TMBIM5 is the Ca ²⁺ /H ⁺ antiporter of mammalian mitochondria

Article2 November 2022Open Access Source DataTransparent process TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria Shane Austin Shane Austin orcid.org/0000-0002-8698-6055 Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Contribution: Conceptualization, Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Ronald Mekis Ronald Mekis Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Sami E M Mohammed Sami E M Mohammed orcid.org/0000-0001-8129-030X Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Mariafrancesca Scalise Mariafrancesca Scalise orcid.org/0000-0003-3860-6844 Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Wen-An Wang Wen-An Wang orcid.org/0000-0003-3871-0174 Department of Cell Physiology & Metabolism, University of Geneva, Geneva, Switzerland Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Michele Galluccio Michele Galluccio Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy Contribution: ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Christina Pfeiffer Christina Pfeiffer Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Contribution: ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Tamara Borovec Tamara Borovec Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: ​Investigation, Methodology Search for more papers by this author Katja Parapatics Katja Parapatics CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Methodology Search for more papers by this author Dijana Vitko Dijana Vitko CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Methodology Search for more papers by this author Nora Dinhopl Nora Dinhopl Department of Pathobiology, Institute of Pathology, University of Veterinary Medicine, Vienna, Austria Contribution: Visualization, Methodology, Writing - review & editing Search for more papers by this author Nicolas Demaurex Nicolas Demaurex orcid.org/0000-0002-9933-6772 Department of Cell Physiology & Metabolism, University of Geneva, Geneva, Switzerland Contribution: Conceptualization, Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Keiryn L Bennett Keiryn L Bennett CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Conceptualization, Data curation, Formal analysis, Methodology, Writing - review & editing Search for more papers by this author Cesare Indiveri Cesare Indiveri orcid.org/0000-0001-9818-6621 Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Bari, Italy Contribution: Conceptualization, Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Karin Nowikovsky Corresponding Author Karin Nowikovsky [email protected] orcid.org/0000-0001-8435-8410 Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: Conceptualization, Resources, Data curation, Supervision, Funding acquisition, ​Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Shane Austin Shane Austin orcid.org/0000-0002-8698-6055 Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Contribution: Conceptualization, Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Ronald Mekis Ronald Mekis Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Sami E M Mohammed Sami E M Mohammed orcid.org/0000-0001-8129-030X Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Mariafrancesca Scalise Mariafrancesca Scalise orcid.org/0000-0003-3860-6844 Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Wen-An Wang Wen-An Wang orcid.org/0000-0003-3871-0174 Department of Cell Physiology & Metabolism, University of Geneva, Geneva, Switzerland Contribution: Data curation, Formal analysis, Validation, ​Investigation, Visualization, Methodology, Writing - review & editing Search for more papers by this author Michele Galluccio Michele Galluccio Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy Contribution: ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Christina Pfeiffer Christina Pfeiffer Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Contribution: ​Investigation, Methodology, Writing - review & editing Search for more papers by this author Tamara Borovec Tamara Borovec Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: ​Investigation, Methodology Search for more papers by this author Katja Parapatics Katja Parapatics CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Methodology Search for more papers by this author Dijana Vitko Dijana Vitko CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Methodology Search for more papers by this author Nora Dinhopl Nora Dinhopl Department of Pathobiology, Institute of Pathology, University of Veterinary Medicine, Vienna, Austria Contribution: Visualization, Methodology, Writing - review & editing Search for more papers by this author Nicolas Demaurex Nicolas Demaurex orcid.org/0000-0002-9933-6772 Department of Cell Physiology & Metabolism, University of Geneva, Geneva, Switzerland Contribution: Conceptualization, Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Keiryn L Bennett Keiryn L Bennett CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Contribution: Conceptualization, Data curation, Formal analysis, Methodology, Writing - review & editing Search for more papers by this author Cesare Indiveri Cesare Indiveri orcid.org/0000-0001-9818-6621 Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Bari, Italy Contribution: Conceptualization, Data curation, Writing - original draft, Writing - review & editing Search for more papers by this author Karin Nowikovsky Corresponding Author Karin Nowikovsky [email protected] orcid.org/0000-0001-8435-8410 Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria Contribution: Conceptualization, Resources, Data curation, Supervision, Funding acquisition, ​Investigation, Methodology, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Shane Austin1,8,†, Ronald Mekis1,2,†, Sami E M Mohammed2,†, Mariafrancesca Scalise3, Wen-An Wang4, Michele Galluccio3, Christina Pfeiffer1, Tamara Borovec1,2, Katja Parapatics5, Dijana Vitko5, Nora Dinhopl6, Nicolas Demaurex4, Keiryn L Bennett5, Cesare Indiveri3,7 and Karin Nowikovsky *,1,2 1Department of Internal Medicine I and Comprehensive Cancer Center, Medical University of Vienna, Vienna, Austria 2Department of Biomedical Sciences, Institute of Physiology, Pathophysiology and Biophysics, University of Veterinary Medicine Vienna, Vienna, Austria 3Department DiBEST (Biologia, Ecologia, Scienze della Terra) Unit of Biochemistry and Molecular Biotechnology, University of Calabria, Arcavacata di Rende, Italy 4Department of Cell Physiology & Metabolism, University of Geneva, Geneva, Switzerland 5CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria 6Department of Pathobiology, Institute of Pathology, University of Veterinary Medicine, Vienna, Austria 7CNR Institute of Biomembranes, Bioenergetics and Molecular Biotechnologies (IBIOM), Bari, Italy 8Present address: Department of Biological & Chemical Sciences, The University of the West Indies, Cave Hill Campus, Cave Hill, Barbados † These authors contributed equally to this work *Corresponding author. Tel: +43 125077/4573; E-mail: [email protected] EMBO Reports (2022)23:e54978https://doi.org/10.15252/embr.202254978 PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial Ca2+ ions are crucial regulators of bioenergetics and cell death pathways. Mitochondrial Ca2+ content and cytosolic Ca2+ homeostasis strictly depend on Ca2+ transporters. In recent decades, the major players responsible for mitochondrial Ca2+ uptake and release have been identified, except the mitochondrial Ca2+/H+ exchanger (CHE). Originally identified as the mitochondrial K+/H+ exchanger, LETM1 was also considered as a candidate for the mitochondrial CHE. Defining the mitochondrial interactome of LETM1, we identify TMBIM5/MICS1, the only mitochondrial member of the TMBIM family, and validate the physical interaction of TMBIM5 and LETM1. Cell-based and cell-free biochemical assays demonstrate the absence or greatly reduced Na+-independent mitochondrial Ca2+ release in TMBIM5 knockout or pH-sensing site mutants, respectively, and pH-dependent Ca2+ transport by recombinant TMBIM5. Taken together, we demonstrate that TMBIM5, but not LETM1, is the long-sought mitochondrial CHE, involved in setting and regulating the mitochondrial proton gradient. This finding provides the final piece of the puzzle of mitochondrial Ca2+ transporters and opens the door to exploring its importance in health and disease, and to developing drugs modulating Ca2+ exchange. Synopsis TMBIM5 mediates mitochondrial Ca2+/H+ exchange and interacts with the K+/H+ exchanger LETM1 to maintain mitochondrial pH, Ca2+ and K+ homeostasis. The K+/H+ exchanger LETM1 was proposed to mediate mitochondrial Ca2+/H+ exchange. Transmembrane BAX Inhibitor Motif containing protein 5 (TMBIM5) interacts with LETM1 and maintains mitochondrial K+/H+ exchange. TMBIM5, but not LETM1, mediates pH-dependent mitochondrial Ca2+ extrusion. TMBIM5 regulates cell metabolism by setting the mitochondrial proton gradient. Introduction Ion homeostasis is critical for mitochondrial function. The dynamic balance of cations is achieved by a set of integrated transport systems for K+, Na+, and Ca2+. Loss of this balance between cation uptake and release has consequences for the organelle and ultimately the cell, and includes mitochondrial swelling, disrupted cristae structure, deregulated bioenergetics, and may result in cell death. Intracellularly, mitochondria are major sinks of Ca2+, an ion of comparatively low concentration to K+ and Na+. The role of mitochondrial Ca2+ buffering has been extensively studied (Giorgi et al, 2018; Pallafacchina et al, 2018), yet some of the players in maintaining Ca2+ balance have not been identified (De Stefani et al, 2016; Urbani et al, 2020). One of the missing pieces in this molecular puzzle is the Na+-independent Ca2+ efflux pathway, a putative Ca2+/H+ exchanger (CHE). This exchanger, whose existence has been postulated since the 1970s (Carafoli et al, 1974) is critical for maintaining mitochondrial Ca2+ levels and pH homeostasis. One of the CHE candidate proteins is LETM1. LETM1, a single transmembrane domain-containing protein, has initially been characterized as the mitochondrial K+/H+ exchanger (KHE) (Nowikovsky et al, 2004, 2007; Hasegawa & van der Bliek, 2007; McQuibban et al, 2010; Hashimi et al, 2013). The proposal that LETM1 could also be a CHE was based on a Drosophila S2 genome-wide RNAi screen of modulators of mitochondrial Ca2+ transport (Jiang et al, 2009). Subsequent studies have confirmed an involvement of LETM1 in Ca2+ and K+ transport, but key questions remained, perhaps the most important being how a single transmembrane protein can mediate a process of ion exchange (Nowikovsky & Bernardi, 2014; Austin & Nowikovsky, 2019, 2021). It appeared possible that LETM1 acts as a multimer, or as part of a protein complex. The first possibility is supported by cryo-EM structures of LETM1 oligomers, which facilitated pH-dependent Ca2+-movement in a cell-free system (Shao et al, 2016). Whether LETM1 is part of a protein complex remains unaddressed. Here, we searched for partners of LETM1 and found the interactor Transmembrane BAX Inhibitor Motif containing protein 5 (TMBIM5), also called Mitochondrial Morphology and Cristae Structure 1 (MICS1), a member of the TMBIM family, which has been implicated in the regulation of intracellular Ca2+ by a number of studies (Hung et al, 2011; Carrara et al, 2012; Lisak et al, 2015; Rojas-Rivera & Hetz, 2015; Liu, 2017; Kim et al, 2021). TMBIM5/MICS1 is the only species with a mitochondrial localization (Oka et al, 2008) while other TMBIM family members are localized to the ER, Golgi, and plasma membrane (Rojas-Rivera & Hetz, 2015). Importantly, TMBIM5 was reported as a regulator of Ca2+ and apoptosis (Oka et al, 2008; Lisak et al, 2015). Here, we demonstrate that TMBIM5 is the long-sought mitochondrial CHE, a crucial component of mitochondrial Ca2+ homeostasis. Results TMBIM5/MICS1 interacts with LETM1 To determine the interactome of LETM1, we generated HEK293 cell lines with inducible expression of LETM1 fused to hemagglutinin and streptavidin (SH) as originally described in Glatter et al (2009) and Rudashevskaya et al (2013). We applied a powerful two-step tandem affinity purification (TAP) approach to identify high-confidence interaction partners of LETM1-SH from whole cell lysates and one-step affinity purification (AP) coupled with mass spectrometry (MS) from isolated mitochondria (Fig 1A). In the latter approach, only the streptavidin component of the tag was utilized and the protein complex eluted with biotin. As few studies have used the limited amounts of material from isolated organelles for AP-MS, we first assessed the reliability of our one-step method to investigate organellar interactomes. As a benchmark, the inner mitochondrial membrane protein mitochondrial Ca2+ uniporter (MCU) was fused to SH. The members of the published core interactome were identified except for the tertiary interactor MICU2 (Sancak et al, 2013; Fig 1B and Appendix Fig S1A). Thus, the method was sufficiently robust to cover approximately 75% of the MCU mitochondrial core interactome and therefore likely to detect other mitochondrial interactomes with similar accuracy. Next, we determined the LETM1 interactome using TAP- and AP-MS from both whole cells and isolated mitochondria (Dataset EV1), obtaining 31 overlapping proteins (Appendix Fig S1B), including TBK1, a protein previously observed to interact with LETM1 in similar AP-MS studies (Li et al, 2011). We compared these 31 proteins to nonspecific interactors from similar AP-MS experiments using the data from the CRAPome (Mellacheruvu et al, 2013b) and were able to identify 12 high-confidence interactors with scores greater than 0.95 (Fig 1C and Appendix Fig S1C). Figure 1. LETM1 and TMBIM5 interact Scheme illustrating workflow for miniaturized AP-MS experiments, left to right: whole cells or isolated mitochondria were lysed or solubilized, respectively. The cell/mitochondrial lysates were used for affinity purification (AP) using the STREP tag and tandem affinity purification (TAP) using STREP and HA tag found on the bait protein. Eluates of the AP and control experiments were reduced, alkylated, and digested by trypsin. Peptides are purified on a C18 stage tip and then run on an LTQ Orbitrap Velos. Protein identifications were made by internal tools using MASCOT and Phenyx and the removal of nonspecific interactors done using the CRAPome. Created with Biorender.com. MCU was selected as a model protein, the functional complex consists of the five proteins above (MCU, MCUb, MICU1, MICU2, EMRE). Note that an additional tissue-specific tertiary interaction partner (MICU3) is only expressed at very low levels in HEK293 cells (Diego De Stefani, personal communication). Illustration adapted from Sancak et al (2013). All high-confidence interaction partners of LETM1 are shown as nodes. Node color indicates SAINT score, a probability-based measure of interaction confidence. See also Appendix Fig S1A–C. Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane protein complexes. The middle and right panel of the co-IPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN–PAGE conducted in TMBIM5WT and KO. Source data are available online for this figure. Source Data for Figure 1 [embr202254978-sup-0005-SDataFig1.pptx] Download figure Download PowerPoint Of immediate interest was TMBIM5, an inner mitochondrial membrane protein with 8 predicted transmembrane helices (https://alphafold.ebi.ac.uk/entry/Q9H3K2). Similar to LETM1, TMBIM5 is involved in the regulation of mitochondrial structure (Oka et al, 2008; Seitaj et al, 2020). The interaction of TMBIM5 and LETM1 was confirmed with co-IP and reverse co-IP experiments with TMBIM5 and LETM1 antibodies (Fig 1D left panels). Probing for mitochondrial Prohibitin demonstrated this interaction was not an unspecific enrichment of membrane-associated proteins. Furthermore, immunoblots of blue native gel electrophoresis indicated that LETM1 and TMBIM5 both migrated equally at the estimated mass of ~400 and ~700 kDa (Fig 1D right and Appendix Fig S1D). In the absence of TMBIM5, the signals for both TMBIM5 and LETM1 at ~700 kDa markedly decreased and those at ~400 kDa became weaker for TMBIM5 but not for LETM1. These data suggest that the proteins oligomerize in protein complexes of ~400 and 700 kDa and that LETM1 requires TMBIM5 to oligomerize in the latter. TMBIM5-containing complexes in HeLa mitochondria were comparable, and their levels markedly increased when LETM1 was knocked-down, suggesting that TMBIM5 may compensate for the decrease in LETM1, without changing the mass of the complex (Fig EV1). Figure 2. TMBIM5KO causes mitochondrial matrix swelling and cristae disorganization Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones. Proliferation assay of HEK293 cells in the function of TMBIM5. Graph shows the mean ± SD of three individual counts, One-way ANOVA with the Dunnett's multiple comparisons test performed against TMBIM5WT *P = 0.0155. Live imaging of HEK293 TMBIM5WT and KO cells stained with MitoTracker Green FM. Scale bars: 10 μm. Alteration of the mitochondrial ultrastructure shown by transmission electron microscopy, red arrow pointing to the dilated matrix. Wider mitochondria in the middle and right panel compared with controls, a middle panel showing the strongest phenotype of matrix width and cristae forms. Scale bars: 1 μm. Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT, and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, one-way ANOVA with the Bonferroni's multiple comparisons test performed against TMBIM5WT *P < 0.05, **P < 0.008, two-way ANOVA with the Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***P = 0.0009, ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 2 [embr202254978-sup-0006-SDataFig2.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. TMBIM5 and LETM1 are present in protein complexes of the same molecular weightImmunoblotting analysis of blue native PAGE of isolated mitochondria from HeLa WT, TMBIM5KO, or LETM1KD cells, using the indicated antibody, arrows indicate TMBIM5 complexes. Download figure Download PowerPoint TMBIM5 depletion impairs mitochondrial bioenergetics and morphology We generated TMBIM5 stable knockdown (KD) cells by short hairpins targeting various exons. TMBIM5KD cells had up to 80% reduced TMBIM5 levels compared with scrambled controls with matching decrease in LETM1 (Fig EV2A). The proliferation rate of TMBIM5KD cells in a glucose-containing medium was reduced marginally and only the final time point being significantly affected (Fig EV2B). TMBIM5KD respiratory parameters were not significantly affected (Fig EV2C and D), in contrast to severely compromised galactose-dependent respiration (Fig EV2E and F), indicating that TMBIM5 impacts mitochondrial function. Click here to expand this figure. Figure EV2. TMBIM5KD decreases LETM1 and mitochondrial bioenergetics A. Western blot analysis of LETM1 and TMBIM5 in HEK293 TMBIM5WT cells with a scramble shRNA and two different TMBIM5 knockdowns, HSP60 served as a loading control. B. Proliferation curve of TMBIM5WT scramble controls (scr) compared with TMBIM5KD cells (KD) over 4 days. Data are means ± SEM (scr, KD2 n = 3, KD1 n = 5) (biological replicates), at 96 h statistical analysis using an unpaired student's t-test (*P < 0.05). C–F. Cellular bioenergetics of TMBIM5KD cells in various nutrient conditions. Oxygen consumption rate of WT cells with a scrambled control (WT) and TMBIM5KD cells (KD) grown in (C) 25 mM glucose, (E) 10 mM galactose for 24 h before measurement. Data are representative of at least three independent experiments (biological replicates). Shown are the mean data of triplicate measurements ± SEM. Inhibitors as indicated: (A) oligomycin (0.5 μM), (B, C) FCCP (0.2 μM each), (D) antimycin A/rotenone (0.5 μM). (D, F) Bar charts of XF experiment traces (C, E), data are means of multiple time points after experiment start or drug addition of at least three independent experiments ± SEM (biological replicates). Statistical analysis using an unpaired student's t-test (**P < 0.01, ***P < 0.001). Source data are available online for this figure. Download figure Download PowerPoint To address the specific function of TMBIM5 in mitochondrial morphology and cation homeostasis, we generated TMBIM5 knockout (KO) HEK293 and HeLa cells by CRISPR/Cas9 genome editing. At the gene expression level, we obtained HEK293 and HeLa knockout KO individual clones with entirely abrogated transcript levels of TMBIM5. At the protein level, the total knockout was confirmed in HeLa cells clone IIIF3 (HeLa TMBIM5KO) and in HEK293 cells clone IIF1 (HEK293 TMBIM5KO1). In several other clones, translation was not entirely abolished, like in HEK293 clone IE12 (HEK293 TMBIM5KO2; Fig 2A and E). These clones were used in parallel when indicated to exclude off-target effects or check for gene dose effects. Cell growth was somewhat slowed under TMBIM5 depletion especially in TMBIM5KO2 (Fig 2B). As previously shown for HeLa and HAP cells (Oka et al, 2008; Seitaj et al, 2020), compared with wild-type (WT) cells HEK293 TMBIM5KO1 and TMBIM5KO2 displayed fragmented and less elongated mitochondria, respectively (Fig 2C). Electron micrographs showed TMBIM5KO mitochondria with swollen sections and altered cristae structures, cristae being also affected in the incomplete TMBIM5KO2 (Fig 2D, arrows). LETM1 levels were somewhat reduced in TMBIM5KO (Fig 2E and F). Since TMBIM5 is involved in cristae structures (Oka et al, 2008), and OPA1 controls cristae volume and junction organization, critical for mitochondrial cytochrome c retention (Olichon et al, 2003; Del Dotto et al, 2017), we investigated whether cristae structure and OPA1-cleavage pattern were coupled. OPA1 subunits c and e, both cleavage products of OMA1, appeared to increase in HEK293 TMBIM5KO compared with controls (Fig 2E and F). Consistent with the autocatalytic degradation of activated OMA1, TMBIM5KO had significantly reduced levels of OMA1 (Fig 2E and F). Furthermore, DRP1 was upregulated (Fig 2E and F), matching the shift toward mitochondrial fission and stress-sensitive activation of OMA1 and OMA1-dependent OPA1 cleavage. Thus, TMBIM5KO affected the dynamics of OPA1-dependent cristae structures (Fig 2E and F). Mitochondrial KHE requires LETM1 and TMBIM5 The interaction of TMBIM5 with the mitochondrial KHE component LETM1 (Nowikovsky et al, 2012), raised the question of whether TMBIM5 contributes to KHE activity. Light scattering methods have been classically used to monitor the swelling of mitochondria (Mitchell, 1966; Bernardi, 1999). Previous studies have established that potassium acetate- (KOAc-) induced passive swelling is impaired in LETM1KD mitochondria. HeLa and HEK293 TMBIM5KO mitochondria showed a reduced initial optical density compared with TMBIM5WT, suggesting enlarged mitochondria. TMBIM5KO mitochondria also swell significantly less than TMBIM5WT in KOAc media (Fig 3A–D) as also seen for LETM1KD mitochondria (Fig 3E and F) and Austin et al (2017). Re-expression of TMBIM5 in HEK293 TMBIM5KO cells restored the swelling amplitude to WT levels (Fig 3A and B). Thus, TMBIM5KO led to swollen mitochondria and lower KHE activity, perhaps by reducing LETM1 levels and/or function, or by disrupting the osmotic balance through the overload of another ion. Figure 3. TMBIM5 and LETM1 are involved