TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I

Significance The oxidation of energy rich compounds generates a proton motive force, a chemical potential difference for protons, across the inner membranes of the mitochondria. The proton motive force drives the turning of the rotor in the membrane domain of the ATP synthase. This rotation provides the energy to synthesize the ATP required to sustain life. The assembly in the inner organellar membrane of human ATP synthase from 27 nuclear encoded proteins and 2 mitochondrially encoded subunits involves the formation of intermediate modules representing the F1-catalytic domain, the peripheral stalk, and the c8-ring in the membrane part of the rotor. The assembly of the c8-ring requires the participation of two membrane-associated proteins, TMEM70 and, as we demonstrate, TMEM242.

cells were obtained by screening clones from single cells with an antibody against TMEM70. As no antibody against TMEM242 was available for identifying HAP1-ΔTMEM242 cells by a similar approach, clonal cells with deletions in TMEM242 were identified by screening the targeted regions of the genes by amplifying them by PCR and by sequencing the amplified fragments. The three disrupted alleles of TMEM242 in HEK293 cells were characterized in similar way.
TMEM70. The 32 nucleotide deletion in exon I of TMEM70 in HAP1-∆TMEM70 cells resulted in the possibility of a transcript encoding a C-terminally truncated 75 amino acid protein in which the Cterminal 28 amino acids do not correspond to the sequence of TMEM70 (SI Appendix Fig. S4A and B). In HEK293-Δδ.∆TMEM70 cells a 78 nucleotide deletion in TMEM70 exon I removed the translational initiation codon (SI Appendix Fig. S10A), and there was no potential second initiation codon in exon I downstream of the deletion. Neither cell line expressed TMEM70 (SI Appendix Fig. S5).

TMEM242.
A 113 nucleotide deletion in HAP1-∆TMEM242 cells removed the majority of exon I of TMEM242, including the translation initiation codon, and 10 nucleotides from the 5' upstream sequence (SI Appendix Fig. S4C). There is no additional initiation codon in exon I, and no significant level of an TMEM242 transcript was observed (SI Appendix Fig. S5B). HAP1-∆TMEM70 cells provided the parent for the HAP1-∆TMEM70.∆TMEM242 clone, where a 120 nucleotide deletion in TMEM242 removed most of exon I, including the translation initiation codon and 14 nucleotides of the upstream 5' sequence (SI Appendix Fig. S4D), preventing any significant level of transcription (SI Appendix Fig. S5B). In the polyploid HEK293-Δδ.ΔTMEM242 cells, three different modifications of TMEM242 were identified (SI Appendix Fig. S10B-D). In two instances, each individual gRNA produced a different nucleotide deletion (SI Appendix Fig. S10B and C) where 2 or 30 nucleotides, respectively, preceding the translational initiation codon were deleted in the 5' upstream region before exon I plus the first base of exon I. Additional deletions of 1 or 7 nucleotides at the 3' end of exon I, each created a frame-shift and a premature translation termination codon, resulting in a potential transcript encoding a 42 or 40 amino acid polypeptide, with 13 and 12 mismatched amino acids, respectively, in the C-terminal region (SI Appendix Fig. S10B and C). In the third instance, a 120 nucleotide deletion removed 17 nucleotides from the upstream 5' sequence of TMEM242 and the majority of exon I, including the translational initiation codon (SI Appendix Fig. S10D). Transcripts from the HEK293-Δδ.ΔTMEM242 cells (SI Appendix Fig. S5B) were sequenced across the exon I-II boundary and five out of thirteen of them corresponded to one of the gene deletions (Fig. S10C). The remainder corresponded to a deletion across the exon I-II boundary. No sequence was obtained for the second transcript (Fig. S10B), presumably because it is rare. The HEK293-Δδ.∆TMEM70.∆TMEM242 clone, derived from HEK293-Δδ.∆TMEM70 cells, arose from a 115 nucleotide deletion in TMEM242 including 12 nucleotides from the upstream 5' sequence, plus the majority of exon I including the translational initiation codon (SI Appendix Fig. S10E). No significant level of transcription was observed (SI Appendix Fig. S5B).

Sources of Antibodies. See
Expression of Tagged Mitochondrial Proteins. Two expression plasmids were prepared with the coding sequences for subunit c-t encoded in ATP5MC3 and g-t in ATP5MG, cloned between the Hind III and Xho I sites of plasmid pcDNA5 TM /FLP/TO (Thermo Fisher Scientific). The coding sequences for TMEM70-t, TMEM242-t, or TMEM242-Nt, were introduced into the same plasmid between its BamH I and Xho I sites. The proteins were expressed in HEK293 Flp-In TM T-Rex TM , HEK293-∆δ Flp-In TM T-Rex TM cells, and HeLa Flp-In TM T-Rex TM cells. Expression of proteins was induced for 72-96 h with doxycycline (20 ng/mL). TMEM242-t and TMEM242-Nt were each expressed transiently in HeLa cells. After 48 h of growth, the cells were fixed with formaldehyde, permeabilized and examined by confocal microscopy.
Confocal Microscopy. TMEM242-t and TMEM242-Nt were detected in transiently transfected HeLa cells with a mouse anti-FLAG primary antibody (Table S3) and a goat anti-mouse secondary antibody labelled with the fluorescent dye, Alex-Fluro ® 488. TOM20 was detected with rabbit polyclonal anti-TOM20 primary antibody (Table S3) and the Alex-Fluro ® 647 fluorescently labelled goat anti-rabbit secondary antibody. Fluorescently labelled cells in the presence of ProLong Gold Antifade Mountant (Thermo Fisher Scientific) plus the nuclear stain, 4′,6-diamidino-2-phenylindole (Invitrogen) were imaged with a Zeiss LSM 880 confocal microscope equipped with LSM 880 software. Images were acquired with a Zeiss 63 x oil immersion objective, set at zoom 2.4. Images were edited in Affinity designer.
Mitochondrial Membrane Association of TMEM242. TMEM242-t and TMEM242-Nt were expressed stably in Flp-In TM T-REx TM HEK293 and Flp-In TM T-REx TM HeLa cells, respectively. Cells were disrupted by hypotonic lysis and homogenization, and fractionated by differential centrifugation. Purified mitochondria were resuspended at 4 o C in 0.1 M Na2CO3, pH 11.5, to a protein concentration of 0.1 mg/mL and kept at 4 o C for 30 min. The suspension was centrifuged (4ºC; 233,000xg; 1 h), and the supernatant was retained. The pellet consisting of mitochondrial membranes was rinsed at 4ºC with milli-Q water, resuspended in 0.1 M Na2CO3 at 4 o C, and then extracted with increasing concentrations of deoxycholate for 30 min. The residual extracted pellet was recentrifuged. The various pellets and supernatants were fractionated by SDS-PAGE and specific proteins were detected with antibodies (Table S3).
Membrane Topology of TMEM242. Purified mitochondria were resuspended in 1 x MSH buffer and the agitated suspension was digested at 25 o C for 30 min with various concentrations of trypsin. Digestion was terminated by the addition of a five-fold molar excess of bovine pancreatic trypsin inhibitor (Sigma) and cOmplete TM proteinase inhibitor cocktail (Roche). Mitochondria that had been solubilized with 1% (w/v) n-dodecyl-β-D-maltoside (DDM) were digested with trypsin in a similar way.
Protein Quantitation. Relative quantitation of proteins in SILAC samples (8) was performed as described before (13) by analysis of tryptic and chymotryptic peptide digests of proteins. Peptide mass data were analyzed with MaxQuant version 1.6.5.0 or 1.6.10.43, and the integrated Andromeda search engine (14,15). The procedures for processing and analysis of the data have been described previously (13). The identification of subunit c in the MS analyses of SILAC samples depends principally on the observation of two chymotryptic peptides, residues 1-8 and 37-47. To a lesser extent, larger partial products containing these peptides are observed also. Peptide 37-47 contains trimethylated Lys-43, and therefore the data interrogation scheme includes this modification (13). Subunit c has only two potential tryptic cleavage sites and none of the tryptic peptides is recovered consistently from the liquid chromatography (LC) procedure incorporated in the LC-MS analyses, although tryptic peptide 8-38 is observed sometimes.  with an N-terminal FLAG tag for 96 h with doxycycline, 20 ng/mL in (D), and 2 ng/mL in (E). The mitochondria were digested for 30 min at 25ºC with trypsin (at concentrations in µg/100 μg protein indicated above the panels); above the tracks, -and + indicate, respectively, samples that were trypsinized directly, and those that were first lysed with 1% (w/v) DDM and then trypsinized. Digestion was terminated by the addition of a 5-fold molar excess of bovine pancreatic trypsin inhibitor. The sizes of protein markers are shown on the right. Proteins were detected with antibodies against the following: Strep II or FLAG tags; amino acids 264-617 in the C-terminal region of ATAD3, located in the mitochondrial matrix (16); the mitochondrial outer membrane protein TOM20; the NDUFB10 subunit of complex I which is exposed in the IMS; the NDUFS1 subunit of complex I and the β-subunit of ATP synthase, both of which are located on the matrix side of the IMM; CS, citrate synthase found in the matrix. On the right, the letter f denotes protein fragments that are protected from trypsinization by the IMM.    Samples were prepared from a 1:1 mixture of HAP1-WT cells and HAP1-ΔTMEM70, HAP1-ΔTMEM242 or HAP1-ΔTMEM70.ΔTMEM242 cells that had been differentially SILAC-labelled. The experiments were performed twice with reciprocal SILAC labelling orientations. The protein ratio is derived from a minimum of two peptide ratios from each experiment, except for subunit c (in panel A, both experiments) and for ATP6 in experiment 1 in (C), where values are from a single peptide ratio. In (B), no ratio for subunit c was obtained. In (C) the TMEM70 experiment 1 ratio was re-calculated using the median peptide value, replacing the 'plateau' value from MaxQuant. The ratios for proteins obtained in both experiments are plotted as a single point on a scatter plot as the log base 2 value. In (B), the data point for DIS3-like exonuclease 2 (-2.49, 8.39) is outside the axes, in the upper left 'contaminant' quadrant. Protein ratios are given in SI Appendix, Datasets S4-S20.     In the upper part of panels (B-E), alignment of the sequences of the targeted gene region with the intact sequence of TMEM242. Carets indicate the PAM (protospacer adjacent motif) sequences for each guide RNA, and solid lines the target sequences for guide RNAs. In the wild type TMEM242, exon I and parts of the 5' upstream and downstream intron A (grey boxes) are shown. The arrow indicates the translational initiation codons in exon I, and deleted nucleotides are denoted by dashed lines. In the lower parts of panels (B and C), mutated protein sequences are displayed beneath the parental wild-type sequence. Asterisks, denotes amino acid changes; dashes, deleted amino acids. In panels (D and E), the deletion removed most of exon I including the translational initiator methionine codon, and part of the 5'-upstream sequences, and therefore presumably no protein was produced.   respectively, expressed in HEK293 cells, (C) c-t expressed in HEK293-Δδ cells. TMEM70-t, TMEM242t and subunit c-t were detected with an anti-Strep II antibody, and other proteins with the specific antibodies indicated on the left of the panels. The top panels in A-C were probed twice, once with a Strep II antibody and then with an antibody for TMEM70. Extracts of mitochondria (ex) with digitonin (digitonin:protein, 10:1, w/w) were applied under gravity to a Strep II column, and the flow-through (fl) collected. The column was washed five times with 1 bed vol of buffer (w1-w5). Bound proteins were eluted with seven further portions of 0.5 bed vols of elution buffer (e1-e7) containing 10 mM desthiobiotin; (D) BN-PAGE analysis of eluates e3 and e5; complexes were detected with specific antibodies against the proteins indicated beneath the panels, and subunit c-t, TMEM70-t and TMEM242-t with an anti-FLAG antibody. c1, complexes containing subunit c-t and TMEM70-t and/or TMEM242-t; c2, complexes of subunit c-t lacking TMEM70-t and TMEM242-t; o, oligomers of TMEM70; MCIA, mitochondrial complex I assembly complex. The positions of molecular mass markers are indicated on the right.   Omega v 1.2.4. The symbols *, :, and . denote identical, strongly conserved, and weakly conserved residues, respectively. The N-terminal methionine residue of TMEM242 is N-acetylated (Uniprot Q9NWH2). (B and C), secondary structure predictions for TMEM242 and mature TMEM70, respectively, produced with PSIPRED 4.0 (19) and JPred 4 (23), where dashes, H, and E denote random coil, αhelix, and β-strand, respectively, and grey boxes transmembrane α-helical regions predicted by a combination of analyses from HMMTOP (20), TMHMM (21) and MEMSAT-SVM in the PSIPRED server (22). Fig. S17. Topologies and secondary structures of TMEM242 and TMEM70. The brown horizontal lines denote the boundaries of the IMM. Red rods and blue arrows represent α-helices and β-strands, respectively. Random coils at the N-and C-termini and between α-helices and β-strands are depicted as black lines. The predicted secondary structures are taken from Fig. S16. The topology of TMEM70 has been described previously (24).

Table S1. Target sites for gRNAs for the disruption of TMEM70 and TMEM242
gRNA Sequence

Dataset S1 (separate file). Proteins identified in SILAC experiments comparing affinity purified subunit c-t and subunit g-t expressed in HEK293-Δδ cells. This information is the output from
Perseus after processing of MaxQuant SILAC peptide pair data. Samples were affinity purified from mitoplasts prepared from a 1:1 mixture of Flp-In™ T-REx™ HEK293-Δδ cells overexpressing either subunit c-t or subunit g-t, and fractionated by SDS-PAGE. Gel sections were digested with trypsin or chymotrypsin. Experiment 1 refers to heavy isotope labelled subunit c-t cells mixed with light labelled subunit g-t cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database, experiment 2 ratios were inverted, and the protein ratios rendered as base two logarithmic values. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio is derived from a minimum of two peptide ratios from each experiment. Ratios for subunits c-t and g-t, and endogenous subunit c and subunit g were calculated manually with data from different migration positions on the gel (see Datasets S2 and S3).

Dataset S2 (separate file). Peptide data for ATP synthase subunit c obtained in SILAC experiments comparing affinity purified subunit c-t and subunit g-t expressed in HEK293-Δδ cells.
This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. Samples were affinity purified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of Flp-In™ T-REx™ HEK293-Δδ cells overexpressing either subunit c-t or subunit g-t.
Peptide data are derived from chymotrypsin digests. Experiment 1 refers to heavy isotope labelled subunit c-t cells mixed with light labelled subunit g-t cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. Gel section 10 corresponds to peptides from subunit c-t, and gel section 12 to peptides from endogenous subunit c. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios were included also so as to determine the protein ratio, represented by the median peptide ratio.

Dataset S3 (separate file). Peptide data for ATP synthase subunit g obtained in SILAC experiments comparing affinity purified subunit c-t and subunit g-t expressed in HEK293-Δδ cells.
This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. Samples were affinity purified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of Flp-In™ T-REx™ HEK293-Δδ cells overexpressing either subunit c-t or subunit g-t.
Peptide data were derived from both trypsin and chymotrypsin digests. Experiment 1 refers to heavy isotope labelled subunit c-t cells mixed with light labelled subunit g-t cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. Gel sections 9 and 10 correspond to peptides from subunit g-t, and gel section 11 to the peptides from the endogenous subunit g. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios were included also so as to determine the protein ratio, represented by the median peptide ratio.

Dataset S4 (separate file). Proteins identified in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and and HAP1-ΔTMEM70 cells. This information is the output from
Perseus after processing of MaxQuant SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70 cells, and digested in-solution with either trypsin or chymotrypsin. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70 cells mixed with light labelled WT cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database. Experiment 2 ratios were inverted, and the protein ratios rendered as base two logarithmic values. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio is derived from a minimum of two peptide ratios from each experiment, except for the ATP synthase subunit c protein ratio for both experiments, which was obtained from a single peptide value. ATP5IF1 ratios were calculated manually with data for unique peptides from the N-terminus of one mature form (IF1-M1) of the protein (see Dataset S5).

Dataset S5 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and HAP1-ΔTMEM70 cells.
This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1 WT and HAP1-ΔTMEM70 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70 cells mixed with light labelled WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information is included. When fewer than three MULTI-MSMS ratios were obtained ISO-MSMS ratios were included also so as to determine the protein ratio, represented by the median peptide ratio.

Dataset S6 (separate file). Proteins identified in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM70 cells. This information is the output from Perseus after processing
MaxQuant SILAC peptide pair data. Digitonin solubilized mitoplasts were prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70 cells, fractionated by SDS-PAGE and gel sections were digested with trypsin. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70 cells mixed with light labelled WT cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database, experiment 2 ratios were inverted, and the protein ratios rendered base two logarithmic. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio was derived from a minimum of two peptide ratios from each experiment, except for the TMEM242 protein ratio in experiment 2, which was obtained from a single peptide value. ATP5IF1 ratios were calculated manually with data from unique peptides at the N-terminus of one mature form, IF1-M1 (see Dataset S7). Subunit 11 of the cytochrome bc1 complex (UQCRFS1 mitochondrial targeting sequence) and Rieske subunit ratios were determined individually by manual calculation of the peptide data for each component (see Dataset S8), and FMC1 specific peptide data were employed in the manual recalculation of the protein ratio (see Dataset S9).

Dataset S7 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM70 cells.
This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts prepared with a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70 cells mixed with light labelled WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included.

Dataset S8 (separate file). Peptide data for the Rieske subunit and subunit 11 of cytochrome bc1 obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM70 cells.
This information is obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts prepared with a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are included also so as to determine the protein ratio, represented by the median peptide ratio.

Dataset S9 (separate file). Peptide data for protein FMC1 homolog obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM70 cells.
This information is obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts prepared with a 1:1 mixture of HAP1-WTand HAP1-ΔTMEM70 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included.

Dataset S10 (separate file). Proteins identified in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and HAP1-ΔTMEM242 cells. This information is the output from
Perseus after processing of MaxQuant SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM242 cells, and digested in-solution with either trypsin or chymotrypsin. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database. Experiment 2 ratios were inverted, and the protein ratios rendered as base two logarithmic values. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio was derived from a minimum of two peptide ratios from each experiment, except for MT-ATP6 (experiment 1) where the protein ratio was from a single peptide value. ATP5IF1 ratios were calculated manually with data for unique peptides from the N-terminus of one mature form (IF1-M1) of the protein (see Dataset S11). The TMEM70 experiment 1 ratio was re-calculated with the median peptide value, replacing the 'plateau' value from MaxQuant (see Datasheet S12).
Dataset S11 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and HAP1-ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1-WTand HAP1-ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM242 cells mixed with light labelled wild-type cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included.
Dataset S12 (separate file). Peptide data for TMEM70 obtained in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and HAP1-ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are included also so as to determine the protein ratio, represented by the median peptide ratio.

Dataset S13 (separate file). Proteins identified in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM242 cells.
This information is the output from Perseus after processing of MaxQuant SILAC peptide pair data. Digitonin solubilized mitoplasts were prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM242 cells, fractionated by SDS-PAGE and gel sections were digested with trypsin. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database, experiment 2 ratios were inverted, and the protein ratios rendered as base two logarithmic values. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio was derived from a minimum of two peptide ratios from each experiment. ATP5IF1 ratios were calculated manually with data for unique peptides from the Ntermini of three mature forms (IF1-M1, -M2 and M3) of the protein (see Dataset S14). The ratios for subunit 11 (UQCRFS1 mitochondrial targeting sequence) and the Rieske subunit of the cytochrome bc1 complex were determined individually by manually calculating the peptide data for each component (see Dataset S15).
Dataset S14 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts prepared with a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM242 cells mixed with light labelled wild-type cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are also included to determine the protein ratio, represented by the median peptide ratio.
Dataset S15 (separate file). Peptide data for Rieske subunit and subunit 11 of cytochrome bc1 obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts prepared with a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are also included to determine the protein ratio, represented by the median peptide ratio.
Dataset S16 (separate file). Proteins identified in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. This information is the output from Perseus after processing of MaxQuant SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells, and digested in-solution with either trypsin or chymotrypsin. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70.ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database, experiment 2 ratios were inverted, and the protein ratios rendered base two logarithmic. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio is derived from a minimum of two peptide ratios from each experiment. ATP5IF1 ratios were calculated manually with data for unique peptides from the N-termini of one mature form (IF1-M1) of the protein (see Dataset S17).
Dataset S17 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing immunopurified ATP synthase from HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. ATP synthase was immunopurified from digitonin solubilized mitoplasts prepared from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70.ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are included also so as to determine the protein ratio, represented by the median peptide ratio. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database, experiment 2 ratios were inverted, and the protein ratios rendered as base two logarithmic values. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio was derived from a minimum of two peptide ratios from each experiment. ATP5IF1 ratios were calculated manually with data for unique peptides from the N-termini of two mature forms (IF1-M1 and M3) of the protein (see Dataset S19). Ratios for subunit 11 (UQCRFS1 mitochondrial targeting sequence) and the Rieske subunit of cytochrome bc1 were determined individually by manually calculating the peptide data for each component (see Dataset S20).
Dataset S19 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70.ΔTMEM242 cells mixed with light labelled HAP1-WT, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained ISO-MSMS, ratios are included also to determine the protein ratio, represented by the median peptide ratio.
Dataset S20 (separate file). Peptide data for the Rieske subunit and subunit 11 of cytochrome bc1 obtained in SILAC experiments comparing mitoplasts from HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. The peptide data are from digitonin protein extracts of mitoplasts from a 1:1 mixture of HAP1-WT and HAP1-ΔTMEM70.ΔTMEM242 cells. Experiment 1 refers to heavy isotope labelled HAP1-ΔTMEM70.ΔTMEM242 cells mixed with light labelled HAP1-WT cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are included also so as to determine the protein ratio, represented by the median peptide ratio.
Dataset S21 (separate file). Proteins identified in SILAC experiments comparing mitoplasts from HEK293 parental, HEK293-Δδ and HEK293-Δδ.ΔTMEM70 cells. Relative protein abundance was determined by quantitative mass spectrometry and a 3-way SILAC labelling procedure. Heavy labelled (H; Arg-10, Lys-8), medium labelled (M; Arg-6, Lys-4) and light (L; Arg-0, Lys-0) cells were mixed Dataset S23 (separate file). Proteins identified in SILAC experiments comparing affinity purified TMEM242-t and METTL12-t expressed in HEK293 cells. This information is the output from Perseus after processing of MaxQuant SILAC peptide pair data. Samples were affinity purified from mitoplasts from a 1:1 mixture of Flp-In™ T-REx™ HEK293 cells overexpressing either TMEM242-t or METTL12t, fractionated by SDS-PAGE and gel sections were digested with trypsin or chymotrypsin. Experiment 1 refers to heavy isotope labelled TMEM242-t cells mixed with light labelled METTL12-t cells, and experiment 2 is vice versa. Perseus processing removed proteins identified in both a decoy database (created in MaxQuant by reversing protein entries) and a contaminant database. Experiment 2 ratios were inverted, and the protein ratios were rendered as base two logarithmic values. Only protein groups (listed under "Protein names") with ratios determined in both experiments are included. The protein ratio was derived from a minimum of two peptide ratios from each experiment. ATP5IF1 ratios were calculated manually with data for unique peptides from the N-termini of one mature form (IF1-M1) of the protein (see Dataset S24).
Dataset S24 (separate file). Peptide data for IF1 obtained in SILAC experiments comparing affinity purified TMEM242-t and METTL12-t expressed in HEK293 cells. This information was obtained from the MaxQuant evidence file after the processing of SILAC peptide pair data. Samples were affinity purified from digitonin solubilized mitoplasts from a 1:1 mixture of Flp-In™ T-REx™ HEK293 cells overexpressing either TMEM242-t or METTL12-t. Peptide data were derived from both trypsin and chymotrypsin digests. Experiment 1 refers to heavy isotope labelled TMEM242-t cells mixed with light labelled METTL12-t cells, and experiment 2 is vice versa. Only peptide data that provided ratio information are included. When fewer than three MULTI-MSMS ratios were obtained, ISO-MSMS ratios are included also so as to determine the protein ratio, represented by the median peptide ratio.
Dataset S25 (separate file). Proteins identified in SILAC experiments comparing affinity purified TMEM70-t and METTL12-t expressed in HEK293 cells. This information is the output from Perseus after processing of MaxQuant SILAC peptide pair data. Samples were affinity purified from mitoplasts from a 1:1 mixture of Flp-In™ T-REx™ HEK293 cells overexpressing either TMEM70-t or METTL12-t,