 Log f.c. f.c. Log Mito. other Log Mito. Log f.c. other f.c. NDUFS4 -3.9 NDFIP1 -1.7 ALAS1 2.0 GNAO1 2.1 NDUFA7 -3.7 NDFIP2 -1.6 MAOA 1.8 RAB9B 1.9 NDUFA12 -3.3 FGFR3 -1.4 GLDC 1.5 INA 1.6 NDUFB4 -2.5 UNC5B -1.4 TRMT2B 1.4 FAM105A 1.6 NDUFB8 -2.4 RNF130 -1.4 PCK2 1.3 CDH2 1.5 NDUFS1 -2.4 SSNA1 -1.3 CISD1 1.3 ZNF462 1.4 NDUFA10 -2.3 TMEM132A -1.3 RAB32 1.3 EEF1A2 1.3 NDUFS6 -2.1 IFNGR1 -1.3 ACSS1 1.3 METTL7A 1.3 NDUFS5 -2.1 NEO1 -1.2 ABAT 1.3 TAPBP 1.3 NDUFV1 -1.9 NOTCH1 -1.2 CHCHD7 1.3 ARMCX3 1.2 MT-ND2 -1.8 UNC5C -1.2 CHCHD10 1.1 ANXA2 1.2 NDUFA2 -1.8 METAP2 -1.2 MFN1 1.1 HSPB1 1.2 NDUFC2 -1.7 UBAP2 -1.1 CHCHD2 1.1 CTNNB1 1.2 MT-ND5 -1.7 LRIG3 -1.1 CMC4 1.1 EMP3 1.2 NDUFV2 -1.6 ZDHHC20 -1.1 CAMK2G 1.2 NDUFC1 -1.6 SMARCAD1 -1.1 TMEM261* 1.1 NDUFA9 -1.6 SERINC1 -1.1 S100A10 1.1 NDUFB9 -1.5 RABL6 -1.1 MRE11A 1.1 NDUFB7 -1.5 BCL2L12 -1.0 CTNNA1 1.1 NME4 -1.4 SCD -1.0 TAP2 1.1 HMGCS1 -1.3 UGCG -1.0 SELM 1.1 NDUFV3 -1.3 CAMK2D 1.1 MGARP -1.3 GGH 1.0 FTH1 -1.2 TMBIM6 1.0 NDUFA6 -1.1 PXDN 1.0 DLD -1.1 ITPR3 1.0 AHCYL1 -1.1 BCORL1 1.0 d Oxidative phosphorylation Metabolism GO:0006260 GO:0030554 DNA Adenyl 2 2 2 2 replication nucleotide 2 binding Log f.c. Log f.c. Log f.c. Log f.c. Log f.c.

NDUFS4 -3.9 RRM1 -0.7 ALAS1 2.0 SET -0.9 NDUFA10 -2.3 NDUFA7 -3.7 GTF2F2 -0.7 MAOA 1.8 RRM2 -0.8 FGFR3 -1.4 NDUFA12 -3.3 CLNS1A -0.7 GLDC 1.5 MCM3 -0.8 NME4 -1.4 NDUFB4 -2.5 NDUFS7 -0.7 ABAT 1.3 MCM2 -0.8 DLD -1.1 NDUFB8 -2.4 PTPRS -0.7 SPR 0.9 MCM6 -0.8 SMARCAD1 -1.1 NDUFS1 -2.4 PAK4 -0.7 ALDH6A1 0.8 MCM5 -0.7 TYRO3 -0.9 NDUFA10 -2.3 EIF3A -0.7 ERO1LB 0.7 PRIM2 -0.7 CDC42BPB -0.9 NDUFS6 -2.1 SLC1A3 -0.6 GPT2 0.7 MCM4 -0.7 KIF4A -0.9 NDUFS5 -2.1 NDUFS2 -0.6 FDXR 0.7 RRM1 -0.7 RIOK2 -0.9 TTK -0.8 NDUFV1 -1.9 HRAS -0.6 HMGCL 0.7 MCM7 -0.7 ANKRD17 -0.8 NDUFA2 -1.8 CSNK1D -0.6 AIFM1 0.6 GINS3 -0.6 EPHA7 -0.8 NDUFC2 -1.7 LLGL1 -0.6 DHTKD1 0.6 GLUL -0.8 NDUFV2 -1.6 NDUFS3 -0.6 ACAD11 0.6 Translation HSPA1A -0.8

NDUFC1 -1.6 RPS15 -0.6 AASS 0.6 2 HSPA1B -0.8 NDUFA9 -1.6 NDUFA5 -0.6 PPOX 0.6 Log f.c. PFAS -0.8 NDUFB9 -1.5 CD2AP -0.6 ACADS 0.6 MCM3 -0.8 NDUFB7 -1.5 SHC1 -0.6 LEPREL2 0.6 MRPS36 -0.9 MCM2 -0.8 FGFR3 -1.4 CSNK1G2 -0.6 OAT 0.6 NACA -0.8 NDUFV3 -1.3 GTF2F1 -0.6 ASCC3 -0.8 HBS1L -0.8 MCM6 -0.8 FTH1 -1.2 PAK2 -0.6 EEF1A1 -0.8 STEAP3 -0.8 NDUFA6 -1.1 MDN1 -0.6 Transporters EEF1A1P5 -0.8 DDX20 -0.8 DLD -1.1 PTPRF -0.6 2 EIF1 -0.7 GRK6 -0.7 SMARCAD1 -1.1 ABCE1 -0.6 RPS21 -0.7

Log f.c. MSH3 -0.7 SCD -1.0 KDM3B -0.6 EIF4A1 -0.7 MCM5 -0.7 NDUFB3 -1.0 CSNK1G1 -0.6 TAPBP 1.3 EIF3G -0.7 MCM4 -0.7 GJA1 -0.9 TAP2 1.1 RPSA -0.7 EIF4A1 -0.7 TYRO3 -0.9 MAOA 1.8 TAP1 0.8 EIF3H -0.7 RRM1 -0.7 FAR1 -0.9 GLDC 1.5 ATP11C 0.6 EIF2B2 -0.7 EIF2B2 -0.7 NXN -0.9 PXDN 1.0 ATP6V1H 0.6 EIF3F -0.7 GTF2F2 -0.7 CDC42BPB -0.9 SDHAF2 1.0 ABCB8 0.6 EIF3K -0.7 MCM7 -0.7 NDUFA11 -0.9 SPR 0.9 ATP6V1G1 0.6 EIF3B -0.7 PAK4 -0.7 PTPN23 -0.9 H6PD 0.8 EEF2 -0.7 MAT2A -0.7 SET -0.9 ALDH6A1 0.8 EEF1D -0.7 HSP90AB2P -0.6 RIOK2 -0.9 PCYOX1L 0.7 GO:0005544 EIF3A -0.7 CSNK1D -0.6 Calcium dependent FASN -0.8 ERO1LB 0.7 EIF3L -0.7 phospholipid 2 HSP90AB1 -0.6 TTK -0.8 FDXR 0.7 binding EIF3C -0.7 CSNK1G2 -0.6 EPHA7 -0.8 AIFM1 0.6 Log f.c. EIF3CL -0.7 PAK2 -0.6 DAG1 -0.8 GO:0030554DHTKD1 Adenyl0.6 nucleotideGO:0005544 binding Calcium dependent phospholipid binding ANXA2 1.2 RPS15 -0.6 MDN1 -0.6 GOPC -0.8 ACAD11 0.6 ANXA2P2 1.2 RPS19 -0.6 ABCE1 -0.6 NDUFS8 -0.8 CPOX 0.6 ANXA4 0.8 RPS28 -0.6 PAPSS1 -0.6 RRM2 -0.8 AASS 0.6 CPNE1 0.7 EIF5A -0.6 CSNK1G1 -0.6 MCM2 -0.8 PPOX 0.6 EIF4A1 -0.7 EIF5AL1 -0.6 GEMIN4 -0.8 ACADS 0.6 EIF2B2 -0.7 RPS12 -0.6 STEAP3 -0.8 LEPREL2 0.6 EIF3M -0.6 DDX20 -0.8 GPD2 0.6 RPS17 -0.6 GRK6 -0.7 DHRS1 0.6 EIF3E -0.6 ALDOA -0.7 SCCPDH 0.6 RPS10 -0.6 PRIM2 -0.7 NSDHL 0.6 EIF3I -0.6

Stroud ED Fig. 5 a

NDUFV3KO NDUFA7KO NDUFA12KO NDUFS4KO NDUFS6KO NDUFA2KO NDUFA6KO

NDUFA5KO NDUFA3KO NDUFA8KO NDUFA13KO NDUFA1KO NDUFS5KO NDUFA10KO

NDUFC1KO NDUFC2KO NDUFA9KO NDUFB1KO NDUFB5KO NDUFB10KO NDUFB11KO

NDUFB4KO NDUFB7KO NDUFB3KO NDUFB2KO NDUFB9KO NDUFB8KO NDUFB6KO

b

NDUFA11KO NDUFAB1KO

Log2 ratio vs HEK293T -2 -1 0 12

n.d.

Stroud ED Fig. 6 Stroud EDFig.7

HEK293T

NDUFV3KO

NDUFS6KO

NDUFA2KO KO celllines NDUFA8KO

NDUFA1KO

NDUFS5KO

NDUFC1KO

NDUFB10KO

NDUFB11KO

NDUFB7KO 216 12 mean reads Log row 2 MRPL46 MRPS2 ATP5h ATP5B NDUFA4 COX4I1 UQCRFS1 UQCRC1 SDHA NDUFAB1 NDUFA11 NDUFB9 NDUFB8 NDUFB7 NDUFB3 NDUFB6 NDUFB2 NDUFB4 NDUFB11 NDUFB10 NDUFB5 NDUFB1 NDUFA9 NDUFS5 NDUFC2 NDUFC1 NDUFA10 NDUFA1 NDUFA13 NDUFA8 NDUFA3 NDUFS8 NDUFS7 NDUFS3 NDUFS2 NDUFA5 NDUFV3 NDUFV2 NDUFV1 NDUFS6 NDUFS4 NDUFS1 NDUFA12 NDUFA7 NDUFA6 NDUFA2 −2 Difference row mean (log from 0 2 ) 2

transcript b a Stroud EDFig.8

-Log p-value -Log10 p-value -Log10 p-value 10 SDS-PAGE 0 2 4 0 2 4 0 2 4 12 -4 -4 -4 Log

Log HEK293T Log KO

2 NDUFAF2 2 ratioNDUFAF2 2

ratioNDUFAF4 IB: SDHA IB: IB: NDUFAF2 ratioNDUFAF6 -2 -2 -2 0 0 0 KO KO KO /HEK293T /HEK293T /HEK293T 2 2 2 34

HEK293T SDS-PAGE KO 4 4 NDUFAF1 4 567 TIMMDC1KO -Log10 p-value -Log10 p-value KO 0 2 4 0 2 4 NDUFAF6

-4 -4 NDUFAF4KO IB: SDHA IB: IB: NDUFAF4 IB: TIMMDC1 IB: NDUFAF1 Log Log ND2 ND1/Q N mod. ND4 n.s. ND5 other mod. N/A 2 2 ratioNDUFAF1 ratioTIMMDC1 -2 -2 0 0 KO KO /HEK293T /HEK293T 2 2 4 4 c 2- 0 -1 -2 Log NDUFAF4 NDUFAF1 NDUFAF2 NDUFAF6 TIMMDC1 HEK293T 2 ratiovs 12 KO KO KO KO KO n.d. a BN-PAGE, IB: c NDUFA9 UQCRC1 COX2 4 KO3 KO3 KO7 KO3 KO7 KO7 d HEK293T ATP5SL ATP5SL HEK293T ATP5SL ATP5SL HEK293T ATP5SL ATP5SL

kDa CI+III CI 669 ATP5SLKO 2 CIII p-value

440 10 N mod. CIV ND1/Q -Log TX100 134 ND2 ND4 ND5 mod. N/A other SC CI 0 n.s. 669 CIII -2 0 2 440 CIV KO Digitonin Log2 ratio ATP5SL /HEK293T 134 4 DMAC1KO 123 456 789 b BN-PAGE, IB: NDUFB6 UQCRC1 COX4 KO1 KO1 KO3 KO1 KO3 KO3 2 p-value 10 -Log HEK293T DMAC1 DMAC1 HEK293T DMAC1 DMAC1 HEK293T DMAC1 DMAC1 Log2 ratio vs HEK293T CI+III kDa -2 -1 0 12 CI n.d. 669

CIII 0

TX100 440 CIV -2 0 2 KO 134 Log2 ratio DMAC1 /HEK293T

f g [35S]-met., pulse/chase, 2D-PAGE, autoradiography CISC FLAG DMAC1 HEK293T DMAC1KO 669 kDa 440669 134 440669 134 CIII 440 COX1 Digitonin 37 ND4 CIV ND4 ND4 134 ND2 ND2 Cyt b

IFA: FLAG ND2 25 ND1 123 456 789 ND1 ND1 20 COX2/3 15 ATP6 1h pulse e ND3 10 SDS-PAGE, IB: ATP8/ND4L

Na2CO3 Swelling − − + + − − ND5 − − − − + + ND5 COX1 TX-100 IFA: TOMM20 37

elle t ND4 − + − + − + ND4 PK Total P SN ND2 Cyt b TOMM20 ND2 ND1 ND2 25 ND1 ND1 MIC10 20 COX2/3 FLAG ATP6

DMAC1 24h chase 15 Merge NDUFAF1 ND3 ND3 NDUFS2 10 ND4L ATP8/ND4L 123456789 I ◊ V III IV ◊ V III # IV

Stroud ED Fig. 9

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: Stroud, DA; Surgenor, EE; Formosa, LE; Reljic, B; Frazier, AE; Dibley, MG; Osellame, LD; Stait, T; Beilharz, TH; Thorburn, DR; Salim, A; Ryan, MT

Title: Accessory subunits are integral for assembly and function of human mitochondrial complex I

Date: 2016-10-06

Citation: Stroud, D. A., Surgenor, E. E., Formosa, L. E., Reljic, B., Frazier, A. E., Dibley, M. G., Osellame, L. D., Stait, T., Beilharz, T. H., Thorburn, D. R., Salim, A. & Ryan, M. T. (2016). Accessory subunits are integral for assembly and function of human mitochondrial complex I. NATURE, 538 (7623), pp.123-+. https://doi.org/10.1038/nature19754.

Persistent Link: http://hdl.handle.net/11343/198409

File Description: Accepted version