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**Figure legends**

**Figure 1. Subcellular localization of TMEM70.** A, scheme representing the domain organization of TMEM70 with amino-acid residues numbering above. MTS: Mitochondrial Targeting Sequence. TM: Transmembrane segment. Positions of immunogenic peptides are indicated (P1, P2). B, 143B-rho+ cells were fractionated by differential centrifugation as detailed in the materials and methods section. Fractions enriched in nuclei (Nuc), mitochondria (Mito), microsomes (Micro), and cytosol (Cyto) were analyzed by SDS-PAGE and western blot using antibodies against indicated proteins. Protein markers of different cellular compartments/machineries were probed: Fibrillarin (nucleus), Su.Beta (mitochondria), ERGIC p58 (ER-Golgi), Actin (cytoskeleton). Endogenous TMEM70 was detected almost exclusively in the crude mitochondrial fraction as for Su.Beta. C, isolated mitochondria from 143B-rho+ cells were subjected to carbonate (Na2CO3 pH11.5) or salt (KCl 500 mM) extraction. Proteins from supernatants (Sup) and pellets were separated by SDS-PAGE and analyzed by western blot. Endogenous TMEM70 behaved like an integral membrane protein similarly to the outer membrane protein VDAC, by contrast with cytochrome c peripherally associated with the inner membrane and as expected released in the soluble fraction. D, isolated mitochondria from 143B-rho+ cells were subjected to a protease protection assay. Mitochondria were resuspended in hypotonic (Swelling +) or isosmotic buffer (Swelling -) in the presence (+PK) or absence (-PK) of Proteinase K. Protein pellets were separated by SDS-PAGE and analyzed by western blot. TMEM70, like cytochrome c, became accessible to proteinase k after rupturing the outer membrane by osmotic swelling while the matrix-localized ATP synthase subunit Beta remained intact, showing that the integrity of the inner membrane was preserved. E, topological model of TMEM70 within mitochondria. (IM) inner membrane; (IMS) intermembrane space. N- and C-termini of TMEM70 are indicated.

**Figure 2. Submitochondrial localization of TMEM70 by super resolution and expansion microscopy.** A, 143B-rho+ cells expressing TMEM70 cDNA were fixed and immunostained with antibodies against Mitofilin/Mic60 (upper panel), or TMEM70 (lower panel). Confocal (left) and STED super-resolution (right) images are shown with insets magnified on the right panels. B, 143B-rho+ cells expressing TMEM70-FLAG were fixed, immunostained with antibodies against Mitofilin/Mic60 (red) and FLAG (green), counter-stained with DAPI (blue), and subjected to expansion microscopy. A representative image is shown (left panel) with an inset magnified on the right panel in separate channels (upper right) and overlay (lower right). C, cultured human skin fibroblasts were fixed, immunostained with antibodies against TMEM70 (green), and subjected to expansion microscopy. A representative image is shown on the left and the inset magnified on the top right corner. D, scheme representing the relative localization of Mic60 (in red) and TMEM70 (in green) in the inner membrane sub-compartments. OM: outer membrane; IBM: inner boundary membrane; ICM: inner cristae membrane. Scale bars: 5 µm full size images, 500 nm zoomed images (C, 1µm). Representative results from different fields and independent experiments (n ≥ 3) are shown.

**Figure 3. Inducible knockdown of TMEM70.** HEK293T cells were transduced with constructs allowing the doxycycline-inducible expression of a mix of two shRNAs targeted to TMEM70 mRNA. A-C, cells were cultured in the presence of doxycycline for indicated time periods or left without the drug during equal time and harvested. Mitochondria were isolated from the
**Figure legends**

**Figure 1. Subcellular localization of TMEM70.** A, scheme representing the domain organization of TMEM70 with amino-acid residues numbering above. MTS: Mitochondrial Targeting Sequence. TM: Transmembrane segment. Positions of immunogenic peptides are indicated (P1, P2). B, 143B-rho+ cells were fractionated by differential centrifugation as detailed in the materials and methods section. Fractions enriched in nuclei (Nuc), mitochondria (Mito), microsomes (Micro), and cytosol (Cyto) were analyzed by SDS-PAGE and western blot using antibodies against indicated proteins. Protein markers of different cellular compartments/machineries were probed: Fibrillarin (nucleus), Su.Beta (mitochondria), ERGIC p58 (ER-Golgi), Actin (cytoskeleton). Endogenous TMEM70 was detected almost exclusively in the crude mitochondrial fraction as for Su.Beta. C, isolated mitochondria from 143B-rho+ cells were subjected to carbonate (Na2CO3 pH11.5) or salt (KCl 500 mM) extraction. Proteins from supernatants (Sup) and pellets were separated by SDS-PAGE and analyzed by western blot. Endogenous TMEM70 behaved like an integral membrane protein similarly to the outer membrane protein VDAC, by contrast with cytochrome c peripherally associated with the inner membrane and as expected released in the soluble fraction. D, isolated mitochondria from 143B-rho+ cells were subjected to a protease protection assay. Mitochondria were resuspended in hypotonic (Swelling +) or isosmotic buffer (Swelling -) in the presence (+PK) or absence (-PK) of Proteinase K. Protein pellets were separated by SDS-PAGE and analyzed by western blot. TMEM70, like cytochrome c, became accessible to proteinase k after rupturing the outer membrane by osmotic swelling while the matrix-localized ATP synthase subunit Beta remained intact, showing that the integrity of the inner membrane was preserved. E, topological model of TMEM70 within mitochondria. (IM) inner membrane; (IMS) intermembrane space. N- and C-termini of TMEM70 are indicated.

**Figure 2. Submitochondrial localization of TMEM70 by super resolution and expansion microscopy.** A, 143B-rho+ cells expressing TMEM70 cDNA were fixed and immunostained with antibodies against Mitofilin/Mic60 (upper panel), or TMEM70 (lower panel). Confocal (left) and STED super-resolution (right) images are shown with insets magnified on the right panels. B, 143B-rho+ cells expressing TMEM70-FLAG were fixed, immunostained with antibodies against Mitofilin/Mic60 (red) and FLAG (green), counter-stained with DAPI (blue), and subjected to expansion microscopy. A representative image is shown (left panel) with an inset magnified on the right panel in separate channels (upper right) and overlay (lower right). C, cultured human skin fibroblasts were fixed, immunostained with antibodies against TMEM70 (green), and subjected to expansion microscopy. A representative image is shown on the left and the inset magnified on the top right corner. D, scheme representing the relative localization of Mic60 (in red) and TMEM70 (in green) in the inner membrane sub-compartments. OM: outer membrane; IBM: inner boundary membrane; ICM: inner cristae membrane. Scale bars: 5 µm full size images, 500 nm zoomed images (C, 1µm). Representative results from different fields and independent experiments (n ≥ 3) are shown.

**Figure 3. Inducible knockdown of TMEM70.** HEK293T cells were transduced with constructs allowing the doxycycline-inducible expression of a mix of two shRNAs targeted to TMEM70 mRNA. A-C, cells were cultured in the presence of doxycycline for indicated time periods or left without the drug during equal time and harvested. Mitochondria were isolated from the
cell pellets and analyzed as follows. A, mitochondria were lysed, and proteins were separated by SDS-PAGE and analyzed by western blot. B, quantitation of signal intensities from A. Signals were normalized to VDAC1 used as a loading control. For each protein tested, an average of normalized signals in the presence of doxycycline at the different time points is shown as a percentage of the average signal obtained in the absence of doxycycline; +/- S.D. (error bars). C, digitonin extracts from the isolated mitochondria were subjected to BN-PAGE and ATPase activity was revealed in-gel (white precipitate). The position of ATP synthase monomers (M) and dimers (D) are indicated to the right. D, scheme of the human ATP synthase structure. The general structure of the complex is represented on the scheme with the different types of subunits colored and named. E, cells were cultured in the presence of doxycycline for the indicated time periods and harvested. Total protein extracts were separated by SDS-PAGE and analyzed by western blot. Signal densities were quantified and expressed as percentage of signal obtained in the absence of doxycycline. Results are mean +/- S.E.M (error bars). (**, p < 0.01; ***, p < 0.001; n = 4-6; one-way ANOVA).

**Figure 4. Characterization of TMEM70-knockout cells generated from HEK293T. A,** isolated mitochondria from different TMEM70-KO clones and HEK293T (WT) as a control were lysed and proteins were separated by SDS-PAGE and analyzed by western blot. The faint signal indicated by * just below TMEM70 band is an unspecific cross-reaction of anti-TMEM70 serum. B, TMEM70-KO (T3AA2; T2BB9) and HEK293T (WT) permeabilized cells were subjected to oxygen consumption measurements after addition of ADP, Oligomycin, and CCCP. The results shown, expressed in nmolO2/min/106cells, are mean +/- S.D. of 6 to 9 measurements from 3 to 4 biological replicates, respectively. C, the ratio of ADP-stimulated rate over Oligomycin-inhibited rate was calculated for each measurement and the average ratios +/- S.D. (error bars) for each cell line are represented in the bar graph with corresponding numerical values indicated within each bar (***, p < 0.001; n = 6-9; student's t-test). D, digitonin extracts from isolated mitochondria of HEK293T (WT), T3AA2 TMEM70 knock-out (KO), and the same KO cell line expressing exogenous TMEM70 (KO+TMEM70) were subjected to BN-PAGE and ATPase activity was revealed in-gel. ATP synthase monomers (M) and dimers (D) are detected in these conditions. E, isolated mitochondria from HEK293T (WT) and TMEM70 knock-out (KO) cell lines were lysed and proteins were separated by SDS-PAGE and analyzed by western blot. F, total protein extracts from KO cell lines transduced with the indicated constructs were separated by SDS-PAGE and analyzed by western blot. G, quantitation of relative signal intensities of several ATP synthase subunits from E, F, and additional samples for each condition. The results are expressed as the fold change in levels of each subunit when TMEM70 is expressed as compared to KO. Data are mean +/- S.D. (error bars). (***, p < 0.001; n = 6-16; student's t-test). H, TMEM70 KO cells expressing TMEM70 cDNA (KO + TMEM70) or not (KO) were fixed, sliced and imaged by transmission electron microscopy. Representative images are shown together with magnified insets.

**Figure 5. Characterization of patient skin fibroblasts bearing the c.497_498del mutation in TMEM70.** A, scheme representing the truncated version of TMEM70 resulting from the c.497_498del mutation leading to premature stop codon p.Tyr166Cysfs*7. MTS: Mitochondrial Targeting Sequence. TM: Transmembrane segment. B, isolated mitochondria from cultured fibroblasts of the patient (Pat.) and a control individual (Ctrl) were lysed and
cell pellets and analyzed as follows. A, mitochondria were lysed, and proteins were separated by SDS-PAGE and analyzed by western blot. B, quantitation of signal intensities from A. Signals were normalized to VDAC1 used as a loading control. For each protein tested, an average of normalized signals in the presence of doxycycline at the different time points is shown as a percentage of the average signal obtained in the absence of doxycycline; +/- S.D. (error bars). C, digitonin extracts from the isolated mitochondria were subjected to BN-PAGE and ATPase activity was revealed in-gel (white precipitate). The position of ATP synthase monomers (M) and dimers (D) are indicated to the right. D, scheme of the human ATP synthase structure. The general structure of the complex is represented on the scheme with the different types of subunits colored and named. E, cells were cultured in the presence of doxycycline for the indicated time periods and harvested. Total protein extracts were separated by SDS-PAGE and analyzed by western blot. Signal densities were quantified and expressed as percentage of signal obtained in the absence of doxycycline. Results are mean +/- S.E.M (error bars). (**, p < 0.01; ***, p < 0.001; n = 4-6; one-way ANOVA).

**Figure 4. Characterization of TMEM70-knockout cells generated from HEK293T. A,** isolated mitochondria from different TMEM70-KO clones and HEK293T (WT) as a control were lysed and proteins were separated by SDS-PAGE and analyzed by western blot. The faint signal indicated by * just below TMEM70 band is an unspecific cross-reaction of anti-TMEM70 serum. B, TMEM70-KO (T3AA2; T2BB9) and HEK293T (WT) permeabilized cells were subjected to oxygen consumption measurements after addition of ADP, Oligomycin, and CCCP. The results shown, expressed in nmolO2/min/106cells, are mean +/- S.D. of 6 to 9 measurements from 3 to 4 biological replicates, respectively. C, the ratio of ADP-stimulated rate over Oligomycin-inhibited rate was calculated for each measurement and the average ratios +/- S.D. (error bars) for each cell line are represented in the bar graph with corresponding numerical values indicated within each bar (***, p < 0.001; n = 6-9; student's t-test). D, digitonin extracts from isolated mitochondria of HEK293T (WT), T3AA2 TMEM70 knock-out (KO), and the same KO cell line expressing exogenous TMEM70 (KO+TMEM70) were subjected to BN-PAGE and ATPase activity was revealed in-gel. ATP synthase monomers (M) and dimers (D) are detected in these conditions. E, isolated mitochondria from HEK293T (WT) and TMEM70 knock-out (KO) cell lines were lysed and proteins were separated by SDS-PAGE and analyzed by western blot. F, total protein extracts from KO cell lines transduced with the indicated constructs were separated by SDS-PAGE and analyzed by western blot. G, quantitation of relative signal intensities of several ATP synthase subunits from E, F, and additional samples for each condition. The results are expressed as the fold change in levels of each subunit when TMEM70 is expressed as compared to KO. Data are mean +/- S.D. (error bars). (***, p < 0.001; n = 6-16; student's t-test). H, TMEM70 KO cells expressing TMEM70 cDNA (KO + TMEM70) or not (KO) were fixed, sliced and imaged by transmission electron microscopy. Representative images are shown together with magnified insets.

**Figure 5. Characterization of patient skin fibroblasts bearing the c.497_498del mutation in TMEM70.** A, scheme representing the truncated version of TMEM70 resulting from the c.497_498del mutation leading to premature stop codon p.Tyr166Cysfs*7. MTS: Mitochondrial Targeting Sequence. TM: Transmembrane segment. B, isolated mitochondria from cultured fibroblasts of the patient (Pat.) and a control individual (Ctrl) were lysed and
proteins were separated by SDS-PAGE and analyzed by western blot. C, total protein extracts from control fibroblasts (Ctrl) and patient fibroblasts transduced with the empty vector (Pat.) or expressing exogenous TMEM70 (Pat.+TMEM70) were separated by SDS-PAGE and analyzed by western blot. Contaminant signal surrounded by * represents residual signal from Su.d in the subsequent decoration of TMEM70. D, patient fibroblasts expressing TMEM70 (Pat.+TMEM70) or GFP as a control (Pat.) were fixed, sliced and imaged by TEM. Representative images are shown together with magnified insets.

**Figure 6. TMEM70 targets specifically the subunit c of the ATP synthase.** A, isolated mitochondria from 143B cell line containing (Rho+) or not (Rho°) mitochondrial DNA were lysed and proteins were separated by SDS-PAGE and analyzed by western blot. B, total protein extracts from 143B cell line containing (Rho+) or not (Rho°) mitochondrial DNA expressing the indicated constructs were separated by SDS-PAGE and analyzed by western blot. C, HEK293T cells were transduced with shRNA constructs to knock-down TMEM70 (sh-TMEM70) and/or ATP5B (sh-β). Non-transduced HEK293T were used as control. Total protein extracts were prepared, separated by SDS-PAGE and analyzed by western blot.

**Figure 7. TMEM70 is required for proper accumulation of subunit c after transient block of mitochondrial protein import with CCCP.** TMEM70 knock-out (KO), and the same KO cell line expressing exogenous TMEM70 (KO+TMEM70) were transiently transfected with a C-terminally FLAG-tagged Su.c (Su.c-FLAG), or Su.d (Su.d-FLAG) as a control. 8 hours after transfection, the cells were treated with 10µM CCCP during 14 hours to block mitochondrial protein import before the construct is significantly expressed. Cells were then washed and incubated further without CCCP for the indicated time to trigger mitochondrial import of Su.c-FLAG or Su.d-FLAG. Cells were then harvested in PBS containing protease inhibitors. Total protein extracts were separated by SDS-PAGE and analyzed by western blot using anti-FLAG antibodies. A, 60 µg of total protein extracts from cells expressing Su.c-FLAG were analyzed. The signal corresponding to the tagged protein is indicated (Su.c-FLAG) together with a degradation product (deg.) accumulated during CCCP treatment (see Fig. S4B). Representative results are shown (top panel). Signal densities were quantified and expressed as percentage of signal obtained in KO+TMEM70 cells at latest time point for each replicate (lower panel). Data are mean +/- S.D. (error bars) (n = 3-5). See other replicates on Fig. S5A. B, 40 µg of total protein extracts from cells expressing Su.d-FLAG were analyzed. The signal corresponding to the tagged protein is indicated (Su.d-FLAG) together with a degradation product (deg.) accumulated during CCCP treatment (see Fig. S5B). Representative results are shown (top panel). Signal densities were quantified and expressed as percentage of signal obtained in KO+TMEM70 cells at latest time point for each replicate (lower panel). Data are mean +/- S.D. (error bars) (n = 3). See other replicates on Fig. S5B.

**Figure 8. Radioactive pulse-chase labeling reveals a very rapid turnover of subunit c in the absence of TMEM70.** TMEM70 knock-out (KO), and the same KO cell line expressing exogenous TMEM70 (KO+TMEM70) were transiently transfected with a C-terminally FLAG-tagged Su.c construct (Su.c-FLAG) or left untransfected (NT). 24 hours later, the cells were pulse-labeled for 20 min with 35S-Met/Cys as detailed under the Material and Methods section. Cells were then washed and incubated further with cold Met/Cys for the indicated chase time. Cells were then harvested in PBS containing protease inhibitors, lysed in 0.5% Triton X-100 buffer and subjected to immunoprecipitation using anti-FLAG magnetic beads.
proteins were separated by SDS-PAGE and analyzed by western blot. C, total protein extracts from control fibroblasts (Ctrl) and patient fibroblasts transduced with the empty vector (Pat.) or expressing exogenous TMEM70 (Pat.+TMEM70) were separated by SDS-PAGE and analyzed by western blot. Contaminant signal surrounded by * represents residual signal from Su.d in the subsequent decoration of TMEM70. D, patient fibroblasts expressing TMEM70 (Pat.+TMEM70) or GFP as a control (Pat.) were fixed, sliced and imaged by TEM. Representative images are shown together with magnified insets.

**Figure 6. TMEM70 targets specifically the subunit c of the ATP synthase.** A, isolated mitochondria from 143B cell line containing (Rho+) or not (Rho°) mitochondrial DNA were lysed and proteins were separated by SDS-PAGE and analyzed by western blot. B, total protein extracts from 143B cell line containing (Rho+) or not (Rho°) mitochondrial DNA expressing the indicated constructs were separated by SDS-PAGE and analyzed by western blot. C, HEK293T cells were transduced with shRNA constructs to knock-down TMEM70 (sh-TMEM70) and/or ATP5B (sh-β). Non-transduced HEK293T were used as control. Total protein extracts were prepared, separated by SDS-PAGE and analyzed by western blot.

**Figure 7. TMEM70 is required for proper accumulation of subunit c after transient block of mitochondrial protein import with CCCP.** TMEM70 knock-out (KO), and the same KO cell line expressing exogenous TMEM70 (KO+TMEM70) were transiently transfected with a C-terminally FLAG-tagged Su.c (Su.c-FLAG), or Su.d (Su.d-FLAG) as a control. 8 hours after transfection, the cells were treated with 10µM CCCP during 14 hours to block mitochondrial protein import before the construct is significantly expressed. Cells were then washed and incubated further without CCCP for the indicated time to trigger mitochondrial import of Su.c-FLAG or Su.d-FLAG. Cells were then harvested in PBS containing protease inhibitors. Total protein extracts were separated by SDS-PAGE and analyzed by western blot using anti-FLAG antibodies. A, 60 µg of total protein extracts from cells expressing Su.c-FLAG were analyzed. The signal corresponding to the tagged protein is indicated (Su.c-FLAG) together with a degradation product (deg.) accumulated during CCCP treatment (see Fig. S4B). Representative results are shown (top panel). Signal densities were quantified and expressed as percentage of signal obtained in KO+TMEM70 cells at latest time point for each replicate (lower panel). Data are mean +/- S.D. (error bars) (n = 3-5). See other replicates on Fig. S5A. B, 40 µg of total protein extracts from cells expressing Su.d-FLAG were analyzed. The signal corresponding to the tagged protein is indicated (Su.d-FLAG) together with a degradation product (deg.) accumulated during CCCP treatment (see Fig. S5B). Representative results are shown (top panel). Signal densities were quantified and expressed as percentage of signal obtained in KO+TMEM70 cells at latest time point for each replicate (lower panel). Data are mean +/- S.D. (error bars) (n = 3). See other replicates on Fig. S5B.

**Figure 8. Radioactive pulse-chase labeling reveals a very rapid turnover of subunit c in the absence of TMEM70.** TMEM70 knock-out (KO), and the same KO cell line expressing exogenous TMEM70 (KO+TMEM70) were transiently transfected with a C-terminally FLAG-tagged Su.c construct (Su.c-FLAG) or left untransfected (NT). 24 hours later, the cells were pulse-labeled for 20 min with 35S-Met/Cys as detailed under the Material and Methods section. Cells were then washed and incubated further with cold Met/Cys for the indicated chase time. Cells were then harvested in PBS containing protease inhibitors, lysed in 0.5% Triton X-100 buffer and subjected to immunoprecipitation using anti-FLAG magnetic beads.
**A**, The eluates were separated by SDS-PAGE and the radioactive signal was acquired using a phosphorimager. Main signals corresponding to the precursor (precursor) and mature (Su.c-FLAG) forms of the tagged protein are indicated. Endogenous Su.c that was co-immunoprecipitated could also be detected (Su.c endo.). Representative results are shown (n = 3). See other replicates on Fig. S6. **B**, Signal densities of the different bands were quantified and expressed as percentage of signal obtained in KO+TMEM70 cells at initial time point. Data are mean +/- S.D. (error bars) (n = 3).

**Figure 9. Physical interaction between TMEM70 and the subunit c of the ATP synthase.** **A**, immunoprecipitation of TMEM70-FLAG. Mitochondria isolated from TMEM70-KO cells overexpressing TMEM70-FLAG, or TMEM70 as a control, were used in immunoprecipitation assays using anti-FLAG M2 magnetic beads. Digitonin extractions were performed in order to preserve protein-protein interactions. Extraction supernatants (S) represent the input material incubated with the beads while insoluble pellet (P) were set apart for analysis. The unbound material come with the flow-through (FT) while bound proteins are retained on the beads and further eluted (E) natively by competition with free 3xFLAG peptide. Portions (indicated at the bottom of the panel) of each fraction were analyzed by SDS-PAGE and western blot. The signals surrounded by * represent residual signal from Su.d in the subsequent decoration of TMEM70. **B-H**, mitochondria were isolated for each cell line and subjected to digitonin extraction. The extracts were analyzed by BN-PAGE in a first dimension. An arrow above each panel indicates the migration direction. Some lanes were used for in-gel ATPase activity assay where shown (ATPase). All other lanes of the BN-PAGE were cut and analyzed by SDS-PAGE in a second dimension (an arrow on the right side of each panel indicates the migration direction). Proteins were transferred to nitrocellulose and analyzed by western blot. Position of fully (CV) or partially (Vestigial CV) assembled ATP synthase, F1-c8 subcomplex, F1-ATPase, and free Su.Beta, or TMEM70-containing complexes (*), are indicated below each panel with approximate calculated molecular weight in KDa above panels when applicable. **BC**, analysis of original HEK293T (B) and TMEM70 KO (C). The high molecular weight part of the 4-16% BN-PAGE lanes was cut in order to fit lanes till bottom in the SDS-PAGE 2D well. **D-G**, the low molecular weight part of the 3-12% BN-PAGE lanes was cut in order to fit lanes till top in the SDS-PAGE 2D well. **DE**, analysis of TMEM70 KO expressing TMEM70-FLAG (D) or TMEM70 (E). These samples were loaded twice on the BN-PAGE. One lane was used for 2D western blot analysis and the second was used to reveal in-gel ATPase activity that is shown at the top of each panel. **FG**, analysis of 143B cell line containing (Rho+, F) or not (Rho°, G) mitochondrial DNA. **H**, native eluates from the immunoprecipitation of TMEM70-FLAG described in A were analyzed by two-dimensional electrophoresis and western blot, as described above for mitochondrial extracts. TMEM70-Su.c complexes display different sizes (*), probably reflecting increasing incorporation of Su.c molecules.

**Figure 10. Radioactive pulse-chase labeling reveals transient interactions of subunit c with TMEM70 before its incorporation in the ATP synthase.** TMEM70 knock-out cell lines expressing exogenous TMEM70 (KO+TMEM70 or KO+TMEM70-FLAG as indicated) were pulse labeled for 20 min with 35S-Met/Cys and further incubated for the indicated chase time, in identical conditions as Figure 8. Cells were then harvested in ice-cold PBS containing protease inhibitors, lysed and subjected to immunoprecipitation. The eluates were separated by SDS-PAGE and the radioactive signals were acquired using a phosphorimager.
**A**, The eluates were separated by SDS-PAGE and the radioactive signal was acquired using a phosphorimager. Main signals corresponding to the precursor (precursor) and mature (Su.c-FLAG) forms of the tagged protein are indicated. Endogenous Su.c that was co-immunoprecipitated could also be detected (Su.c endo.). Representative results are shown (n = 3). See other replicates on Fig. S6. **B**, Signal densities of the different bands were quantified and expressed as percentage of signal obtained in KO+TMEM70 cells at initial time point. Data are mean +/- S.D. (error bars) (n = 3).

**Figure 9. Physical interaction between TMEM70 and the subunit c of the ATP synthase.** **A**, immunoprecipitation of TMEM70-FLAG. Mitochondria isolated from TMEM70-KO cells overexpressing TMEM70-FLAG, or TMEM70 as a control, were used in immunoprecipitation assays using anti-FLAG M2 magnetic beads. Digitonin extractions were performed in order to preserve protein-protein interactions. Extraction supernatants (S) represent the input material incubated with the beads while insoluble pellet (P) were set apart for analysis. The unbound material comes with the flow-through (FT) while bound proteins are retained on the beads and further eluted (E) natively by competition with free 3xFLAG peptide. Portions (indicated at the bottom of the panel) of each fraction were analyzed by SDS-PAGE and western blot. The signals surrounded by * represent residual signal from Su.d in the subsequent decoration of TMEM70. **B-H**, mitochondria were isolated for each cell line and subjected to digitonin extraction. The extracts were analyzed by BN-PAGE in a first dimension. An arrow above each panel indicates the migration direction. Some lanes were used for in-gel ATPase activity assay where shown (ATPase). All other lanes of the BN-PAGE were cut and analyzed by SDS-PAGE in a second dimension (an arrow on the right side of each panel indicates the migration direction). Proteins were transferred to nitrocellulose and analyzed by western blot. Position of fully (CV) or partially (Vestigial CV) assembled ATP synthase, F1-C8 subcomplex, F1-ATPase, and free Su.Beta, or TMEM70-containing complexes (*), are indicated below each panel with approximate calculated molecular weight in KDa above panels when applicable. **BC**, analysis of original HEK293T (B) and TMEM70 KO (C). The high molecular weight part of the 4-16% BN-PAGE lanes was cut in order to fit lanes till bottom in the SDS-PAGE 2D well. **D-G**, the low molecular weight part of the 3-12% BN-PAGE lanes was cut in order to fit lanes till top in the SDS-PAGE 2D well. **DE**, analysis of TMEM70 KO expressing TMEM70-FLAG (D) or TMEM70 (E). These samples were loaded twice on the BN-PAGE. One lane was used for 2D western blot analysis and the second was used to reveal in-gel ATPase activity that is shown at the top of each panel. **FG**, analysis of 143B cell line containing (Rho+, F) or not (Rho°, G) mitochondrial DNA. **H**, native eluates from the immunoprecipitation of TMEM70-FLAG described in A were analyzed by two-dimensional electrophoresis and western blot, as described above for mitochondrial extracts. TMEM70-Su.c complexes display different sizes (*), probably reflecting increasing incorporation of Su.c molecules.

**Figure 10. Radioactive pulse-chase labeling reveals transient interactions of subunit c with TMEM70 before its incorporation in the ATP synthase.** TMEM70 knock-out cell lines expressing exogenous TMEM70 (KO+TMEM70 or KO+TMEM70-FLAG as indicated) were pulse labeled for 20 min with 35S-Met/Cys and further incubated for the indicated chase time, in identical conditions as Figure 8. Cells were then harvested in ice-cold PBS containing protease inhibitors, lysed and subjected to immunoprecipitation. The eluates were separated by SDS-PAGE and the radioactive signals were acquired using a phosphorimager.
**A**, Extracts were obtained with digitonin (2 g/g protein) and immunoprecipitations were done using anti-FLAG magnetic beads. Two major specific (absent in KO+TMEM70 control) signals could be identified corresponding to TMEM70-FLAG and co-immunoprecipitated endogenous Su.c as indicated. Representative results are shown (top panel). Signal densities of the two bands were quantified. The ratios between Su.c and TMEM70-FLAG signals were calculated at each time point to normalize and expressed as percentage of this ratio at initial time point. Data are mean +/- S.D. (error bars) (n = 3). **B**, Extracts were obtained with digitonin (9 g/g protein) and immunoprecipitations were done using anti-ATP synthase immunocapture antibodies. Several bands could be detected among which Su.c as indicated (Su.c endo.). Representative results are shown (top panel). Signal densities of Su.c were quantified at different time points and expressed as percentage of signal obtained at initial time point. Data are mean +/- S.D. (error bars) (n = 3). See other replicates and immunodecorations using anti-Su.c on Fig. S11.

**Figure 11.** Working model for the TMEM70-dependent assembly of the c-ring of the ATP synthase within mitochondrial cristae. The biogenesis of the ATP synthase is represented with a special focus on the c-ring. The Su.c precursor (in green) is imported into mitochondria through the protein translocases (grey cylinders) across the outer membrane (OM) and the inner boundary membrane (IBM). The Su.c precursor gets processed by the Matrix Processing Peptidase (Proteolytic maturation). TMEM70 (in red; T) located in the inner cristae membrane (ICM) interacts with Su.c within higher order oligomers in order to stabilize it and form the c-ring. TMEM70 complexes release directly or disassemble to liberate the c-ring and take in charge new Su.c. The c-ring next assembles with premade F1 subcomplex and the additional subunits are added to form ATP synthase monomers, dimers, and oligomers.
A, Extracts were obtained with digitonin (2 g/g protein) and immunoprecipitations were done using anti-FLAG magnetic beads. Two major specific (absent in KO+TMEM70 control) signals could be identified corresponding to TMEM70-FLAG and co-immunoprecipitated endogenous Su.c as indicated. Representative results are shown (top panel). Signal densities of the two bands were quantified. The ratios between Su.c and TMEM70-FLAG signals were calculated at each time point to normalize and expressed as percentage of this ratio at initial time point. Data are mean +/- S.D. (error bars) (n = 3). B, Extracts were obtained with digitonin (9 g/g protein) and immunoprecipitations were done using anti-ATP synthase immunocapture antibodies. Several bands could be detected among which Su.c as indicated (Su.c endo.). Representative results are shown (top panel). Signal densities of Su.c were quantified at different time points and expressed as percentage of signal obtained at initial time point. Data are mean +/- S.D. (error bars) (n = 3). See other replicates and immunodecorations using anti-Su.c on Fig. S11.

**Figure 11. Working model for the TMEM70-dependent assembly of the c-ring of the ATP synthase within mitochondrial cristae.** The biogenesis of the ATP synthase is represented with a special focus on the c-ring. The Su.c precursor (in green) is imported into mitochondria through the protein translocases (grey cylinders) across the outer membrane (OM) and the inner boundary membrane (IBM). The Su.c precursor gets processed by the Matrix Processing Peptidase (Proteolytic maturation). TMEM70 (in red; T) located in the inner cristae membrane (ICM) interacts with Su.c within higher order oligomers in order to stabilize it and form the c-ring. TMEM70 complexes release directly or disassemble to liberate the c-ring and take in charge new Su.c. The c-ring next assembles with premade F1 subcomplex and the additional subunits are added to form ATP synthase monomers, dimers, and oligomers.
A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

C

<figure><img src="image_5.png" /></figure>

D

<figure><img src="image_6.png" /></figure>

E

<figure><img src="image_7.png" /></figure>
A

<figure><img src="image_2.png" /></figure>

B

<figure><img src="image_3.png" /></figure>

C

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

E

<figure><img src="image_6.png" /></figure>
A

Mitofilin - Mic60

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

TMEM70

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

B

Mic60 / TMEM70-FLAG / DAPI

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

C

TMEM70

<figure><img src="image_18.png" /></figure>

D

<figure><img src="image_19.png" /></figure>
A

Mitofilin - Mic60

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

TMEM70

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

B

Mic60 / TMEM70-FLAG / DAPI

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

C

TMEM70

<figure><img src="image_17.png" /></figure>

D

<figure><img src="image_18.png" /></figure>
A

<figure><img src="image_20.png" /></figure>

B

<figure><img src="image_21.png" /></figure>

C

<figure><img src="image_22.png" /></figure>

D

<figure><img src="image_23.png" /></figure>

E

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>
A

<figure><img src="image_19.png" /></figure>

B

<figure><img src="image_20.png" /></figure>

C

<figure><img src="image_21.png" /></figure>

D

<figure><img src="image_22.png" /></figure>

E

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>
A

<figure><img src="image_26.png" /></figure>

B

<table><thead><tr><th>Respiratory Activity</th><th>WT</th><th>T3AA2</th><th>T2BB9</th></tr></thead><tbody><tr><th>ADP-stimulated</th><td>3.9<br/>+/- 0.79</td><td>2.48<br/>+/- 0.47</td><td>2.19<br/>+/- 0.46</td></tr><tr><th>Oligomycin-inhibited</th><td>0.92<br/>+/- 0.16</td><td>1.10<br/>+/- 0.13</td><td>1.34<br/>+/- 0.24</td></tr><tr><th>CCCP-uncoupled</th><td>8.38<br/>+/- 1.93</td><td>7.33<br/>+/- 1.07</td><td>7.35<br/>+/- 0.85</td></tr></tbody></table>

C

<figure><img src="image_27.png" /></figure>

D

<figure><img src="image_28.png" /></figure>

E

<figure><img src="image_29.png" /></figure>

F

<figure><img src="image_30.png" /></figure>

G

<figure><img src="image_31.png" /></figure>

H

<figure><img src="image_32.png" /></figure>

KO

<figure><img src="image_33.png" /></figure>

KO + TMEM70

<figure><img src="image_34.png" /></figure>

<figure><img src="image_35.png" /></figure>
A

<figure><img src="image_25.png" /></figure>

B

<table><thead><tr><th>Respiratory Activity</th><th>WT</th><th>T3AA2</th><th>T2BB9</th></tr></thead><tbody><tr><th>ADP-stimulated</th><td>3.9<br/>+/- 0.79</td><td>2.48<br/>+/- 0.47</td><td>2.19<br/>+/- 0.46</td></tr><tr><th>Oligomycin-inhibited</th><td>0.92<br/>+/- 0.16</td><td>1.10<br/>+/- 0.13</td><td>1.34<br/>+/- 0.24</td></tr><tr><th>CCCP-uncoupled</th><td>8.38<br/>+/- 1.93</td><td>7.33<br/>+/- 1.07</td><td>7.35<br/>+/- 0.85</td></tr></tbody></table>

C

<figure><img src="image_26.png" /></figure>

D

<figure><img src="image_27.png" /></figure>

E

<figure><img src="image_28.png" /></figure>

F

<figure><img src="image_29.png" /></figure>

G

<figure><img src="image_30.png" /></figure>

H

<figure><img src="image_31.png" /></figure>

KO

<figure><img src="image_32.png" /></figure>

KO + TMEM70

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>
A

<figure><img src="image_36.png" /></figure>

B

<figure><img src="image_37.png" /></figure>

C

<figure><img src="image_38.png" /></figure>

D

Pat.

<figure><img src="image_39.png" /></figure>

Pat.+TMEM70

<figure><img src="image_40.png" /></figure>
A

<figure><img src="image_35.png" /></figure>

B

<figure><img src="image_36.png" /></figure>

C

<figure><img src="image_37.png" /></figure>

D

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>
A

<figure><img src="image_41.png" /></figure>

B

<figure><img src="image_42.png" /></figure>

C

<figure><img src="image_43.png" /></figure>
A

<figure><img src="image_40.png" /></figure>

B

<figure><img src="image_41.png" /></figure>

C

<figure><img src="image_42.png" /></figure>
A

<figure><img src="image_44.png" /></figure>

<figure><img src="image_45.png" /></figure>

B

<figure><img src="image_46.png" /></figure>

<figure><img src="image_47.png" /></figure>
A

<figure><img src="image_43.png" /></figure>

<figure><img src="image_44.png" /></figure>

B

<figure><img src="image_45.png" /></figure>

<figure><img src="image_46.png" /></figure>
A

<figure><img src="image_48.png" /></figure>

B

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>

<figure><img src="image_51.png" /></figure>
A

<figure><img src="image_47.png" /></figure>

B

<figure><img src="image_48.png" /></figure>

<figure><img src="image_49.png" /></figure>

<figure><img src="image_50.png" /></figure>
<figure><img src="image_52.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_57.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_58.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_59.png" /></figure>
<figure><img src="image_51.png" /><figcaption>B</figcaption></figure>

<figure><img src="image_52.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_53.png" /><figcaption>D</figcaption></figure>

<figure><img src="image_54.png" /><figcaption>E</figcaption></figure>

<figure><img src="image_55.png" /><figcaption>F</figcaption></figure>

<figure><img src="image_56.png" /><figcaption>G</figcaption></figure>

<figure><img src="image_57.png" /><figcaption>H</figcaption></figure>

<figure><img src="image_58.png" /></figure>
A

<figure><img src="image_60.png" /></figure>

B

<figure><img src="image_61.png" /></figure>
A

<figure><img src="image_59.png" /></figure>

B

<figure><img src="image_60.png" /></figure>
<figure><img src="image_62.png" /></figure>

Su.c
precursor

x 8 Su.c

Topology

c-ring
Assembly

Complete ATP synthase
Assembly
<figure><img src="image_61.png" /></figure>