Cell lines
The following cell lines were purchased from the American Type Culture Collection: A549 (tissue, lung cancer; gender, male), U373MG (tissue, glioblastoma; gender, male), LS174T (tissue, colon cancer; gender, female), and 293 (tissue, embryonic kidney). A549 cells were cultured in RPMI 1640 (Sigma). U373MG, LS174T, and 293 cells were cultured in DMEM (Sigma). All media were supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a humidified chamber with 5% CO2. These cell lines have not been authenticated.
Mice
Animal experiment protocols were approved by the Committee for Ethics in Animal Experimentation (approved protocol No. T17-043), and experiments were conducted in accordance with Guidelines for Animal Experiments of the National Cancer Center. C57BL/6J WT mice were obtained from CLEA Japan (Tokyo, Japan). Mieap-knockout (Mieap−/−) mice were generated using the Cre/loxP recombination system, as previously reported32. Briefly, floxed and trapped alleles were generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT, located between exons 5 and 8 of the mouse Mieap gene, which is located on chromosome 5. Mieap homozygous (Mieap−/−) deficient mice were generated by mating breeding pairs of Mieap heterozygous (Mieap+/−) mice. p53-deficient mice were a gift from Dr. S. Aizawa, Center for Developmental Biology, RIKEN70.
For generation of N-terminal or C-terminal mNeonGreen-tagged Mieap KI mice, targeting vectors containing the cassette of the 5’UTR-mNeonGreen-Linker-Exon1-intron1-FRT-NEO-FRT-intron1 or the intron10-exon11-intron11-exon12-intron12-exon13-Linker-mNeonGreen-FRT-NEO-FRT-3’UTR were constructed to express N-terminal or C-terminal mNeonGreen-tagged Mieap protein in mice, respectively. Targeting vectors were introduced into ES cells by electroporation. After geneticin selection, positive ES clones were screened by PCR and Southern blot analysis. The positive clones were injected into C57/B6 mouse blastocysts. The obtained heterozygous and homozygous mice were subjected to analysis.
All mice were housed at 22 ± 2°C with a 12 h light/dark cycle with free access to food, CE-2 (CLEA Japan), and water.
Establishment of KD cell lines
We established a Mieap-KD cell line using LS174T, as previously described29. Mieap expression was inhibited in this cell line by retroviral expression of short-hairpin RNA (shRNA) against the Mieap sequence (Supplementary Fig. 15a, b). We also established LS174T-cont cells using a retroviral vector with a target sequence for EGFP, or an empty retroviral vector, and A549-cont cells using an empty retroviral vector.
Plasmid construction
Constructs containing Mieap
For construction of the plasmid containing N-terminal EGFP-tagged Mieap, the nucleotide sequence of Mieap was PCR-amplified using primers N-EGFP-Mieap-F and N-EGFP-Mieap-R. PCR products were digested with Kpn I and ligated into pEGFP-C1 (Clontech) cut with the same enzyme. For construction of the plasmid containing C-terminal EGFP-tagged Mieap, the nucleotide sequence of Mieap, excluding the stop codon, was PCR-amplified using the primers C-EGFP-Mieap-F and C-EGFP-Mieap-R. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Inserted products were excised using Hind III restriction sites, and ligated into pEGFP-N1 (Clontech), cut with the same enzyme. N-terminal EGFP-tagged Mieap was used as EGFP-Mieap, except for Fig. 1a – d and Supplementary Movie 3 where the C-terminal EGFP-tagged Mieap was used.
Plasmids containing N-FLAG-Mieap (pN-FLAG-Mieap) were constructed as follows. The nucleotide sequence of Mieap was PCR-amplified using the primers, N-FLAG-Mieap-F and N-FLAG-Mieap-R. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Inserted products were excised using the Kpn I restriction sites and ligated into pre-digested pcDNA3.1 (+) (Thermo Fisher Scientific) cut with the same enzyme. The nucleotide sequence of Mieap was excised from the plasmid using the Hind III and Xho I restriction sites, and ligated into pre-digested pCMV-Tag2A (Agilent) cut with the same enzyme.
The plasmid containing C-FLAG-Mieap (pC-FLAG-Mieap) was constructed as follows. The nucleotide sequence of Mieap, excluding the stop codon, was PCR-amplified using the primers, C-EGFP-Mieap-F and C-EGFP-Mieap-R, the same primers used for construction of the plasmid containing C-terminal EGFP-tagged Mieap. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Inserted products were excised using the Hind III restriction site and ligated into pre-digested p3xFLAG-CMV-14 (Sigma Aldrich) cut with the same enzyme.
Prior to construction of plasmids containing EGFP-Mieap deletion mutants, point mutations in Bgl II, Sac I, EcoR I, and Pst I restriction sites of the multiple cloning site of pEGFP-Mieap were introduced using QuikChange Site-Directed Mutagenesis Kits (Agilent) with primers Mut-F1, Mut-R1, Mut-F2 and Mut-R2, which were confirmed by DNA sequencing.
For construction of plasmids containing EGFP-MieapΔCC (pEGFP-MieapΔCC), the nucleotide sequence of pEGFP-Mieap between two Pst I restriction sites was deleted by digestion with Pst I. The remainder was self-ligated, additionally deleting c.810C using the QuikChange Site-Directed Mutagenesis Kit (Agilent) with primers Mut-F3 and Mut-R3 to make the deletion mutation in-frame.
For construction of plasmids containing EGFP-MieapΔ275 (pEGFP-MieapΔ275), the nucleotide sequence of pEGFP-Mieap between the Bgl II and Sma I restriction sites was deleted by digestion using Bgl II and Sma I. After blunting with T4 DNA polymerase (Thermo Fisher Scientific), the remainder was self-ligated.
For construction of plasmids containing EGFP-MieapΔ496 (pEGFP-MieapΔ496), the nucleotide sequence of pEGFP-Mieap was deleted between the EcoR I and Kpn I restriction sites by digestion using EcoR I and Kpn I. After blunting with T4 DNA polymerase (Thermo Fisher Scientific), the remainder was self-ligated.
For construction of plasmids containing TagRFP-T-Mieap (pTagRFP-T-Mieap), the nucleotide sequence of pEGFP-Mieap between the Nhe I and Xho I restriction sites containing EGFP was replaced with nucleotide sequence of pTagRFP-T-EEA1 (Addgene #42635) between the Nhe I and Xho I restriction sites containing TagRFP-T, by digestion using Nhe I and Xho I.
For construction of plasmids containing GST-Mieap (pGST-Mieap), the nucleotide sequence of Mieap (amino acids 99-298) was PCR-amplified using the primers, GST-Mieap-F and GST-Mieap-R. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Products were digested with EcoR I and Xho I, and ligated into pGEX5X-2 (Cytiva).
For construction of plasmids containing Mieap ΔCC (pMieap ΔCC), pEGFP-Mieap ΔCC was digested at the Kpn I restriction sites to obtain the nucleotide sequence of Mieap ΔCC, and ligated into pcDNA3.1 (+) (Thermo Fisher Scientific) cut with the same enzyme, Kpn I.
For construction of plasmids containing Mieap Δ274 (pMieap Δ274), the nucleotide sequence of Mieap Δ274 was PCR-amplified from pEGFP- Mieap Δ275 using the primers, Δ274-F and Δ274-R. PCR products were digested with Kpn I, and ligated into pcDNA3.1 (+) (Thermo Fisher Scientific) cut with the same enzyme, Kpn I.
For construction of plasmids containing Mieap Δ496 (pMieap Δ496), pEGFP-Mieap Δ496 was subjected to inverse PCR using the primers, ΔEGFP-F and ΔEGFP-R to delete the nucleotide sequence of EGFP from pEGFP-Mieap Δ496, and the product was self-ligated using KOD-Plus-Mutagenesis Kit (TOYOBO).
Prior to construction of plasmids containing TagRFP-T-Mieap deletion mutants, the nucleotide sequence of TagRFP-T was PCR-amplified using the primers, TagRFP-T-F and TagRFP-T-R. PCR products were digested with Hind III and EcoR V, and ligated into pcDNA3.1 (+) (Thermo Fisher Scientific) cut with the same enzymes (pcDNA-N-TagRFP). The nucleotide sequence of Mieap was PCR-amplified using the primers, G35-F and G35-R. PCR products were digested with EcoRV and PspOMI, and ligated into pcDNA-N-TagRFP cut with the same enzymes (pG35).
For construction of plasmids containing TagRFP-T-Mieap ΔCC (pTagRFP-T-Mieap ΔCC), pG35 was subjected to inverse PCR using the primers, ΔCC-F and ΔCC-R, and the product was self-ligated using KOD-Plus-Mutagenesis Kit (TOYOBO).
For construction of plasmids containing TagRFP-T-Mieap Δ275 (pTagRFP-T-Mieap ΔCC), pTagRFP-T-Mieap was subjected to inverse PCR using the primers, Δ275-F and Δ275-R, and the product was self-ligated using KOD-Plus-Mutagenesis Kit (TOYOBO).
For construction of plasmids containing TagRFP-T-Mieap Δ496 (pTagRFP-T-Mieap Δ496), pG35 was subjected to inverse PCR using the primers, Δ496-F and Δ496-R, and the product was self-ligated using KOD-Plus-Mutagenesis Kit (TOYOBO).
All primers are listed in Supplementary Data 2.
Other constructs
For construction of plasmids containing EGFP-BNIP3 (pEGFP-BNIP3), plasmids containing FLAG-BNIP3 (pCMV-Tag2B-BNIP3) were constructed in advance. For construction of the pCMV-Tag2B-BNIP3, the nucleotide sequence of BNIP3 was PCR-amplified using the primers, BNIP3-F and BNIP3-R. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Inserted products were digested with EcoR I and Xho I, and ligated into the pre-digested pCMV-Tag2B (Agilent) cut with the same enzyme. The nucleotide sequence of pCMV-Tag2B-BNIP3 was digested at the EcoR I and Xho I restriction sites, and subsequently blunted with T4 DNA polymerase (Thermo Fisher Scientific). pEGFP-C1 (Clontech) was digested with Bgl II, blunted with T4 DNA polymerase, self-ligated, digested with EcoR I and Sma I, and ligated with the fragment of pCMV-Tag2B-BNIP3.
For construction of plasmids containing EGFP-NIX (pEGFP-NIX), plasmids containing FLAG-NIX (pCMV-Tag2B-NIX) were constructed in advance. For construction of the pCMV-Tag2B-NIX, the nucleotide sequence of NIX was PCR-amplified using the primers, NIX-F and NIX-R. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Inserted products were digested with EcoR I and Xho I, and ligated into pre-digested pCMV-Tag2B (Agilent) cut with the same enzyme. The nucleotide sequence of pCMV-Tag2B-NIX was digested at the EcoR I and Xho I restriction sites, and subsequently blunted with T4 DNA polymerase (Thermo Fisher Scientific). pEGFP-C1 (Clontech) was digested with Bgl II, blunted with T4 DNA polymerase, self-ligated, digested with EcoR I and Sma I, and ligated with the fragment of pCMV-Tag2B-NIX.
Plasmids containing EGFP-cytochrome c (pEGFP-cytochrome c) were constructed as follows. The nucleotide sequence of cytochrome c was PCR-amplified using the primers (Cytochrome c -F and Cytochrome c -R) as reported by Goldstein et al.71. PCR products were ligated into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and sequenced. Inserted products were excised using the EcoR I and BamH I restriction sites, and ligated into pEGFP-C1 (Clontech) cut with the same enzymes.
For construction of the plasmid backbone containing EGFP (pN-EGFP), the nucleotide sequence of EGFP, excluding the stop codon, was PCR-amplified using the primers, N-EGFP-F and N-EGFP-R. PCR products were digested with Hind III and BamH I, and ligated into pcDNA3.1 (+) (Thermo Fisher Scientific) cut with the same enzymes.
For construction of the plasmid backbone containing EGFP (pC-EGFP), the nucleotide sequence of EGFP was PCR-amplified using the primers, C-EGFP-F and C-EGFP-R. PCR products were digested with BamH I and Not I, and ligated into pcDNA3.1 (+) (Thermo Fisher Scientific) cut with the same enzymes.
For construction of plasmids containing EGFP-ATP5F1B (pEGFP-ATP5F1B), the nucleotide sequence of ATP5F1B, excluding the stop codon, was PCR-amplified using the primers, ATP5F1B-F1, ATP5F1B-R1, ATP5F1B-F2, and ATP5F1B-R2. PCR products were digested with Hind III and BamH I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-PHB2 (pEGFP-PHB2), the nucleotide sequence of PHB2, excluding the stop codon, was PCR-amplified using the primers, PHB2-F and PHB2-R. PCR products were digested with Nhe I and Kpn I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-ATP5F1A (pEGFP-ATP5F1A), the nucleotide sequence of ATP5F1A, excluding the stop codon, was PCR-amplified using the primers, ATP5F1A-F and ATP5F1A-R. PCR products were digested with Nhe I and Hind III and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-TAMM41 (pEGFP-TAMM41), the nucleotide sequence of TAMM41, excluding the stop codon, was PCR-amplified using the primers, TAMM41-F and TAMM41-R. PCR products were digested with Nhe I and BamH I, and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-PGS1 (pEGFP-PGS1), the nucleotide sequence of PGS1, excluding stop codon, was PCR-amplified using the primers, PGS1-F and PGS1-R. PCR products were digested with Hind III and BamH I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-PTPMT1 (pEGFP-PTPMT1), the nucleotide sequence of PTPMT1, excluding the stop codon, was PCR-amplified using the primers, PTPMT1-F and PTPMT1-R. PCR products were digested with Nhe I and Hind III and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-CRLS1 (pEGFP-CRLS1), the nucleotide sequence of CRLS1 was PCR-amplified using the primers, CRLS1-F and CRLS1-R. PCR products were digested with BamH I and Not I and ligated into pN-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-PLA2G6 (pEGFP-PLA2G6), the nucleotide sequence of PLA2G6, excluding the stop codon, was PCR-amplified using the primers, PLA2G6-F and PLA2G6-R. PCR products were digested with Nhe I and Kpn I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-TAZ (pEGFP-TAZ), the nucleotide sequence of TAZ, excluding the stop codon, was PCR-amplified using the primers, TAZ-F and TAZ-R. PCR products were digested with Nhe I and Kpn I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-PRELI (pEGFP-PRELI), the nucleotide sequence of PRELI, excluding the stop codon, was PCR-amplified using the primers, PRELI-F and PRELI-R. PCR products were digested with Nhe I and BamH I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-LONP1 (pEGFP-LONP1), the nucleotide sequence of LONP1, excluding the stop codon, was PCR-amplified using the primers, LONP1-F and LONP1-R. PCR products were digested with Hind III and BamH I and ligated into pC-EGFP cut with the same enzymes.
For construction of plasmids containing EGFP-PLD6 (pEGFP-PLD6), the nucleotide sequence of PLD6, excluding the stop codon, was PCR-amplified using the primers, PLD6-F and PLD6-R. PCR products were digested with Nhe I and Kpn I and ligated into pC-EGFP cut with the same enzymes.
All primers are listed in Supplementary Data 2.
Transfection
For transfection, cells were seeded (2×105 cells/dish) in 35-mm glass bottom dishes. Plasmids (2 μg /dish) were transfected using FuGENE6 transfection reagent (Promega), according to the manufacturer’s instructions.
Recombinant adenovirus construction
Ad-Mieap was derived from viruses as previously reported29,35,72. Replication-deficient recombinant viruses Ad-EGFP-Mieap, Ad-N-FLAG-Mieap, Ad-C-FLAG-Mieap, Ad-EGFP-MieapΔCC (Δ104-270), Ad-EGFP-MieapΔ275, Ad-EGFP-MieapΔ496, Ad-TagRFP-T-Mieap, Ad-mApple-TOMM20 derived from mApple-TOMM20-N-10 (Addgene #54955), Ad-EGFP-BNIP3, Ad-EGFP-NIX, Ad-AcGFP1-Mito, Ad-DsRed2-Mito, and Ad-EGFP-cytochrome c were generated from the corresponding plasmid vectors and purified as described previously73. Briefly, DNA fragments obtained by restriction of each plasmid vector were blunted using T4 DNA polymerase, ligated into the SmiI site of the cosmid, pAxCAwtit (Takara), which contains the CAG promoter and the entire genome of type 5 adenovirus, except the E1 and E3 regions. Recombinant adenoviruses were generated by in vitro homologous recombination in the 293 cell line with the cDNA-inserted pAxCAwtit and the adenovirus DNA terminal–protein complex. Viruses were propagated in the 293 cell line and purified by two rounds of CsCl density centrifugation. Viral titers were determined with a limiting dilution bioassay using 293 cells.
Adenoviral infection
Infection of cell lines was carried out by adding viral solutions to cell monolayers, incubating them at 37°C for 120 min with brief agitation every 20 min. This was followed by addition of culture medium and return of the infected cells to the 37°C incubator.
Immunocytochemistry
For immunocytochemistry, cells were grown on 8-well chamber slides (1-4×104 cells/well) at 37°C in conventional culture medium, and fixed in paraformaldehyde (Supplementary Fig. 1b, 2%; Supplementary Fig. 1c, 4%) for 15 min at room temperature. Slides were incubated with Triton X-100 (Supplementary Fig. 1b, 0.1% for 2 min; Supplementary Fig. 1c, 0.5% for 10 min), and washed 3x with phosphate-buffered saline (PBS) at room temperature. Cells were blocked with 3% bovine serum albumin (BSA) in PBS (Supplementary Fig. 1b, for 3 h; Supplementary Fig. 1c, for 2 h), and sequentially incubated with rabbit polyclonal anti-Mieap antibody (1:200), mouse monoclonal anti-GFP antibody (1:200), or mouse monoclonal anti-FLAG antibody (1:1000) for 2 h at room temperature. After washing 3x with PBS, slides were incubated with Alexa Fluor 546 goat anti-rabbit IgG antibody (1:200) or Alexa Fluor 546 goat anti-mouse IgG antibody (1:200) at room temperature (Supplementary Fig. 1b, for 2 h; SSupplementary Fig. 1c, for 1 h). Slides were washed 3x with PBS. Then they were mounted with VECTASHIELD H-1000 (Vector Laboratories) and observed using a FLUOVIEW FV3000 confocal laser scanning microscope (Olympus).
Histological analysis
Hematoxylin and eosin (HE) staining was performed using Eosin (CS701, Dako) and Hematoxylin (S2020, Dako). Immunohistochemistry (IHC) was performed as described previously32. Briefly, for antigen retrieval, paraffin-fixed sections were autoclaved in citric acid buffer (pH 6.0) at 121 °C for 10 min. Sections were treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity and incubated with 5% bovine serum albumin (BSA) in 50 mM Tris-buffered saline (pH 7.4) containing 0.05% Triton X-100 (T-TBS) for 1 h at room temperature to block non-specific protein binding sites. Sections were then incubated at 4 °C with the primary antibodies, rabbit polyclonal anti-mouse Mieap antibody (1:1000) 32, and mouse monoclonal anti-cytochrome c (ab13575, 1 µg/mL) in TBS-T. After overnight incubation, sections were incubated with EnVision + Dual Link System-HRP reagents (Dako) for 1 h according to the manufacturer’s instructions at room temperature and treated with 0.02% DAB (DOJINDO) in 0.05 M Tris-HCl buffer (pH 7.6). Finally, sections were counterstained with Hematoxylin (Dako). For immunofluorescence (IF), rabbit polyclonal anti-mouse Mieap antibody (1:1000)32, and mouse monoclonal anti-cytochrome c (ab13575, 1 µg/mL) were used for primary antibodies. Alexa Fluor 594 goat antirabbit IgG antibody (1:200) and Alexa Fluor 488 goat anti-mouse IgG antibody (1:100) were used for secondary antibodies. Nuclear staining was performed with Hoechst 33342 (10 μg/mL) or TO-PRO-3 (200 nM).
Transmission electron microscopy (TEM)
A549-cont and U373MG cells (4×104 cells/24-well plate) were infected with Ad-Mieap. On day 1 after infection, cells were fixed in phosphate buffered 2.5% glutaraldehyde and subsequently post-fixed in 1% OsO4 at 4°C for 2 h. Then, specimens were dehydrated in a graded ethanol series and embedded in epoxy resin. Ultrathin sections (75 nm) were cut with an ultramicrotome. Ultrathin sections stained with uranyl acetate and lead staining solution were observed on a transmission electron microscope H-7500 (Hitachi) at 80 kV.
LS174T (control and Mieap-KD) cells cultured under normal conditions were also processed for TEM as mentioned above, with the following modifications. 2% glutaraldehyde was used for prefixation. 2% OsO4 was used instead for post-fixation. 80-90-nm ultrathin sections were cut and observed on a transmission electron microscope H-7600 (Hitachi) at 100 kV.
Kidney and liver specimens were collected from an 18-week-old WT mouse and a 16-week-old Mieap−/− male mouse. BAT specimens were collected from a 40-week-old WT and a 40-week-old Mieap−/− male mouse. Specimens cut into approximately 3 × 3 × 3 mm3 were also processed for TEM and processed in the same fashion as the aforementioned A549-cont cells. However, a mixture of 2% paraformaldehyde and 2% glutaraldehyde was used for prefixation.
Post-embedding immunoelectron microscopy
A549-cont cells (2×105 cells/35-mm glass bottom dish) were infected with Ad-Mieap. On day 1 after infection, cells were fixed with 4% paraformaldehyde and 0.025% glutaraldehyde in 0.1 M PBS (pH 7.4) for 1 h at 4°C. After fixation, cells were washed with 0.1 M PBS (pH 7.4) for 16 h at 4°C, dehydrated in a graded ethanol series, and infiltrated with LR White resin. Polymerization was performed in TAAB embedding capsules (TAAB) inverted on glass-bottom dishes for 3h at 60°C. Ultrathin sections (75 nm) were collected on nickel grids. After blocking with 3% BSA in PBS for 1 h, sections were incubated with anti-Mieap antibody (1:200) diluted in PBS with 0.05% Triton X-100 for 2h at RT. Sections were washed 8x with 0.15% glycine in PBS, and incubated with goat anti-rabbit IgG 10-nm gold antibody (1:50) diluted in PBS with 0.05% Triton X-100 for 2h at RT. Sections were washed 8x in PBS and fixed with 1% glutaraldehyde in PBS for 5 min. Sections were washed 8x in distilled water. Grids were embedded in a mixture containing 2.7% polyvinyl alcohol and 0.3% uranyl acetate. Sections on grids were observed on a transmission electron microscope H-7500 (Hitachi) at 75 kV.
Amino acid sequence analyses of Mieap protein
We analyzed the phylogenetic spread of Mieap orthologs using OrthoDB v10 (https://www.orthodb.org/)37. Multiple sequence alignment for Mieap orthologs was performed using Genetyx ver. 10. Prediction of IDRs in the amino acid sequence of Mieap was done using VL3-BA39 on the PONDR server (http://www.pondr.com/) and collated with meta-prediction of IDRs using DisMeta (http://www-nmr.cabm.rutgers.edu/bioinformatics/disorder/)38. Prediction of coiled-coil regions was done using COILS (https://embnet.vital-it.ch/software/COILS_form.html)40. Hydrophobicity of Mieap was analyzed according to the Kyte-Doolittle index42 using ProtScale (https://web.expasy.org/protscale/)74. The linear net charge per residue of Mieap was analyzed using CIDER (http://pappulab.wustl.edu/CIDER/)41.
Analyses of confocal microscopy image data
Throughout the study, confocal microscopy images were taken with a FLUOVIEW FV3000 confocal laser scanning microscope (Olympus). For validation of the spatial relationship between Mi-BCs and mApple-TOMM20, additional images were taken using a SpinSR10 spinning disk confocal super resolution microscope (Olympus). For Z-stack and time-lapse imaging, a montage of differential interference contrast (DIC) and fluorescence images was created using MetaMorph ver. 7.8 (Molecular Devices). 3D reconstruction was performed using cellSens Imaging Software (Olympus). Line-scan profiles were acquired using MetaMorph ver. 7.8 (Molecular Devices).
FRAP experiments
EGFP-Mieap, EGFP-MieapΔCC, EGFP-MieapΔ275, and EGFP-MieapΔ496 were expressed in A549-cont cells to generate condensates by infection with Ad-EGFP-Mieap, Ad-EGFP-MieapΔCC, Ad-EGFP-MieapΔ275, and Ad-EGFP-MieapΔ496, respectively. FRAP experiments were performed on a FLUOVIEW FV3000 confocal laser scanning microscope (Olympus), using a 60x/1.4 NA oil immersion objective (Olympus). Condensates were subjected to spot-bleaching or full-bleaching (bleaching entire condensates). For spot-bleaching, the bleaching area was unified to a diameter of 1.38 µm. Condensates were imaged for 6 s, acquiring 30 images prior to spot-bleaching or 50 s, acquiring 5 images prior to full-bleaching. Photobleaching employed a 488-nm laser at 10% laser power with 11.6 µs/µm exposure time or 1.4% laser power with 1.4 µs/µm exposure time. Time-lapse images were acquired at 0.2-ms intervals for 60 s or 10 s intervals for 15 min. Spot-bleaching data for each construct were acquired from 15 different condensates. Full-bleaching data of each construct were acquired from 10 different condensates.
Calculation of intensity ratio (IR)
To evaluate partitioning of EGFP-Mieap and deletion mutant proteins, EGFP-Mieap WT, ΔCC, Δ275, and Δ496 were expressed in A549-cont cells to generate condensates by infection with Ad-EGFP-Mieap WT, ΔCC, Δ275, and Δ496, respectively. EGFP intensity of condensates and cytoplasm was measured. Because EGFP intensity of these condensates was higher than the intensity of 0.4 mg/mL His-EGFP solution for standard curve, we chose intensity ratio (IR) rather than partition coefficient (PC) for the parameter of this partitioning experiments52. IR was calculated as (Icondensates-Ibackground)/(Icytoplasm-Ibackground), where Icondensates, Icytoplasm, and Ibackground are the mean intensities of condensates, cytoplasm, and PBS acquired by the identical conditions (laser wavelength, 488 nm; laser transmissivity, 0.01%; detection wavelength, 500–600 nm; voltage, 350 V) on a FLUOVIEW FV3000 confocal laser scanning microscope (Olympus). IR data were obtained from 40 cells for each construct.
Expression and purification of GST and GST-Mieap
Escherichia coli (BL21) cells transformed with expression vectors were grown in 200 mL of Luria-Bertani medium at 37°C until the OD600 was between 0.55-0.6. Protein expression was induced with 100 µM IPTG, and bacteria were subsequently incubated for 3 h at 25°C. After harvesting bacteria by centrifugation at 3000 × g for 10 min at 4 °C, pellets were lysed with lysis buffer (1% Triton X-100 buffered in PBS supplemented with 1 mM Phenylmethylsulfonyl fluoride), and sonicated (20 × 30 s bursts with 10 s rest between bursts). Insoluble material was removed by centrifugation at 10,000 rpm for 30 min at 4 °C. Supernatant was incubated with glutathione-Sepharose 4B (Cytiva) pre-equilibrated with lysis buffer at 4°C overnight. After the beads were washed twice with lysis buffer, proteins were eluted with elution buffer (50 mM glutathione diluted in 50 mM Tris–HCl, pH 8.0), and dialyzed at 4°C overnight against PBS.
Lipid-binding analysis
For lipid-binding analysis, protein-lipid interactions on lipid-spotted membranes were evaluated with fat blot assays49. Natural CL, PC, and PE derived from bovine heart (Olbracht Serdary Research Laboratories) were diluted with chloroform/methanol/1N HCl (80:80:1). 1 μL of each diluted lipid was spotted onto PVDF membranes (Cytiva) for antigen-antibody reactions using anti-Mieap antibody or nitrocellulose membranes (Cytiva) for antigen-antibody reactions using an anti-GST antibody to align spots with increasing amounts of lipids ranging from 0-667 pmol. Here, approximate molarities of CL, PC, and PE calculated from molecular weights of tetralinoleoyl CL, distearoyl PC, and distearoyl PC were used, respectively. After membranes were blocked with blocking buffer (3% fatty acid-free BSA diluted in 50 mM Tris–HCl, 150 mM NaCl, pH 7.5) for 1 h, membranes were incubated with 2.5 μg/mL of GST-Mieap or GST protein diluted in blocking buffer containing 0.1% Tween 20 overnight. Membranes were incubated with primary antibody (rabbit anti-Mieap antibody or rabbit anti-GST antibody) diluted in blocking buffer containing 0.06% Tween 20 (1:1000) for 3.5 h, and subsequently a secondary antibody (goat anti-rabbit antibody conjugated to horseradish-peroxidase) diluted in blocking buffer containing 0.06% Tween 20 (1:10000) for 1 h. ECL Western Blotting Detection Reagents (Cytiva) was used to detect HRP and chemiluminescence was visualized with an ImageQuant LAS 4000 system (Cytiva).
Lipid preparation
Lipid preparation was performed as described previously75,76. Briefly, total lipids were extracted from samples using the Bligh-Dyer method77. An aliquot of the organic phase was added to an equal volume of methanol before being loaded onto a DEAE-cellulose column (Wako Chemical) pre-equilibrated with chloroform. After successive washes with chloroform/methanol (1:1, v/v), acidic phospholipids were eluted with chloroform/methanol/HCl/water (12:12:1:1, v/v), followed by evaporation to dryness to yield a residue was soluble in methanol.
Mass spectrometric analyses of CL
Analyses were performed on an LC/MS/MS system consisting of a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) equipped with an electrospray ionization source and an UltiMate 3000 system (Thermo Fisher Scientific). Lipid samples were separated on a Waters X-Bridge C18 column (3.5 μm, 150 mm × 1.0 mm i.d.) at 40°C using a solvent step-gradient as follows: mobile phase A (isopropanol/methanol/water (5:1:4, v/v/v) supplemented with 5 mM ammonium formate and 0.05% ammonium hydroxide (28% in water))/mobile phase B (isopropanol supplemented with 5 mM ammonium formate and 0.05% ammonium hydroxide (28% in water)) ratios of 60%/40% (0 min), 40%/60% (1 min), 20%/80% (9 min), 5%/95% (11-30 min), 95%/5% (31-35 min) and 60%/40% (45 min). Flow rate was 25 μL/min. Source and ion transfer parameters applied were as follows. Spray voltage was 3.0 kV. For negative ionization modes, the sheath gas and capillary temperatures were maintained at 60 and 320 °C, respectively. The Orbitrap mass analyzer was operated at a resolving power of 70,000 in full-scan mode (scan range: 200–1800 m/z; automatic gain control (AGC) target:3e6) and of 35,000 in the Top 20 data-dependent MS2 mode (stepped normalized collision energy: 20, 30 and 40; isolation window: 4.0 m/z; AGC target: 1e5). Identification of CL molecular species was performed using LipidSearch 4.2 software (Mitsui Knowledge Industry).
Real-time ATP rate assay
LS174T-cont and Mieap-KD cells were seeded at a density of 2.5×104 cells/well (n=9) on a Seahorse XF24 Cell Culture Microplate. Cells were incubated at 37°C in a humidified chamber with 5% CO2. 18 h after seeding, culture medium was replaced with XF DMEM medium pH 7.4 supplemented with 25 mM glucose and 2 mM L-glutamine through three washes.
HCT116 cells were seeded at a density of 0.8×106 cells/60-mm dish (n=9). Cells were incubated at 37°C in a humidified chamber with 5% CO2. 24 h after seeding, cells were treated with Ad-Mieap or Ad-empty. 24 h after infection, cells were reseeded at a density of 4×104 cells/well (n=9) on a SeahorseXF24 Cell Culture Microplate. 20 h after reseeding, culture medium was replaced with XF DMEM medium pH 7.4 supplemented with 25 mM glucose and 2 mM L-glutamine through three washes.
After cells were incubated at 37 °C in a non-CO2 incubator for 60 min, cell culture plates were loaded into a Seahorse XFe24 Analyzer. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were recorded before and after serial injections of oligomycin and rotenone/antimycin A to yield final concentrations of 0.5 μM.
Flow cytometric analysis
LS174T-cont and Mieap-KD cells cultured under normal conditions were harvested by trypsin-EDTA treatment. After adding complete growth media to inactivate trypsin, cells were centrifuged, washed with PBS, and incubated with 5 μM 2′,7′-dichlorofluorescin-diacetate (Sigma) for 20 min at 37°C. After being washed with PBS, cells were immediately analyzed with an EC800 flow cytometry analyzer (Sony) using the 488-nm line.
Quantification and statistical analysis
FRAP data quantification
Fluorescence recovery rates were calculated using cellSens Imaging Software (Olympus), in which the intensity initially acquired after bleaching was set to 0 and the pre-bleaching intensity was set to 1. The normalized average fluorescence recovery was plotted in JMP 14.2.0 (SAS).
Crista data quantification
For quantification of crista data, crista area and outlines of mitochondrial sections in TEM images were marked manually using Adobe Photoshop CC, where normal crista morphology was identified by the presence of lamellar structures with distinct OsO4 staining. Aberrant crista-like structures that were not observed in mitochondria of WT were excluded. Subsequently, the ratio of crista area per mitochondrial section was calculated from the indicated number of mitochondria in legends of Fig. 8d, l, m, and 9f, using Image J78.
Statistical analysis
Statistical analyses were performed in JMP 14.2.0 (SAS). Levels of significance in Figure 6i, 8a, 8b, 8d, 8h–j, 8l, 8n, 9b–d, 9f, S10b–d, S10f–h were assessed using Student’s two-tailed t-tests. Levels of significance in mass spectrometric analyses for biological replicate pairs shown in Figure 5a, 5b, 8e, and 8f were assessed using the paired two-tailed t-test. p < 0.05 was considered statistically significant. Asterisks were allotted to all the Figures containing statistical analyses as follows: *, p < 0.05; **, p < 0.01, ***, p < 0.001, ****, p < 0.0001.
Data visualization
Visualization of the experimental data subjected to statistical analyses were performed using Graph Builder engine in JMP 14.2.0 (SAS). When the data were visualized using violin plots, box plots were overlaid. The center line in the box indicates the median. The bottom and top of the box indicate the 25th and 75th percentiles. The whiskers extend 1.5 times the interquartile range (IQR) from the top and bottom of the box unless the minimum and maximum values are within the IQR. The values which fall above or below the whiskers are plotted individually as outliers.
Data availability
The datasets generated during the mass spectrometric analyses of cardiolipin are included in Supplementary Data 1, and available in the Metabolomics Workbench repository, [https://www.metabolomicsworkbench.org/data/DRCCMetadata.php?Mode=Project&ProjectID=PR001192], under Project ID PR001192. All other relevant data which support the findings of this study are included in this article and its supplementary information files. Source data are provided with this paper.
Code availability
The codes used for data analysis are available from the corresponding author upon
a request.