The role of the mitochondrial outer membrane protein SLC25A46 in mitochondrial fission and fusion

Mitochondrial fragmentation in SLC25A46 knock-out cells and hyperfusion in pathogenic variants

To investigate the molecular basis for SLC25A46 pathogenicity, we first established a knock-out in a control immortalized human fibroblast cell line using CRISPR/Cas9 editing (Fig 1B). We then created stable cell lines expressing either WT SLC25A46 or one of the three different pathogenic variants (p.T142I, p.R257Q, and p.E335D) that span the range of phenotypic severity (Fig 1B). The expression of the individual SLC25A46 pathogenic variants did not reach WT levels, confirming a previous study showing a negative correlation between residual steady-state protein levels and the age of onset and severity of disease (Abrams et al, 2018) (Figs 1C and S1). The knock-out cell line had a proliferation defect that was rescued by the expression of the WT protein, but not by the expression of the pathogenic variants (Fig 1D).

We next examined mitochondrial morphology in these cell lines as previous studies reported that the loss of the SLC25A46 function led to mitochondrial hyperfusion (Abrams et al, 2015; Janer et al, 2016; Steffen et al, 2017). The complete loss of SLC25A46 in the knock-out resulted in a marked fragmentation of the mitochondrial network (Fig 2A and B). These findings were confirmed by creating a CRISPR/Cas9-induced SLC25A46 knock-out cell line in HeLa cells (Fig S2A), which contrasts with previous results of acute siRNA–mediated knock-down (Janer et al, 2016). Extensive mitochondrial hyperfusion was observed in cells expressing the pathogenic variants and in the patient fibroblast cell line (Fig 2A and B), as previously described (Janer et al, 2016).

To confirm and extend these findings, we quantified mitochondrial interconnectivity by expressing a photoactivatable GFP probe targeted to the mitochondrial matrix (OCT-PAGFP) (Patterson & Lippincott-Schwartz, 2002). As a validation for the method, we treated control fibroblasts with OPA1 siRNA or DRP1 siRNA, which, as expected, led to decreased or increased interconnectivity, respectively. Consistent with the morphological findings, the knock-out of SLC25A46 resulted in reduced mitochondrial interconnectivity and the expression of the pathogenic variants led to increased mitochondrial interconnectivity (Figs 2C and D and S2B). Transmission electron microscopy (TEM) demonstrated severely impaired mitochondrial cristae morphology in SLC25A46 knock-out cells and in cells expressing the pathogenic variants. Mitochondria were swollen, and cristae were either not present or not aligned (Fig 2E). The ultrastructural defects in the knock-out cells were rescued by re-expressing WT SLC25A46. Although the cristae architecture was highly compromised in the knock-out or pathogenic variant–expressing cells, surprisingly assembly of OXPHOS complexes as assessed by BN-PAGE was not compromised (Fig S2C).

SLC25A46 localizes to foci at mitochondrial tips and branches and is present at fusion and fission sites

The clear morphological changes in the mitochondrial network in the SLC25A46 knock-out cell and in cells with pathogenic variants suggest that SLC25A46 may play a role in both mitochondrial fusion and fission. To determine whether SLC25A46 is an active player in these processes, we examined the suborganellar localization of SLC25A46 by expressing SLC25A46-GFP in fibroblasts. Our previous study showed that the C-terminally GFP-tagged protein is functional, as it was able to rescue the cellular phenotypes in SLC25A46 patient fibroblasts (Janer et al, 2016). Immunofluorescence experiments showed that SLC25A46-GFP is focally expressed at most mitochondrial tips and branch points (80%) (Fig 3A and B). To ensure that the localization of SLC25A46-positive foci was not an effect of the overexpression of the protein with a GFP tag, we analyzed endogenous levels of SLC25A46 in fibroblasts by immunofluorescence with an anti-SLC25A46 antibody (Fig S3A and B). Endogenous SLC25A46 was also present in foci at nearly all mitochondrial tips (97%) and branches (94%). SLC25A46 was also focally expressed, often at the tips of mitochondria, in cerebral neurons differentiated from human induced pluripotent stem cells (Fig S3C). We were able to analyze the localization of the highest expressing pathogenic variant R257Q and confirmed that it is also focally present at mitochondrial tips and branches (Fig S3D and E).

We next used live-cell imaging to examine whether SLC25A46 was involved in mitochondrial fusion/fission events. This analysis demonstrated that SLC25A46 is present at virtually all mitochondrial fusion and fission sites (Fig 3C and D and Video 1, Video 2, and Video 3), suggesting an active role in these processes.

Association of SLC25A46 with the proteins of the fusion and fission machinery

Analysis of the protein proximity interaction network of SLC25A46 (Antonicka et al, 2020) indicated that SLC25A46 interacts with both the mitochondrial fusion and fission machinery (Fig 4A and Table S1) and with proteins involved in intracellular vesicular transport. Among the most specific proximity interactors of SLC25A46 compared with all other mitochondrial proteins investigated in the study (Antonicka et al, 2020) were proteins involved in vesicle budding from the membrane and proteins involved in organelle organization. We focused on the investigation of mitochondrial proximity interactors of SLC25A46. We performed immunoprecipitation of endogenous SLC25A46, followed by immunoblotting (Fig 4B). SLC25A46 co-immunoprecipitated with MFN1, MFN2, and OPA1, proteins of the mitochondrial fusion machinery, and one of the proteins of the MICOS complex (MIC25), in agreement with previously reported data (Janer et al, 2016). Reciprocal immunoprecipitation of the OPA1 protein was able to co-immunoprecipitate other proteins of the fusion machinery and SLC25A46, indicating the proximity of these two proteins in mitochondria. Immunofluorescence analysis showed that SLC25A46-positive punctae are in proximity to foci of not only the fusion protein OPA1, but also the fission protein DRP1 (DNM1L) (Fig 4C), a proximity interactor of SLC25A46 (Fig 4A). Stimulated emission depletion (STED) microscopy analysis further demonstrated that SLC25A46 surrounds the OPA1 punctae as expected (Fig 4D), as the proteins do not share the same membrane. These data indicate a close relationship between SLC25A46 and mitochondrial fusion and fission machinery.

MFN2 high molecular oligomeric complexes are decreased in SLC25A46 loss-of-function cells

As mitochondrial morphology is altered in SLC25A46 knock-out cells and in cells with pathogenic variants, we next investigated the fusion and fission machinery in these cells (Fig 5A and B). No substantive differences in the steady-state protein levels of MFN1/2, OPA1, or DRP1 were detected on conventional immunoblots, which contrasts with the findings by Steffen et al (2017) who showed an alteration in MFN1 and MFN2 protein expression in HEK293T SLC25A46 knock-out cells (Figs 5A and S4A). Steffen et al reported a reduced half-life of the pathogenic variant L341P, and we confirmed a reduction in the half-life of the three pathogenic variants we studied (Fig S4B and C). To test whether the pathogenic variants are inserted into the OMM, we performed a PK assay on the most abundant variant R257Q and an alkaline carbonate extraction assay on all pathogenic variants (T142I, R257Q, and E335D) (Fig S5A and B). The PK assay showed that the R257Q variant behaves as the WT protein and as the outer membrane protein MFN2 (Fig S5A). The alkaline carbonate assay showed that all pathogenic variants (T142I, R257Q, and E335D) are integral membrane proteins (Fig S5B).

The SLC25A46 homolog Ugo1p in yeast has been reported to be required for the complex assembly of the MFN1/2 homolog Fzo1p and the OPA1 homolog Mgm1p (Hoppins et al, 2009). As steady-state levels of these proteins were not affected in our model, we investigated whether the assembly of MFN1/2 and OPA1 complexes was changed in SLC25A46 knock-out cells and cells expressing the pathogenic variants. Isolated mitochondria were solubilized with 1% digitonin and separated on Blue-Native PAGE (BN-PAGE), and individual complexes were visualized by specific antibodies (Fig 5B). In control cells, MFN2 and OPA1 migrated predominantly as either oligomers or monomers, whereas MFN1 ran as a monomer (Fig 5B). In SLC25A46 knock-out cells and in the pathogenic variants, the complex formation of either MFN2 or OPA1, but not MFN1, was affected (Fig 5B). The oligomerization of MFN2 was dependent on the steady-state level of SLC25A46 expression (Fig 5C). The knock-out of SLC25A46 led to reduced MFN2 complex formation, which was rescued by the overexpression of either WT or variants. MFN2 oligomerization correlated with the expression level of SLC25A46 (Fig 5C), suggesting that the steady-state level of the SLC25A46 protein is an important determinant of the underlying defect.

OPA1 high molecular weight oligomeric complexes are increased, and their forms are altered in SLC25A46 loss-of-function cells

OPA1 has multiple isoforms, which can be further processed by the two proteases OMA1 and YME1L1. The long, membrane-bound forms are required for the mitochondrial inner membrane fusion, whereas the short, soluble forms are associated with inhibiting fusion and promoting fission (MacVicar & Langer, 2016). BN-PAGE analysis showed a slight increase in OPA1 high molecular weight complex formation in the SLC25A46 knock-out cell line compared with control fibroblasts (Fig 5B, green and blue box). Because BN-PAGE is unable to distinguish between the individual isoforms, we performed a two-dimensional PAGE (BN-PAGE/SDS–PAGE) analysis to separate the long and short forms (Fig 6A). In control cells, both long and short forms of OPA1 were present as monomers (Fig 6A, red box), whereas long forms were predominantly found in the higher molecular weight complex (∼200–400 kD, blue box). In the knock-out of SLC25A46, the long and short forms were also present as monomers and in a 200- to 400-kD complex (Fig 6A, blue box), but to a lesser extent. Interestingly, OPA1 was also present in even larger complexes (>600 kD), where the short forms were more prominent (Fig 6A [green box], Fig 6B–D). Cells re-expressing the WT form of SLC25A46 present a similar OPA1 distribution pattern to the control cells, demonstrating the specificity of this phenotype. A similar pattern for OPA1 complex formation as in the KO cell line (the presence of the >600-kD complex) was detected in the cell line expressing the pathogenic variant p.T142I; however, the long OPA1 forms were predominantly present in the high molecular weight complexes (Fig 6A, green box and blue box, Fig 6B–D). Thus, the presence of short forms in the knock-out and long forms in the pathogenic variant p.T142I in >600-kD complexes (green box) correlates with the morphology observed in these cells, fragmentation versus hyperfusion (Fig 2A). 2D gels also confirmed our observation from BN-PAGE that oligomers of MFN2 are absent in KO and p.T142I cells.

MFN2 oligomerization is influenced by SLC25A46 and OPA1

As SLC25A46 plays a role in the formation of MFN2 and OPA1 oligomers, we investigated the consequence of MFN2 or OPA1 knock-down in the cells lacking SLC25A46 (Fig 7A). Knock-down of OPA1 resulted in an increase of MFN2 steady-state levels in both the control and SLC25A46 knock-out cells (Fig 7A, SDS–PAGE), and this increase was sufficient to rescue the level of MFN2 complex formation in the SLC25A46 knock-out cell line, suggesting that the oligomeric complex of MFN2 is a compensatory response to the loss of OPA1 function. This was not, however, sufficient to alter mitochondrial morphology, as OPA1 knock-down resulted in mitochondrial fragmentation in both control and KO cells (Fig 7B). As expected, we detected a disassembly of OXPHOS complex V when OPA1 was silenced, because complex V requires adequate cristae morphology and therefore is dependent on OPA1 (Cogliati et al, 2013). MFN2 knock-down induced the disassembly of complex V in the absence of SLC25A46 (Fig 7A) and a concomitant fragmentation of the mitochondrial network (Fig 7B). The effect of the MFN2 knock-down in the SLC25A46 knock-out cells compared with control fibroblasts (Fig 7B) indicates a clear dependence of mitochondrial morphology on the presence of SLC25A46.

SLC25A46-dependent changes in the mitochondrial lipid profile

The oligomerization of GTPases and the curvature of the membranes during fusion and fission events are highly dependent on the lipid content of the mitochondrial membrane (Low et al, 2009; Frohman, 2015; Brandt et al, 2016; Brandner et al, 2019). Our previous results showed an alteration in mitochondrial lipid content in a Leigh syndrome patient with the p.T142I mutation (Janer et al, 2016). To test whether the alteration in mitochondrial morphology, cristae structure, and oligomerization of MFN2 and OPA1 are due to an altered lipid content, we performed shotgun mass spectrometry lipidomics on sucrose bilayer purified mitochondria from the control, knock-out, WT-rescue, and the pathogenic variant p.T142I, allowing broad coverage of lipids and absolute quantification (Lipotype GmbH).

From 1,798 identified lipid entities, we found 203 lipids that were significantly different (0.05%) with a fold change of >2 Table S2). Using multi-variant data analysis by principal component analysis, we showed the clustering of the samples of the same cohorts, demonstrating the quality of the data, and the separation of the different cohorts, suggesting a different mitochondrial lipid content (Fig 8A). Hierarchical clustering with heatmap analysis of lipid classes showed a clear difference in quantities of most classes of lipids between control and knock-out mitochondria, which was rescued by the expression of the WT variant but not by the expression of the pathogenic variant T142I (Figs 8B and C and S6 and Table S2). The knock-out of SLC25A46 resulted in a decrease in the three main mitochondrial lipid categories, cardiolipin (CL), phosphatidylethanolamine (PE), and phosphatidylinositol (PI), and an increase in phosphatidylcholine (PC) (Figs 8B and C and S6). Although PC increased in the SLC25A46 knock-out cell line, the ether-linked PC (PC O-) was decreased. In contrast, the decrease in PE was accompanied by an increase in ether-linked PE (PE O-) (Figs 8B and C and S6 and Table S2). CL was overall decreased in the knock-out cell line compared with control fibroblasts, and more specifically, we observed individual changes, some subclasses were decreased (CL 68:4; 0, CL 70:4;0, and CL 72:5;0), whereas others were increased in the knock-out (CL 72:6;0 and CL 72:7;0) (Fig 8D and Table S2). All alterations in the lipid profiles of the knock-out cells were rescued by the expression of the WT protein but not the pathogenic variant (Figs 8 and S6 and Table S2). Interestingly, the levels of 17 different lipid subspecies were significantly different between the knock-out cells and cells expressing the p.T142I pathogenic variant (Fig S6B). These data indicate a close relationship between the function of SLC25A46, mitochondrial lipid content, cristae formation, and mitochondrial morphology mediated through the oligomerization of MFN2 and OPA1.

Cell lines

Fibroblasts were obtained from a cell bank located in the Montreal Children’s Hospital, and the cell line we used was from a female healthy subject, 58 yr old. The cells were immortalized as described previously (Lochmuller et al, 1999). Fibroblasts, the Phoenix packaging cell line, and Flp-In T-REx 293 cells were cultured in high-glucose DMEM (Wisent 319-027-CL) supplemented with 10% FBS, at 37°C in an atmosphere of 5% CO₂. All cell lines were regularly tested for mycoplasma contamination.

CRISPR/Cas9 SLC25A46 knock-out cell line generation

The sgRNA oligomers for SLC25A46 targeting exons 1 and 3 were annealed and inserted into the plasmid pSpCas9(BB)-2A-Puro (PX459) V2.0 from Addgene (62988) via combined ligation (T7 ligase; NEB).

Sequence for sgRNA targeting exon 1 of SLC25A46:

5′-CTCCGTGGGGCCTTCGTACG GGG-3′

Sequence for sgRNA targeting exon 3 of SLC25A46:

5′-GGCGGCGTAGAACAATGCA AGG-3′

Both plasmids (1.5 μg) were transfected into a fibroblast cell line with Lipofectamine 3000 (Thermo Fisher Scientific) reagent according to the manufacturer’s instructions. The day after, transfected cells were selected by addition of puromycin (2.5 μg/ml) for 2 d. Clonal cells were screened for the loss of target protein by immunoblotting, and frameshift mutations were confirmed by genomic sequencing.

Plasmid generation

SLC25A46 was amplified through PCR with Taq polymerase with 7-deaza-GTP/nucleotide mix (NEB) using cDNA from control fibroblasts as a template and cloned into Gateway-modified pBABE-Puro using Gateway Cloning Technology (Invitrogen). The SLC25A46-GFP plasmid was generated by cloning SLC25A46 into a pDEST-pcDNA5-GFP vector. The three pathogenic variants were created using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies) with primers:

p.T142I: c.C425T 5′-cagcattaccatctcactccatttatagtcatcaatattatgtaca-3′

p.R257Q: c.G770A 5′-gagtgcctcatagcaaacaacttcttccgcttctttc-3′

p.E335D: c.A1005T 5′-cgttatactttacccattggatacagttttgcaccgcc-3′

pOCT-PAGFP was previously described in Nagashima et al (2020) and obtained from Addgene. The vector was cloned into a pcDNA-pDEST47 Gateway destination vector (Invitrogen) and flipped into a retroviral expression vector pLXSH.

The sequence of all plasmids was verified by Sanger sequencing (IRIC).

Generating overexpression cell lines

Retroviral constructs were transfected into the Phoenix packaging cell line using the HBS/Ca₃(PO₄)₂ method. Control fibroblasts or SLC25A46 knock-out cells were infected 48 h later by exposure to a virus-containing medium in the presence of 4 μg/ml polybrene as described previously (Pear et al, 1997). Finally, antibiotic selection began 2–3 d later with 4 μg/ml of puromycin or 100 U/ml of hygromycin for the pBABE vector or pLXSH vector-infected cells, respectively.

Neuronal cell culture

Neurons derived from induced pluripotent stem cells were kindly provided by Thomas Durcan’s Lab (EDDU, The Neuro). Immunofluorescence analysis of cortical neurons was conducted on day 14.

siRNA transfection

Stealth RNAi duplex constructs (Invitrogen) were used for transient knock-down of SLC25A46 (S40836; Ambion), and ON-TARGETplus siRNAs were used for OPA1, MFN2, and DRP1 (L-005273, L-012961, and L-012092; Dharmacon) knock-down in fibroblasts. siRNAs were transiently transfected into cells using Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s specifications. The transfection was repeated on day 3, and the cells were harvested on day 6 for immunofluorescence and immunoblot analyses.

Preparation of mitochondria-enriched heavy membranes

Mitochondria-enriched heavy membrane fractions were acquired from two 90% confluent 15-cm plates. The plates were washed twice with PBS, and cells were collected and resuspended in HIM buffer (0.2 M mannitol, 0.07 M sucrose, 0.01 M Hepes, and 1 mM EGTA, pH 7.5) and homogenized with 10 passes of a pre-chilled, zero-clearance homogenizer (Kimble/Kontes). The post-nuclear supernatant was obtained by centrifugation of the samples twice for 10 min at 600g. The heavy membrane fraction was pelleted by centrifugation for 10 min at 10,000g and washed once in HIM buffer. The protein concentration was measured by the Bradford assay.

Alkaline carbonate extraction and proteinase K protection assays

To determine the suborganellar location of the pathogenic variants of SLC25A46, mitochondria were extracted with 100 mM alkaline carbonate at pH 11.5 as previously described (Janer et al, 2016) and the input, pellet, and supernatant were analyzed by SDS–PAGE. To determine the localization of the pathogenic variant R257Q at the membrane, 100 μg of freshly prepared crude mitochondria were incubated with an increasing concentration of proteinase K diluted in the isolation buffer, for 20 min on ice. The reaction was stopped by addition of PMSF (2 mM final), and the cells were incubated on ice for 20 min. Mitochondria were then pelleted at 10,000g for 10 min, resuspended in Laemmli buffer, and analyzed by SDS–PAGE.

Denaturing, native, and second-dimension PAGE

For SDS–PAGE, cells were extracted with 1.5% n-dodecyl-D-maltoside (DDM) in PBS and centrifuged at 20,000g for 20 min, after which 20 μg of protein was run on polyacrylamide gels.

BN-PAGE was used to separate individual protein complexes. Each sample was obtained from 100 μg mitochondria-enriched heavy membrane fractions. Mitochondria were resuspended in 40 μl MB2 buffer (1.75 M aminocaproic acid, 75 mM Bis–Tris, and 2 mM EDTA) and solubilized with 4 μl 10% digitonin solution (4 g digitonin/g protein) for 20 min on ice with reciprocal centrifugation for 20 min at 20,000g. 15 μg of supernatants was separated on a 6–15% polyacrylamide gradient gel as previously described (Leary, 2012), with an Amersham HMW native marker kit (17044501; GE Healthcare) as a molecular weight marker, and blotted onto a polyvinylidene difluoride membrane.

For the second-dimension analysis, BN-PAGE/SDS–PAGE was carried out as detailed previously (Antonicka et al, 2003). Separated proteins were transferred to a nitrocellulose membrane, and immunoblot analysis was performed with the indicated antibodies.

Immunofluorescence

For immunofluorescence analysis, cells were fixed in 4% formaldehyde in PBS at 37°C for 20 min, then washed three times with PBS, followed by permeabilization in 0.1% Triton X-100 in PBS and three washes in PBS. The cells were then blocked with 4% FBS in PBS, followed by incubation with primary antibodies in 4% FBS in PBS, for 1 h at RT. After three washes with PBS, cells were incubated with DAPI and the appropriate anti-species secondary antibodies coupled to Alexa fluorochromes (Invitrogen) (1:3,000) for confocal microscopy or with Abberior STAR antibodies (1:1,000) for STED microscopy for 1 h at RT. After three washes in PBS, coverslips were mounted onto slides using fluorescence mounting medium (Dako).

Confocal imaging and STED super-resolution microscopy

For confocal microscopy, stained cells were imaged using 100× objective lenses (NA1.4) on an Olympus IX81 inverted microscope with appropriate lasers using an Andor/Yokogawa spinning disk system (CSU-X), with a sCMOS camera. The laser power of 10 (for, i.e., anti-PRDX3, MitoTracker, and anti-OPA1) or 20% (anti-SLC25A46 and SLC25A46-GFP) with a dwell time of 100–500 μs was used, depending on the strength of the antibody. Mitochondrial network morphology (Fig 2) was classified in a blinded manner as fused, intermediate, or fragmented. For each condition, more than 60 cells were analyzed. Experiments were done three times independently. Errors bars represent the mean ± SEM, and P-values were calculated using a t test. The fluorescence of PAGFP-expressing cells was stimulated by excitation with the 405 nm laser of a ∼1-μm² square close to the nucleus. An image was taken 3 min after stimulation. The PAGFP-positive area of OCT-PAGFP–infected cells was obtained using the Fiji (Schindelin et al, 2012) macro, which automatically sets the threshold and uses the “Analyze particles” plugin in Fiji. At least 20 cells per condition were analyzed.

For live-cell imaging analysis, cells were incubated with MitoTracker Deep Red FM (Thermo Fisher Scientific) for 15 min before imaging and washed three times. Time-lapse videos were acquired over the course of 1 min with each channel captured every 0.5 s.

STED images were obtained with an Abberior Expert Line STED microscope with two pulsed STED lasers (595 and 775 nm) based on an Olympus IX83 inverted microscope with an Olympus Plan-Apo ×100/1.40 NA oil objective and a pixel size of 20 nm using ImSpector software. The laser power of 90% was used for confocal lasers, and the laser power of 100% was used for the STED laser with dwell times of 5 and 20 μs, respectively.

TEM

The TEM was performed at the Facility for Electron Microscopy Research of McGill University. Cells were washed in 0.1 M Na-cacodylate washing buffer (Electron Microscopy Sciences) and fixed in 2.5% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M Na-cacodylate buffer overnight at 4°C. Cells were washed three times in 0.1 M Na-cacodylate washing buffer for a total of 1 h, incubated in 1% osmium tetroxide (Mecalab) for 1 h at 4°C, and washed with ddH2O three times for 10 min. Then, dehydration in a graded series of ethanol/deionized water solutions from 30 to 90% for 8 min each, and 100% twice for 10 min each, was performed. The cells were then infiltrated with a 1:1 and 3:1 Epon 812 (Mecalab):ethanol mixture, each for 30 min, followed by 100% Epon 812 for 1 h. Cells were embedded in the culture wells with new 100% Epon 812 and polymerized overnight in an oven at 60°C. Polymerized blocks were trimmed, and 15–20 100-nm ultrathin sections per sample were cut with an UltraCut E ultramicrotome (Reichert Jung) and transferred onto 200-mesh Cu grids (Electron Microscopy Sciences). Sections were post-stained for 8 min with 4% aqueous uranyl acetate (Electron Microscopy Sciences) and 5 min with Reynold’s lead citrate (Thermo Fisher Scientific). The samples were imaged with a FEI Tecnai-12 transmission electron microscope (FEI Company) operating at an accelerating voltage of 120 kV equipped with an XR-80C AMT, 8-megapixel CCD camera.

Immunoprecipitation

Before extraction, mitochondria-enriched heavy membrane fractions (200 μg) from control fibroblasts were chemically cross-linked with 1 mM dithiobis-sulfosuccinimidyl propionate (DSP) (Sigma-Aldrich) in HIM buffer, for 2 h on ice. The reaction was stopped by adding glycine, pH 8.0, at 70 mM final concentration for 10 min on ice. Mitochondria were pelleted, rinsed once, and extracted in 200 μl of lysis buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% n-dodecyl-D-maltoside [DDM] [Sigma-Aldrich], and complete protease inhibitors [Roche]) on ice for 30 min. The extract was centrifuged at 20,000g at 4°C for 20 min, and the supernatant was pre-cleared overnight with non-coated Dynabeads Protein A (Invitrogen) to reduce non-specific protein binding to the beads. Binding of indicated antibodies to Dynabeads Protein A (Invitrogen) was performed overnight. Antibodies were then cross-linked to the beads using 20 mM dimethyl pimelimidate (DMP) (Sigma-Aldrich). The immunoprecipitation reaction was performed overnight at 4°C. Beads were washed with lysis buffer, and samples were eluted using 0.1 M glycine, pH 2.5/0.5% DDM, precipitated with trichloroacetic acid, and immunoblotted as indicated above.

Proliferation assay

20,000 cells were seeded in a 24-culture dish at day 0. After 1, 2, 3, and 4 d of culture, cells were trypsinized, homogenized, and counted using a Neubauer counting chamber. Experiments were done in independent triplicates. Errors bars represent the mean ± SEM, and P-values were calculated using two-way ANOVA.

Half-life measurement

Flp-In T-REx 293 cells expressing SLC25A46 or the pathogenic variants with a BirA* tag were created as described earlier (Antonicka et al, 2020). The expression of the construct was induced by addition of 1 μg/ml tetracycline (Sigma-Aldrich) for 24 h. The cells were washed and incubated with medium without tetracycline. The cells were harvested after 0, 6, 9, 12, 15, 18, and 24 h, extracted in 1.5% DDM/PBS, and centrifuged at 20,000g for 20 min, after which 20 μg of protein was run on polyacrylamide gels. The SDS–PAGE was quantified using ImageJ, using the endogenous SLC25A46 expression as a loading control. As the intensity of the signal for BirA*-tagged proteins increased up to 6 h, most likely because of the remaining tetracycline within the cells, the signal at 6 h post-removal of tetracycline was considered as the maximal synthesis rate and was set as time 0 for the half-life measurement. The intensity of the normalized SLC25A46-BirA* signal at this time point was set to 1, to which the signal at the following time points was compared with.

BioID analysis

BioID data published in Antonicka et al (2020) were used for the determination of the specificity of the SLC25A46 proximity interactome. The data were loaded into ProHits-viz software (Knight et al, 2017), and a specificity analysis tool was used (BFDR < 0.01; prey-length–normalized average spectral count was used as an abundance measure). The functional enrichment analysis was performed using g:Profiler (version e106_eg53_p16_65fcd97) with the g:SCS multiple testing correction method with a significance threshold of 0.05 (Raudvere et al, 2019) as part of the ProHits-viz analysis platform (Knight et al, 2017).

Antibodies

Antibodies directed against the following proteins were used in this study: SLC25A46 (G2-SC-515823; Santa Cruz), MFN2 (11925S; Cell Signaling), MFN1 (14739S; Cell Signaling), OPA1 (67589S; Cell Signaling) for IF, OPA1 (612607; BD Bioscience) for WB, DRP1/DLP1 (611113; BD Bioscience), VDAC1 (ab14734; Abcam), CHCHD3/MIC19 (ARP57040-P050; Aviva), CHCHD6/MIC25 (20638-1AP; Proteintech), PRDX3 (in-house), NDUFA9 (ab14713; Abcam), ATP5A1 (ab14748; Abcam), Core1 (ab110252; Abcam), COX4I1 (in-house), and SDHA (ab14715; Abcam); the secondary antibodies used in this study were as follows: anti-rabbit Alexa Fluor 488 (A-21206; Invitrogen), anti-mouse Alexa Fluor 647 (A-31571; Invitrogen), anti-rat Alexa Fluor 594 (A-11007; Invitrogen), Abberior STAR 580 goat anti-rabbit IgG (ST580-1002; STED), and Abberior STAR 635P goat (2-0002-007-5; Abberior GmbH).

Mitochondrial isolation for lipidomics

To obtain at least 100 μg of isolated mitochondria from fibroblasts, 4 × 15 cm plates with 90% confluency were grown for each sample. The plates were rinsed twice with PBS, resuspended in ice-cold 250 mM sucrose/10 mM Tris–HCl (pH 7.4), and homogenized with 10 passes of a pre-chilled, zero-clearance homogenizer (Kimble/Kontes). A post-nuclear supernatant was obtained by centrifugation of the samples twice for 10 min at 600g. The heavy membrane fraction was pelleted by centrifugation for 10 min at 10,000g and washed once in the same buffer. The resuspended membranes were loaded on a 1.7 and 1 M sucrose bilayer and centrifuged in an ultracentrifuge at 75,000g for 30 min. The layer between the 1.7 and 1 M sucrose layers was collected and washed in the 250 mM sucrose/10 mM Tris–HCl buffer. The protein concentration was determined by the Bradford assay.

Lipidomics analysis by the Lipotype Shotgun Lipidomics technology

Purified mitochondria were used for lipidomics analysis by the Lipotype Shotgun Lipidomics platform. The samples were extracted and analyzed through high-resolution Orbitrap mass spectrometry and quantified and identified by Lipotype’s in-house software LipotypeXplorer.

Statistical analysis

All data are reported as means ± SEM or means ± SD as indicated in the figure legend. Statistical significance was determined using the indicated tests. P-values < 0.05 were considered statistically significant and labeled as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.