# In vivo composition of the mitochondrial nucleoid in mice

Rodolfo García-Villegas, Franka Odenthal, Yvonne Giannoula, Nina A Bonekamp, Inge Kühl, Chan Bae Park, Henrik Spåhr, Elisa Motori, Fredrik Levander, Nils-Göran Larsson

► To cite this version:

Rodolfo García-Villegas, Franka Odenthal, Yvonne Giannoula, Nina A Bonekamp, Inge Kühl, et al. In vivo composition of the mitochondrial nucleoid in mice. Biochimica et Biophysica Acta - Molecular Cell Research, 2025, 1872 (6), pp.119955. 10.1016/j.bbamcr.2025.119955. hal-05391884

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Submitted on 1 Dec 2025

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# *In vivo* composition of the mitochondrial nucleoid in mice

Rodolfo García-Villegas<sup>a</sup>, Franka Odenthal<sup>b,c</sup>, Yvonne Giannoula<sup>a</sup>, Nina A. Bonekamp<sup>d</sup>, Inge Kühl<sup>e</sup>, Chan Bae Park<sup>f</sup>, Henrik Spåhr<sup>a</sup>, Elisa Motori<sup>b,c,g</sup>, Fredrik Levander<sup>h</sup>, Nils-Göran Larsson<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

Mitochondrial nucleoid

mtDNA expression

Mitochondrial translation

TFAM

Transgenic mice

## ABSTRACT

Mitochondrial DNA (mtDNA) is compacted into dynamic structures called mitochondrial nucleoids (mt-nucleoids), with the mitochondrial transcription factor A (TFAM) as the core packaging protein. We generated bacterial artificial chromosome (BAC) transgenic mice expressing FLAG-tagged TFAM protein (Tfam-FLAG<sup>BAC</sup> mice) to investigate the mt-nucleoid composition *in vivo*. Importantly, we show that the TFAM-FLAG protein is functional and complements the absence of the wild-type TFAM protein in homozygous Tfam knockout mice. We performed immunoprecipitation experiments from different mouse tissues and identified 12 proteins as core mt-nucleoid components by proteomics analysis. Among these, eight proteins correspond to mtDNA replication and transcription factors, while the other four are involved in the mitoribosome assembly. In addition, we used the Tfam-FLAG<sup>BAC</sup> mice to identify ten proteins that may stabilize TFAM-FLAG upon depletion of the mitochondrial RNA polymerase despite the absence of mtDNA and induction of the LONP1 protease. Finally, we evaluated the changes in mt-nucleoids caused by very high levels of TFAM unraveling nine interactors that could counteract the high TFAM levels to maintain active mtDNA transcription. Altogether, we demonstrate that the Tfam-FLAG<sup>BAC</sup> mice are a valuable tool for investigating the mt-nucleoid composition *in vivo*.

## 1. Introduction

Mammalian mitochondrial DNA (mtDNA) is a small, circular, gene-dense double-stranded DNA molecule that encodes two rRNAs, 22 tRNAs, and 11 mRNAs that are translated into 13 essential proteins of the oxidative phosphorylation (OXPHOS) system [1]. In contrast to the nuclear genome, mtDNA is present in hundreds to thousands of copies depending on the cell type and tissue energy demands [2,3]. Interestingly, mtDNA copy number and the number of mutant copies determine the mitochondrial function and contribute to the heterogeneous phenotypic manifestations caused by mtDNA mutations [2,4]. The mtDNA is compacted into mt-nucleoids, which are nucleoprotein complexes of ~100 nm in diameter [5,6] that typically contain single copies of mtDNA and reside in voids between the mitochondrial cristae [7,8].

The main mt-nucleoid component is TFAM which cooperatively binds and bends mtDNA without sequence specificity and enables mtDNA compaction by cross-strand binding [8,9]. In the absence of TFAM, mtDNA is degraded, whereas moderate overexpression of TFAM leads to an increase in mtDNA copy number [10]. Interestingly, the upregulation of mtDNA copy number has beneficial effects in different mouse models with mitochondrial dysfunction [11–13]. However, very high levels of TFAM are deleterious *in vivo* and block mitochondrial transcription in certain tissues due to hyper-compaction of the mt-nucleoid [14–16]. Moreover, several studies have shown that TFAM not bound to mtDNA is degraded by the LONP1 protease [17,18]. Thus, the TFAM-to-mtDNA ratio is essential for mtDNA expression and maintenance [14–16,19].

Despite considerable efforts, there is no consensus on the protein

<footnote><sup>a</sup> Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden</footnote>

<footnote><sup>b</sup> Institute of Biochemistry, University of Cologne, Zülpicher Str. 47, 50674 Cologne, Germany</footnote>

<footnote><sup>c</sup> Center for Molecular Medicine Cologne (CMMC), Faculty of Medicine and University Hospital Cologne, University of Cologne, Cologne, Germany</footnote>

<footnote><sup>d</sup> Department of Neuroanatomy, Mannheim Center for Translational Neuroscience (MCTN), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany</footnote>

<footnote><sup>e</sup> Department of Cell Biology, Institute of Integrative Biology of the Cell, UMR9198, CEA, CNRS, Université Paris-Saclay, Gif-sur-Yvette, France</footnote>

<footnote><sup>f</sup> Department of Physiology, Ajou University School of Medicine, Suwon 16499, Republic of Korea</footnote>

<footnote><sup>g</sup> Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany</footnote>

<footnote><sup>h</sup> Department of Immunotechnology, National Bioinformatics Infrastructure Sweden (NBIS), Science for Life Laboratory, Lund University, Lund 223 87, Sweden</footnote>

<footnote>* Corresponding author.
E-mail address: nils-goran.larsson@ki.se (N.-G. Larsson).</footnote>
composition of the mt-nucleoid [20]. While proteins implicated in mtDNA transcription and replication are recognized as core (stable) mt-nucleoid components [21], a diverse set of proteins with unclear functional significance has been proposed as components of the mt-nucleoid [21–28]. Two main reasons may explain the discrepancies observed in previous studies. First, an intrinsic variability due to the different cell types and tissues analyzed. Second, the diverse experimental approaches influence the detected components of the mt-nucleoid [20]. Another complication is the proximity of the mitochondrial RNA granules where the processing and degradation of transcripts and mitoribosome assembly occurs [29–32]. Hence, previous studies have identified several RNA processing enzymes and mitoribosome proteins as mt-nucleoid components (Table S1, lists reported mt-nucleoid proteins). In addition, it has been shown that just a fraction of the mt-nucleoids is involved in active replication and transcription of mtDNA [14,19]. A more stable binding of TFAM to mtDNA promoter sequences plays a role in transcription activation by enhancing recruitment of mitochondrial RNA polymerase (POLRMT) [33]. The mechanism for transitioning from inactive to active mt-nucleoids is largely unknown and mt-nucleoid composition may be heterogeneous with some proteins only interacting transiently. These circumstances make the definition of the mt-nucleoid composition a challenging task (Table S1).

In this study, we generated mice expressing TFAM with a single FLAG-tag (Tfam-FLAG<sup>BAC</sup> mice) to study the mt-nucleoid composition in different tissues *in vivo* under physiological conditions. Importantly, we demonstrate that the TFAM-FLAG protein is functional and can complement the embryonic lethal homozygous knockout of Tfam resulting in normal viable mice. The proteomic analysis of three different tissues (liver, kidney, and heart) and the isolation of the mt-nucleoid with two salt concentrations (ionic strengths) allowed us to define 12 proteins as core components of the mt-nucleoid. Furthermore, the Tfam-FLAG<sup>BAC</sup> transgene can be introduced into different mouse models of mitochondrial dysfunction to investigate changes in mt-nucleoid composition. As proof of principle, we analyzed the TFAM-FLAG interactors in a tissue-specific knockout of Polrmt in the heart [34] and detected ten proteins that might stabilize TFAM upon depletion of mtDNA. Also, we analyzed the mt-nucleoid composition in mouse models with very high general overexpression of TFAM-FLAG, i.e. the CAG-TFAM mice [16]. We identified nine TFAM-FLAG interactors only in tissues with active transcription where they may counteract the repressive effect on mitochondrial transcription caused by the very high levels of TFAM. We thus demonstrate that the Tfam-FLAG<sup>BAC</sup> transgenic mice are a valuable tool for investigating the mt-nucleoid composition *in vivo* under different physiological and pathological conditions.

## 2. Methods

### 2.1. Animal models

Transgenic mice containing a bacterial artificial chromosome (BAC) expressing TFAM with a single FLAG-tag (Tfam-FLAG<sup>BAC</sup> mice) were generated following previously described methods [12]. We introduced a single FLAG tag into a BAC clone containing 73 kb and 117 kb genomic sequences upstream and downstream of the Tfam gene. The modified DNA was injected into the pronucleus of fertilized oocytes, and animals containing the Tfam-FLAG BAC transgene were identified by PCR. Tissues were collected from 13-week-old female mice for all experiments. Heart- and skeletal-muscle-specific Polrmt knockout mice (Polrmt<sup>LoxP/LoxP</sup>, +/Ckmm-cre) were generated by expressing cre from the muscle creatinine kinase promoter (Ckmm) as previously described [34] and bred with Tfam-FLAG<sup>BAC</sup> animals resulting in Polrmt<sup>LoxP/LoxP</sup>, +/Ckmm-cre, Tfam-FLAG<sup>BAC</sup> mice henceforth called Polrmt<sup>CKO</sup> mice. Tissues were collected from 4 to 5-week-old Polrmt<sup>CKO</sup> female mice for all experiments. CAG-TFAM mice with ubiquitous strong overexpression of TFAM

were generated as previously reported [16]. Tissues were collected from 16 to 18-day-old CAG-TFAM mice for all experiments; the animals were terminated before weaning with no sex identification performed.

LoxP-Stop-LoxP-Tfam-mKate2 mice with a conditional knock-in expression of the fusion protein TFAM-mKATE2 in the Rosa26 locus were obtained from Taconic. Specifically, a silent mutation creating a PvuI restriction site was inserted in the Tfam ORF together with a Kozak sequence (GCCACC), and the resulting Tfam ORF was separated to the mKate2 ORF by a 5 amino-acid spacer (nucleotide sequence: TTACCGGTCGCCTTT). A loxP-flanked transcription termination cassette containing a combination of polyadenylation signals (STOP) was introduced to allow conditional expression. For the LoxP-Stop-LoxP-CAG-Tfam-mKate2 mice, a targeting vector with a CAG promoter was introduced by recombination-mediated cassette exchange to obtain a stronger expression of the fusion protein. The LoxP-Stop-LoxP-CAG-Tfam-mKate2 mice were further crossed with β-actin-cre mice [34] or with mice expressing cre-recombinase from a tissue-specific promoter to obtain mice with whole body or tissue-specific expression the TFAM-mKATE2 protein, respectively. The DAT-cre mice express cre-recombinase under the control of the dopamine transporter promoter and were used to induce expression of the TFAM-mKATE2 protein in midbrain dopamine neurons.

All mice had an inbred C57BL/6NRj nuclear background. Animals were housed in a 12-h light/dark cycle in standard ventilated cages and fed ad libitum. The study was approved by the Stockholm ethical committee (Stockholms Djurförsöksetiska Nämd) and by the Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen, Germany. Animal work was performed by the Federation of European Laboratory Animal Science Associations guidelines (FELASA).

### 2.2. Cell lines

Mouse embryonic fibroblasts (MEFs) and human embryonic kidney cells (Flp-In-T-Rex HEK293, Thermo Fischer) were grown in DMEM+GlutaMAX medium (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 100 U/mL penicillin and 100ug/mL streptomycin (Gibco) at 37 °C, 5% CO₂.

### 2.3. Generation of stable cell lines

Flp-In T-Rex HEK293 inducible expression cell lines were generated according to the manufacturer's instructions (Thermo Fischer). In brief, cells were co-transfected with the pcDNA5/FRT/TO vector harboring the gene of interest and the pOG44 vector containing FLP recombinase. Transfected cells were selected with 15 ug/mL blasticidin S and 100 ug/mL hygromycin B for three weeks, and positive colonies were expanded. Expression of MRPS38-FLAG was induced by treating the cells overnight with 1 ng doxycycline cyclate (Sigma™).

### 2.4. RNA interference using siRNA

MEFs were seeded 24 h before transfection in 6-well plates. The next day 10 pmoles of Silencer Select siRNA (Thermo Fisher) was transfected with lipofectamine RNAiMAX transfection reagent (Thermo Fisher) according to the manufacturer's instructions. For mitochondrial isolation, 300 pmoles (500 cm² dish) or 75 pmoles (150 cm² dish) of siRNA were transfected using 120 µL or 30 µL of RNAiMAX transfection reagent, respectively. The cells were harvested 72 h after transfection for all downstream experiments.

The following siRNAs (Thermo Fisher Scientific) were used in this study: Silencer™ Select Negative Control No. 1 siRNA (4390843), Silencer™ Select Negative Control No. 2 siRNA (4390846), Mrps10 siRNA 1 (s82270), Mrps10 siRNA 2 (s82272), Aurkaip1 siRNA 1 (Mrps38, s82529), and Aurkaip1 siRNA 2 (Mrps38, s82530).
## 2.5. Immunocytochemistry

Cells were fixed with 4% paraformaldehyde (PFA) in 1× PBS for 10 min at RT. The cells were permeabilized in 0.1% triton (CarlRoth™) in 1× PBS for 5 min at RT and incubated in a blocking buffer composed of 3% bovine serum albumin (BSA, Sigma-Aldrich™) in 1× PBS for 10 min at RT. Primary antibodies diluted in blocking buffer were incubated overnight at 4°C. The next day the cells were washed twice with blocking buffer at RT and incubated with appropriate secondary antibodies diluted in blocking buffer at room temperature for 2 h. Afterwards, the cells were washed three times with 1× PBS for 10 min at RT. Lastly, coverslips were mounted onto glass slides with Aqua Polymount (Polyscience™) and dried overnight. Slides were stored at 4°C until imaging.

## 2.6. STED imaging, processing, and analysis

Imaging was performed as previously described [35] with a Leica TCS SP8 gated STED (gSTED) microscope, equipped with a white light laser and a 93× objective lens (HC PL APO CS2 93× GLYC, NA 1.30). Briefly, confocal images of mitochondria (HSP60) and MRPS38 (FLAG) were taken by exciting the fluorophores at 546 and 647 nm respectively. Confocal images of mitochondria (HSP60) and C1QBP were taken by exciting the fluorophores at 488 and 647 nm respectively. Confocal images of mitochondrial nucleoids (DNA) and TFAM (FLAG) were taken by exciting the fluorophores at 594 and 643 nm respectively. The corresponding fluorescent signals were collected using Hybrid detectors. STED images of mitochondrial nucleoids (DNA) and MRPS38 (FLAG), C1QBP or TFAM (FLAG) were obtained with a 775 nm depletion laser. 2D confocal and gSTED images were acquired sequentially with the optical zoom set to obtain a voxel size of 17 × 17 nm. Excitation was provided at 594 and 640 nm and Hybrid detectors collected signal. Gating between 0.3 and 6 ns was applied. Raw images were first deconvolved with the Huygens software. Image panels were created with Photoshop (Adobe); no digital manipulation was applied, except for adjustment of brightness and contrast. Intensity profiles of DNA and FLAG or C1QBP signals were obtained using the Plot Profile Tool in ImageJ. Antibodies used for imaging in this work: rabbit anti-FLAG (F7425, Merck), mouse anti-DNA (61,014, Progen), goat anti-HSP60 (Sc-1052, Santa Cruz), rabbit anti-C1QBP (HPA026483, Sigma), anti-rabbit Atto647 (40,839, Sigma Aldrich), anti-mouse Alexa 594 (A21044, Invitrogen), and anti-rabbit Alexa647 (Jackson Immuno Research, 711,605,152), anti-goat Alexa488 (705,546,147, Jackson Immuno Research) and anti-rabbit Fluo Tag X2 643 (N2402, NanoTag).

## 2.7. Confocal imaging of brain sections and image processing

Fluorescence immunohistochemistry of fixed brain tissue sections was performed using the free-floating procedure as previously described [36]. For detection of the TFAM fusion protein in dopamine neurons, primary antibodies against RFP (5F8, Chromotek, 1:500) and tyrosine hydroxylase (1:500, Synaptic System) were used, and sections were subsequently incubated with appropriate secondary antibodies conjugated with Alexa 488 and Alexa 549. Slides were thereafter imaged with a laser scanning confocal microscope (TCS SP8-X, Leica Microsystems) equipped with a white light laser and a 405-diode ultraviolet laser. Stack images were acquired with the LAS-X software in sequential mode by exciting the fluorophores and collecting their signal through hybrid detectors (HyDs). Images were further processed using Image J and Adobe Photoshop to adjust brightness and contrast. Adobe Illustrator was used for figure preparation.

## 2.8. mtDNA copy number analysis

DNA was isolated with the Puregene Tissue Kit (QIAGEN) according to the manufacturer's instructions. The DNA was quantified with the

Nanodrop 2000 (Thermo Fisher), and 5 ng was used for qPCR analysis with Taqman 2× Universal PCR master mix and TaqMan probes (Applied Biosystems) for mouse mitochondrial 12s (Mm04260177_s1), *Cox1* (Mm04225243_g1), *Atp6* (Mm03649417_g1), and nuclear 18s (Hs99999901_s1).

## 2.9. Southern blot

Two micrograms of DNA were digested with *SacI* for 3 h at 37°C. Digested DNA was incubated at 95°C for 5 min, separated in 0.8% agarose gels, and transferred to a nitrocellulose membrane (Hybond-N +, Cytiva). The membranes were hybridized with [α-³²P] dCTP (2'-deoxycytidine 5'-triphosphate, PerkinElmer) labeled probes to detect 7S DNA, mtDNA, and 18S rDNA (nuclear DNA) as loading control and exposed to a phosphoimager screen.

## 2.10. RNA isolation and RT-qPCR quantification

Total RNA from snap-frozen tissues or cells was isolated with TRIzol Reagent (Thermo Fisher) following the manufacturer's instructions. The RNA was treated with the TURBO DNA-free kit (Thermo Fischer), and 1–2 µg of RNA was used for cDNA synthesis with the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher) following the manufacturer's instructions. For qPCR quantification, 10 ng of cDNA were mixed with Taqman 2× Universal PCR master mix and TaqMan probes (Applied Biosystems) for mouse 12s (Mm04260177_s1), *Nd1* (Mm04225274_s1), *Cox1* (Mm04225243_g1), *Atp6* (Mm03649417_g1), *Cytb* (Mm04225271_g1), *Nd6* (custom made AIVI3E8), *Mrps38* (Mm00481195_m1), *Mrps10* (Mm00812951_g1), *Tfam* (Mm00447485_m1), *Polg* (Mm00450527_m1), *Polrmt* (Mm00553272_m1), *Ssbp1* (Mm01131763_g1) and *Lrpprc* (Mm00511512_m1). *Actb* (Mm01205647_g1) was used as a reference gene transcript for quantification.

## 2.11. In organello replication and transcription assays

In organello replication and transcription assays were performed as previously described [34]. In brief, 500 µg of freshly isolated mitochondria were washed two times with cold incubation buffer (25 mM sucrose, 75 mM sorbitol, 10 mM Tris-HCl, 10 mM K₂HPO₄, 100 mM KCl, 0.05 mM EDTA, 5 mM MgCl₂, 10 mM glutamate, 2.5 mM malate, 1 mM ADP, 1 mg/mL BSA, pH 7.4). Mitochondria were resuspended in 500 µL of warm incubation buffer and an aliquot of 40 µL was taken for immunoblotting against HSP60, VDAC, and MRPS38. For the replication assay, the incubation buffer was supplemented with 50 µM dCTP, dTTP, dGTP, and 20 µCi of [α-³²P] dATP and incubated 2 h at 37°C. DNA was isolated with the Puregene Tissue Kit (QIAGEN) and analyzed as Southern blot samples. For the transcription assay, the incubation buffer was supplemented with 50 µCi of [α-³²P] UTP and incubated for 2 h at 37°C. Pulse samples were incubated for 10 min with 0.2 mM UTP at 37°C. RNA was isolated by TRIzol reagent (Thermo Fischer), resolved in formaldehyde-agarose gels, and transferred to nitrocellulose membranes (Hybond-N+; GE Healthcare). The membranes were used to expose a phosphoimager screen.

## 2.12. ³⁵S-metabolic labeling of mitochondrial proteins

The mitochondrial translation assay was performed as previously described [37]. In brief, cells grown to 80% confluency in a 6-well plate were incubated for 20 min at 37°C with Cys/Met free DMEM media (Gibco) supplemented with 1 mM Glutamax, 1 mM Sodium pyruvate (Gibco), 10% dialyzed bovine serum and 100 µg/mL of anisomycin (Sigma) to inhibit cytosolic translation. Afterward, the cells were incubated with 3.7 MBq of ³⁵S-L-methionine and cysteine mix (PerkinElmer) for 1 h to label newly synthesized mitochondrial proteins. Cells were harvested by trypsinization and lysed with RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium
deoxycholate, 0.1 % SDS, 140 mM NaCl, 1 × mini complete protease inhibitor (Roche)) for 30 min on ice. Protein content was determined using the BCA assay kit (Pierce) and 30 µg aliquots were separated on pre-cast 12 % SDS-PAGE gels (Invitrogen). The gels were stained with Imperial stain solution (Pierce) and dried before exposure to the phosphoimager screen.

## 2.13. Western blotting

Cells were incubated for 30 min on ice with RIPA buffer to obtain total cell extracts. The lysate was cleared for 10 min at 16,000 g and quantified with the BCA assay kit (Pierce). 30–50 µg of protein were separated in pre-cast Nu-PAGE 4–16 % gels (Invitrogen) with MES or MOPS buffer and transferred to nitrocellulose or PVDF membranes using iBlot (Thermo) or wet tank blotting (25 mM Tris, 192 mM glycine, and 20 % ethanol) at 4 °C for 1.5 h at 300 mA. The membranes were blocked in 5 % milk PBST (Phosphate buffer 10 mM, KCl 27 mM, NaCl 137 mM, Tween 20 0.1 %, pH 7.4) and incubated with primary antibodies overnight at 4 °C. The membranes were washed with PBST, incubated with HRP-conjugated secondary antibodies for 2 h at room temperature, and washed with PBST. Detection was performed with ECL Clarity reagent (BioRad).

The following antibodies were used in this study: mouse anti-GAPDH [6C5] (ab8245, Abcam), rabbit anti-Vinculin (4650, Cell Signaling Technology), Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam), mouse monoclonal anti-MTCO1 (1D6E1A8, Thermo Fisher), rabbit anti-MTCO2 antibody (Custom made), rabbit anti-mtTFA (ab131607, Abcam), rabbit monoclonal anti-HSP60 LK-2 (ADI-SPA-807-E, Enzo Biosciences), mouse anti-VDAC1/Porin [20B12AF2] (ab14734, Abcam), rabbit anti-MRPL12 (HPA022853, Sigma-Aldrich), rabbit polyclonal anti-MRPS18B (16139-1-AP, Proteintech), rabbit polyclonal anti-MRPS15 (17006-1-AP, Proteintech), rabbit anti-MRPS16 (HPA054538, Sigma-Aldrich), rabbit anti-MRPS10 (NBP1-83848, Novus Biologicals), rabbit anti-SSBP1 (HPA002866, Sigma-Aldrich), rabbit anti-LRPPRC (HPA036409, Sigma-Aldrich), rabbit anti-LONP (PA5-51692, Invitrogen), rabbit anti-POLG (ab128899, Abcam), rabbit anti-POLRMT [38] rabbit anti-MRPS38 (Custom made), sheep anti-mouse IgG (NA9340, Cytiva), and donkey anti-rabbit (NA9310V, Cytiva).

## 2.14. Linear density glycerol gradients

The 10 to 45 % linear density glycerol gradient was performed as previously described with minor modifications [32,34]. In brief, 1 to 3 mg of mitochondria were pelleted at 10,000 g at 4 °C for 5 min and solubilized in lysis buffer (5 % glycerol, 20 mM NaCl, 30 mM Hepes (pH 8.0), 1 mM EDTA, 2 mM DTT, and 1.2 %, DDM and EDTA-free complete protease inhibitor cocktail (Roche)) on ice for 10 min. The lysate was cleared at 800 g at 4 °C for 5 min and overlaid on top of a 10 to 45 % linear glycerol gradient (20 mM NaCl, 25 mM Hepes (pH 8.0), 1 mM EDTA, 1 mM DTT, 0.2 % DDM, and EDTA-free complete protease inhibitor cocktail (Roche)) prepared using the Gradient Master (Bio-comp Instruments Inc.) in 14 mm × 89 mm Ultra-Clear centrifuge tubes (Beckman Instruments Inc.). The gradient was centrifuged at 210,000g at 4 °C in a SW41 rotor for 1 h in a Beckman Coulter OptimaL-100 XP ultracentrifuge. Fractions of 800 µL were collected from the top of the gradient. One-third of the fraction was precipitated with 0.02 % Sodium Deoxycholate and 18 % Trichloroacetic acid (TCA) on ice for 20 min for Western blotting analysis. For mtDNA analysis, 1 µL of each fraction was used for PCR amplification. Alternatively, DNA was isolated from two-thirds of the fractions with the Puregene Tissue Kit (QIAGEN) and analyzed as the Southern blot samples.

## 2.15. Mitochondria isolation

Mitochondria from mouse tissues were isolated by differential centrifugation. Freshly dissected tissues were rinsed in ice-cold PBS and minced into small pieces before homogenization in mitochondrial isolation buffer (MIB, 320 mM sucrose, 10 mM Tris-HCl, 1 mM EDTA, pH 7.4) supplemented with BSA 0.2 % using a Potter homogenizer (Schuett). Heart and kidney tissues were then homogenized in 5 mL of MIB. Liver tissue was homogenized in 10 mL of MIB. The homogenates were transferred into Falcon tubes, and the volumes were adjusted to 10 mL for heart and kidney tissues, and to 20 mL for liver tissue. Mitochondria were isolated from the tissue homogenates by differential centrifugation. Cell debris was pelleted at 1000 g for 10 min at 4 °C and the supernatant was centrifuged at 10,000 g for 10 min at 4 °C to obtain crude mitochondria. Mitochondria were resuspended in 1 mL of MIB without BSA supplemented with 100 µg of Proteinase K and incubated for 30 min on ice. After, 5 mM PMSF was added and mitochondria were pelleted at 10,000 g for 10 min at 4 °C, washed with 2 mL of MIB supplemented with 1 M KCl, and pelleted again. Mitochondria were resuspended in MIB supplemented with EDTA-free complete protease inhibitor (Roche).

For mitochondrial purification from cells transfected with siRNAs (see methods Section 2.4), we harvested cells from one 500 cm² dish or four to six 150 mm dishes at 80 to 90 % confluency. The cells were pelleted at 800 g for 5 min at 4 °C and washed twice with cold PBS. The cells were homogenized in 10 mL cell culture MIB (20 mM HEPES pH 7.6, 220 mM mannitol, 70 mM sucrose, 1 mM EDTA) in a glass Dounce homogenizer and transferred into Falcon tubes, and the final volume was adjusted to 20 mL Cell debris was pelleted at 1200 g for 10 min at 4 °C and the supernatant centrifuged at 11,000 g for 10 min at 4 °C to obtain crude mitochondria. Mitochondria were resuspended in MIB.

## 2.16. TFAM-FLAG immunoprecipitation

For analysis of the TFAM-FLAG immunoprecipitation, four mitochondrial sample preparations (containing *Tfam-FLAG* transgene) were compared to four mitochondrial control preparations (without *Tfam-FLAG* transgene) per tissue and ionic strength condition (20 mM or 100 mM NaCl). The number of immunoprecipitation samples and controls performed per mouse model were the following: Tfam-FLAG rescue mice, 24 samples (*Tfam*⁻/⁻, *Tfam-FLAG*BAc), and 24 controls (*Tfam*⁺/⁺). PolrmtcKO mice, 8 samples (*Polrmt*LoxP/LoxP, +/Ckmm-cre, *Tfam-FLAG*BAc), and 8 controls (*Polrmt*LoxP/LoxP, *Tfam-FLAG*BAc). CAG-TFAM mice, 24 samples (*CAG-Tfam-FLAG*), and 24 controls (*Tfam*⁺/⁺). For each immunoprecipitation, 500 µg of crude mitochondria were pelleted and resuspended in 150 µL of lysis buffer (50 mM Tris-HCl pH 7.4, 100 or 20 mM NaCl, 5 mM MgCl₂, 1 % n-dodecyl-β-D-maltoside (DDM) and protease inhibitors (Complete EDTA-free, Roche)). Lysis was conducted on ice before clearing at 16,000 g for 5 min. The supernatant was diluted 10 times with wash buffer (50 mM Tris-HCl pH 7.4, 100 or 20 mM NaCl, and 5 mM MgCl₂ with) and incubated with 100 µL of anti-FLAG M2 beads (Sigma-Aldrich) for 2 h at 4 °C. The beads were previously equilibrated in wash buffer supplemented with 0.1 % DDM. Afterwards, the beads were incubated with wash buffer supplemented with 0.1 % DDM followed by wash buffer without detergent (three times). The beads were resuspended in 200 µL and processed as indicated in sample preparation for proteomics.

## 2.17. Sample preparation for proteomics

Agarose beads were incubated with 15 µL of 8 M urea and 1 µg trypsin (sequencing grade, Promega) in 105 µL of 50 mM Tris-HCl, pH 7.5 at room temperature (RT) for 30 min with shaking at 400 rpm.
The samples were centrifuged at 7000 rpm for 30 s before the supernatant was collected (ca. 120 µL) in new tubes. The beads were washed with 75 µL of 1 M urea in 50 mM Tris-HCl, pH 7.5, and 1 mM dithiothreitol (DTT) and with 100 mM Tris-HCl collecting the supernatant in the same tube (total ca. 195 µL). Digestion in the eluent was continued overnight at RT adding 5 µL of 0.1 µg/µL trypsin with shaking at 400 rpm, while the agarose beads were supplemented with 45 µL of 1 M urea and 1 mM DTT and 1 µg trypsin in 35 µL of 100 mM Tris-HCl and further digested overnight at RT with shaking at 400 rpm. The peptide solutions were alkylated with 2 or 4 µL of 250 mM iodoacetamide in 50 mM of Tris-HCl incubating at RT for 30 min in the dark. The digestion was stopped with 14 µL of formic acid (FA) in the combined peptide solution and samples were cleaned on a C-18 HyperSep plate (Thermo Scientific) and dried in a vacuum concentrator (Eppendorf).

## 2.18. Liquid chromatography-tandem mass spectrometry data acquisition

Peptides were reconstituted in 10 µL of solvent A (0.1 % FA in water) and 2 µL was injected on a 50 cm long EASY-Spray C18 column (Thermo Fisher Scientific) connected to an Ultimate 3000 nanoUPLC system (Thermo Fisher Scientific) using a 90 min long gradient: 4–26 % of solvent B (98 % acetonitrile, 0.1 % FA) in 90 min, 26–95 % in 5 min, and 95 % of solvent B for 5 min at a flow rate of 300 nL/min. Mass spectra were acquired on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer (Thermo Fisher Scientific) ranging from m/z 375 to 1500 at a resolution of R = 120,000 (at m/z 200) targeting 5 × 10⁶ ions for maximum injection time of 100 ms, followed by data-dependent higher-energy collisional dissociation (HCD) fragmentations of precursor ions with a charge state 2+ to 8+, using 30 s dynamic exclusion. The tandem mass spectra of the top 17 precursor ions were acquired with a resolution of R = 30,000, targeting 2 × 10⁵ ions for a maximum injection time of 54 ms, setting quadrupole isolation width to 1.4 Th and normalized collision energy to 28 %.

## 2.19. Proteomics data processing and analysis

The raw mass spectrometry data were processed using MaxQuant [39] version 1.6.10.43 on an Ubuntu server. The search database was the UniProt mouse proteome UP000000589 as of, 10/22/2019 and default parameters were used, including protein level filtering at 1 % false discovery rate. The protein group results tables were used for further analysis. Data analyses were performed in R version 4.3.1 in R markdown notebooks. Data overview, normalization, and initial differential abundance analysis were performed using NormalizerDE [40], version 1.18.1. The first dataset was log2-transformed without further normalization, while the second dataset was normalized using Cyclic Loess from the LIMMA package [41]. Proteins were annotated as mitochondrial using Mitocarta 3.0 [42] by matching gene symbols and synonyms. Differential abundance analyses were performed using LIMMA empirical Bayes statistics [43]. For group-wise comparisons, imputation was performed if a protein was detected (had an abundance value) in at least three replicates in one condition (n = 4), but not in the control condition. Then a low abundance value, corresponding to the lowest intensity value reported for any protein in the batch, was imputed for one of the replicates in the condition with only missing values.

Further data visualization and filtering were performed using OmiCLoupe [44]. The dot plots were generated in R version 4.3.1. Hierarchical clustering heatmaps and correlation plots were generated in Instant Clue [45].

# 3. Results

## 3.1. The TFAM-FLAG rescue mice have a normal mitochondrial function

To investigate mt-nucleoid proteins that regulate mtDNA expression, we added a single FLAG-tag to the carboxyl terminus of TFAM, the main

mtDNA packaging protein, and a bona fide mt-nucleoid component. We generated Tfam-FLAGBAC mice that contain a BAC transgene expressing TFAM-FLAG under the control of its endogenous promoter and regulatory elements (Fig. 1A). To ensure that the TFAM-FLAG protein was functional we crossed Tfam-FLAGBAC mice with heterozygous Tfam knockout mice (Tfam$^{+/-}$, Fig. 1B). The full-body homozygous Tfam knockout mouse (Tfam$^{-/-}$) is not viable and embryos die shortly after implantation [46]. In contrast, we obtained normal live Tfam$^{-/-}$ mice when the Tfam-FLAGBAC transgene was present (Tfam-FLAG rescue mice, Fig. S1A). The TFAM-FLAG protein can thus functionally replace the wild-type TFAM protein and western blots confirmed that the Tfam-FLAG rescue mice exclusively express the TFAM-FLAG protein, which is larger than the TFAM protein (Fig. 1C). The expression of the TFAM-FLAG protein in Tfam-FLAG rescue mice occurs at similar levels as the expression of TFAM in different tissues of wild-type mice (Fig. 1C, Table S2). We also performed STED microscopy of Tfam-FLAG rescue MEFs and found that TFAM-FLAG exclusively colocalizes with the mtDNA (Fig. 1D–E). TFAM regulates mtDNA copy number in mammals [10] and we therefore analyzed the mtDNA copy number by qPCR in all genotypes obtained from crosses between Tfam$^{+/-}$ and Tfam-FLAGBAC mice. As expected, we observed a ~50 % reduction of mtDNA levels in liver tissue from Tfam$^{+/-}$ animals, while the Tfam$^{+/-}$, Tfam-FLAGBAC animals had a two-fold increase in mtDNA levels (Fig. 2A). Importantly, the mtDNA levels in Tfam$^{+/-}$, Tfam-FLAGBAC, and Tfam-FLAG rescue animals were like those of control mice (Tfam$^{+/-}$) in liver (Fig. 2A), kidney (Fig. S1B) and heart (Fig. S1C).

The steady-state levels of OXPHOS subunits remained unchanged in all tissues tested in mice with the Tfam-FLAGBAC transgene (Figs. 2B and S1D, E, Table S4) consistent with normal mitochondrial transcript levels as assessed by RT-qPCR (Fig. S1F, G). Finally, we performed linear density glycerol gradient experiments in liver mitochondria to evaluate the comigration of the TFAM-FLAG protein with the mt-nucleoid [32,34]. We detected mtDNA in the glycerol gradient fractions by PCR amplification with primers specific for Nd1 and confirmed the results by Southern blotting (Fig. S2A, B). The comigration of mtDNA, POLRMT, POLG, and TFAM allowed us to identify mt-nucleoid-containing fractions (Fig. 2C, D). Importantly, we demonstrated that the TFAM-FLAG protein comigrates with the mt-nucleoid in a similar way as does the wild-type TFAM protein (Fig. 2C, D).

In summary, we show that the TFAM-FLAG protein is functional and is localized to the mt-nucleoid and can rescue the absence of wild-type TFAM. Hence, the Tfam-FLAG rescue mouse is suitable for investigating the mt-nucleoid composition in vivo.

## 3.2. Definition of the core mt-nucleoid proteins in vivo

The pool of TFAM comigrating with mtDNA in linear glycerol gradients can be modified by increasing the ionic strength in the buffer [23]. In glycerol gradients with 20 mM NaCl, we observed the comigration of TFAM with mtDNA, POLG, and POLRMT (Fig. 3A). In contrast, in samples with 100 mM NaCl, only residual amounts of TFAM, POLG, and POLRMT were present in the same fractions as the mtDNA (Fig. 3B). We reasoned that immunoprecipitation of TFAM-FLAG at low and high ionic strength would allow us to determine the mt-nucleoid composition in the Tfam-FLAG rescue mice more accurately. Therefore, we performed TFAM-FLAG affinity enrichment experiments in buffers containing 20 mM or 100 mM NaCl for proteomic analysis (Table S2). Analysis of the enriched TFAM-FLAG interactors (Log2 fold change (LFC) > 1.5, adjusted P value (adj.P) < 0.05) in 20 mM NaCl samples from three tissues revealed that around 60 % of the proteins were annotated as mitochondrial according to MitoCarta 3.0 [42]. However, the proportion dropped to 30 % when using 100 mM NaCl (Fig. 3C). This result shows that low ionic strength was better for coimmunoprecipitation of TFAM-FLAG interactors and possible nucleoid proteins. We found that 13 of the 22 proteins enriched in the low ionic strength tissue samples were mitochondrial (Fig. 3D), and among those,
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** The TFAM-FLAG protein is functional and rescues the embryonic lethal phenotype of the *Tfam*⁻/⁻ mice. (A) Schematic depiction of the BAC construct containing *Tfam-FLAG* under its endogenous promoter. Black boxes depict *Tfam* exons and the red box indicates the FLAG tag. (B) Breeding strategy to obtain the *Tfam-FLAG* rescue mice. (C) Western blot analysis of TFAM in liver, heart, and kidney mitochondrial lysates from animals with the indicated genotypes. HSP60 was used as a loading control. N = 3 biological replicates. (D) Representative confocal microscopy images of single and merge channels showing FLAG (to visualize TFAM) and DNA in wildtype (left) and TFAM rescue MEF cells (right). (E) Confocal (top) and STED (bottom) microscopy images of individual (left) or merged (center) FLAG and DNA channels. Fluorescence intensity profile analysis of FLAG and DNA is shown on the right. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

10 were previously reported as mt-nucleoid components (Table S1). Importantly, we found the expected mt-nucleoid interactors such as POLG, POLG2, POLRMT, SSBP1, and TWNK involved in mtDNA replication and transcription [1,21]. Other interactors in our list identified were the prohibitins (PHB and PHB2) that form large complexes in the inner mitochondrial membrane to help maintain mitochondrial ultrastructure [47,48] and were previously reported to interact with the mt-

nucleoid (Table S1). Another interactor was the i-AAA protease YME1L1 [49], which has been implicated in regulating mtDNA metabolism [50] and reported once as an mt-nucleoid component (Table S1). Additionally, we detected components of the mitoribosome, whose assembly occurs in the RNA granules close to the mt-nucleoid [29,31,51]. Specifically, we detected the mitoribosome protein MRPS38 and a mitoribosome assembly factor (RBFA) as TFAM-FLAG interactors [52,53].
<figure><img src="image_4.png" /><figcaption>C</figcaption></figure>

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

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

<figure><img src="image_7.png" /><figcaption>**Fig. 2.** The TFAM-FLAG protein is functional in *Tfam*⁻/⁻ mice.</figcaption></figure>

(A) Steady-state mtDNA levels determined by qPCR in the liver of animals with the indicated genotypes. Probes for 12S and Cox1 were used to measure mtDNA content and normalized to the 18S nuclear DNA reference. The data is presented as a fold change from control animals (*Tfam*⁺/⁺). The boxplots show the median (horizontal line), the 25th to 75th percentile interquartile range (box), and the maximum and minimum observations within the 1.5 interquartile range (vertical lines). N = 6 biological replicates, p < 0.001 = ***, two-tailed unpaired Student's t-test against control animals (*Tfam*⁺/⁺). (B) Western blot of the steady-state levels of oxidative phosphorylation proteins in liver mitochondrial lysates. HSP60 was used as a loading control (N = 3 biological replicates). (C, D) Linear density glycerol gradients performed on liver mitochondria. The mtDNA was amplified in each fraction with specific primers located in *Nd1*. Proteins were analyzed by western blotting to determine comigration with the mtDNA. N = 2 biological replicates. (C) Control animals (*Tfam*⁺/⁺). (D) *Tfam*-FLAG rescue animals. * indicates an un-specific band or signal. Blot images depicting TFAM, POLRMT, POLG, HSP60, and *Nd1* PCR are the same as shown in Fig. S4A.

Finally, we found the calcium-independent phospholipase A2-gamma (PNPLA8), [54], once reported as a TFAM interactor (Table S1), and the aspartate/glutamate antiporter (SLC25A13) which was never reported as an interactor in previous studies [55]. The remaining nine enriched proteins do not have mitochondrial localization and likely are non-specific TFAM-FLAG interactors.

In the high ionic strength samples, we found a higher enriched proportion of non-mitochondrial proteins (Figs. 3C and S3) that potentially correspond to non-specific TFAM-FLAG interactors. To further support the specificity of the TFAM-FLAG interactors found in the low ionic strength samples, we checked the shared enriched proteins (LFC > 1.5, adj.P < 0.05) considering the high ionic strength samples. Interestingly, only POLRMT, SSBP1, and TWNK were shared among all tissues in both ionic strength conditions, indicating that these are the strongest TFAM-FLAG interactors (Fig. 3E). To detect weaker interactors, we considered enriched proteins present in at least five of the six datasets used for the analysis (three tissues and two NaCl concentrations during immunoprecipitation). We found 21 TFAM-FLAG interactors, among them 12 mitochondrial proteins that we denote core mt-nucleoid components based on the reproducibility of their enrichment (Fig. 3C, E). The analysis supported that MRPS38, POLG, POLG2, POLRMT, SSBP1, TWNK, TFAM, and YME1L1 are mt-nucleoid components. In addition, some new TFAM-FLAG interactors were detected, including the mitochondrial transcription elongation factor TEFM, the mitoribosome subunit MRPS10, and two mitoribosome assembly factors DDX28 and C1QBP [29,56–58]. Crucially, all mitochondrial TFAM-

FLAG interactors are previously reported as mt-nucleoid components (Table S1), and eight of them cluster as mt-nucleoid components in a STRING analysis (Fig. 3F). In case of the non-mitochondrial proteins, none, except for Lag3, were shared between 20 mM NaCl samples (Fig. 3D) and in at least five out of six datasets of the three different tissues analyzed with 20 mM or 100 mM NaCl (Fig. 3E). Hence, we conclude that non-mitochondrial proteins likely correspond to non-specific TFAM-FLAG interactors with a predominance of cytosolic ribosome subunits (Fig. 3F).

We performed glycerol gradient centrifugation on mitochondria from each tissue to assess the comigration of C1QBP, DDX28, MRPS10, MRPS38, and YME1L1 with the mt-nucleoid. However, we did not detect these proteins in the fractions containing mtDNA, TFAM, POLRMT, POLG, or SSBP1 (Fig. S4). This suggests that only strong interactors of the mt-nucleoid can be detected by glycerol gradients under the conditions used in our experiments. Even TFAM was detected in the top fractions of the gradient, despite imaging data showing that it consistently colocalizes with mtDNA inside mitochondria [6,19]. An alternative possibility is that C1QBP, DDX28, MRPS10, MRPS38, and YME1L1 might interact with TFAM when it is not bound to mtDNA. However, this seems unlikely, as TFAM is degraded when mtDNA is depleted [38,59].

Finally, we conducted additional confocal microscopy analysis of C1QBP, MRPS10, YME1L1, and DDX28 to assess whether these proteins overlap with mtDNA in HEK cells. Unfortunately, the staining with MRPS10, YME1L1, and DDX28 antibodies showed a non-specific
labeling, which prevented us from drawing definitive conclusions. In contrast, HEK cells stained with a C1QBP antibody revealed a clear mitochondrial localization (Fig. S5A). An intensity profile of the C1QBP signal showed a partial overlapping with the anti-DNA staining in the confocal microscopy images. However, super-resolution STED analysis showed that they were adjacent to each other (Fig. S5B). These results show that C1QBP is located in close proximity to the mt-nucleoid.

We aimed to determine tissue-specific components of the mt-nucleoid. Therefore, we analyzed TFAM-FLAG interactors (LFC > 1.5, adj.P < 0.05) in individual tissues at both ionic strength conditions. In this analysis, we observed an average of 40% mitochondrial proteins as TFAM-FLAG interactors, suggesting a high proportion of unspecific interactions detected (Fig. S6A). Nevertheless, we found 22, 26, and 58 enriched mitochondrial proteins in the liver, heart, and kidney, respectively (Fig. S3, Table S2). A STRING analysis [60] with the mitochondrial tissue-specific TFAM-FLAG interactors revealed enrichment of mt-nucleoid components and proteins related to mitoribosome function in each tissue (Fig. S6B–D). In conclusion, we identified probable tissue-specific TFAM-FLAG interactors, but their functional significance remains to be established.

In summary, the study of the TFAM-FLAG interactors from three tissues of the *Tfam-FLAG* rescue mice allowed us to define 12 mitochondrial proteins as core components of the mt-nucleoid *in vivo*. Seven core mt-nucleoid components are either mtDNA replication factors (POLG, POLG2, SSBP1, and TWNK) or mtDNA transcription factors (TFAM, POLRMT, TEFM). In addition, we found four proteins with mitoribosome-related functions (MRPS10, MRPS38, C1QBP, and DDX28). Therefore, our data support previous reports that mitoribosome assembly occurs in the proximity of the mt-nucleoid.

### 3.3. Depletion of MRPS38 leads to an upregulation of the mtDNA replication and transcription

Among the mitoribosome subunits we identified in our analysis, MRPS38 and MRPS10 were consistently enriched in the mt-nucleoid. Interestingly, MRPS38 had a higher enrichment in the low ionic strength condition than any other mitoribosome subunit (Fig. 3E). Mitoribosome subunits or assembly factors are commonly reported as mt-nucleoid components (Table S1) given the proximity of mitoribosome assembly to the mt-nucleoid [29,31,51]. Therefore, we decided to test if MRPS38 and MRPS10 had a direct influence on mtDNA expression by performing siRNA knockdown experiments in mouse embryonic fibroblasts (MEFs). In addition, we used chloramphenicol (CAP) as a control to evaluate changes in mtDNA expression caused by inhibition of mitochondrial translation. We observed a reduction of *Mrps10* and *Mrps38* mRNA levels by RT-qPCR after three days of transfection using two independent siRNA (Fig. S7A). For MRPS10, we confirmed an efficient protein depletion by western blotting in total cell extracts (Fig. 4A). However, we could not validate MRPS38 protein depletion because available commercial antibodies and a custom-made antibody did not work in total cell extracts but confirmed the down-regulation of *Mrps38* transcript by qPCR (Fig. S7A). As expected, the knockdown of *Mrps10* and *Mrps38* decreased the synthesis of mitochondrially-encoded proteins, although not as efficiently as chloramphenicol (CAP)-treatment of cells (Fig. S7B). However, the steady-state levels of OXPHOS subunits were unchanged, except for a mild reduction of COX2 upon depletion of MRPS38. CAP-treated cells showed a strong decrease in OXPHOS protein levels (Fig. S7C). The mitoribosome proteins remained stable in *Mrps10* and *Mrps38* knockdowns, while the CAP-treated cells showed a mild increase of proteins of the small subunit of the mitoribosome (Fig. S7D).

Unexpectedly, we detected a two-fold increase in mtDNA copy number by qPCR in CAP-treated cells and *Mrps38-1* knockdown (Fig. 4B, C). In contrast, the mtDNA copy number was not affected by depletion of MRPS10 (Fig. 4C). We confirmed by Southern blotting that CAP-treated cells and *Mrps38-1* knockdown have increased mtDNA

levels (Fig. 4D, E). Also, we detected a reduction of the 7S DNA (Fig. 4D, E), a product of abortive mtDNA replication [61]. The decrease of 7S DNA and the increase of mtDNA suggested an augmented *de novo* synthesis of mtDNA that we corroborated by *in organello* replication assays in CAP-treated cells and *Mrps38-1* knockdown (Fig. 4F). In addition, we detected MRPS38 by western blotting in mitochondrial lysates. Notably, the efficiency of *Mrps38-1* downregulation correlates with the upregulation of mtDNA copy number.

Usually, an increase in mtDNA copy number leads to an increase in TFAM levels [2,10]. However, the TFAM protein levels remained unchanged despite the increased mtDNA copy number in *Mrps38-1* knockdown and CAP-treated cells (Fig. 4A, B, Table S2). This suggested that the mt-nucleoid is less compacted in these cells because TFAM is the main packaging protein of the mtDNA [8,9]. The compaction of the mt-nucleoid controls the extent of mtDNA transcription [15,16]. Therefore, we measured the levels of mitochondrial transcripts by RT-qPCR and detected a mild increase of 16s and Nd1 transcripts upon depletion of MRPS38 (Fig. S7E). We observed a strong upregulation of *de novo* transcription in these cells (Fig. 4F). We then analyzed the expression of mtDNA replication and transcription factors observing an increase of *Tfam*, *Tfb2m*, *Ssbp1*, and *Lrpprc* transcript levels upon depletion of MRPS38 (Fig. S7F). However, TFAM, SSBP1, and LRPPRC protein levels were not altered (Fig. S7G). In *Mrps10* knockdowns, we detected a mild increase in *Tfam* and *Tfb2m* transcript levels (Fig. S7F), but no changes were observed in CAP-treated cells. At the protein level, there were no changes in TFAM, SSBP1, and LRPPRC in *Mrps10* knockdowns, while the CAP-treated cells showed a mild increase of SSBP1, MRPS10, and LRPPRC (Fig. S7G).

To validate MRPS38 as an mt-nucleoid component we performed confocal and super-resolution (STED) microscopy (Fig. 4H). Due to the lack of reliable antibodies to label the endogenous MRPS38 protein, we generated stable inducible human embryonic kidney (HEK) cells expressing a *Mrps38-FLAG* construct. Western blot analysis confirmed that the expression of MRPS38-FLAG was only detected in doxycycline-treated cells ruling out any leakage in the expression of the construct in our cell system (Fig. S8A). Importantly, the expression of MRPS38-FLAG was restricted to mitochondria, as demonstrated by confocal microscopy cells labeled with the mitochondrial marker HSP60 (Fig. S8B). After validation of our cell line, we proceed with STED microscopy, by staining cells with an anti-DNA and an anti-FLAG antibody to visualize mt-nucleoids and MRPS38-FLAG respectively. Fluorescence intensity profile analysis of anti-DNA and anti-FLAG signals revealed that the peak intensities of the two structures partially overlapping with confocal microscopy (Fig. 4H, upper panel), were instead adjacent to each other when STED was applied (Fig. 4H, lower panel). Also, MRPS38-FLAG showed a punctuate pattern in STED images (Fig. 4H, lower panel). These results support that MRPS38 interacts with TFAM due to the proximity of the mitoribosome assembly intermediaries [29,31].

In summary, we observed that inhibition of mitochondrial translation with CAP and *Mrps38* knockdown led to the upregulation of *de novo* replication and transcription of mtDNA in MEF cells. Surprisingly, the mitochondrial translation was affected to the same extent in *Mrps10* or *Mrps38* knockdowns, but no changes in mtDNA expression were detected upon depletion of MRPS10.

### 3.4. Analysis of the TFAM-FLAG interactors in the absence of POLRMT

The control of TFAM levels is crucial for mtDNA copy number regulation and mt-nucleoid compaction [8,10]. There are multiple reports that TFAM not bound to mtDNA is degraded by the LONP1 protease [17,62]. Nevertheless, in mice with a knockout of *Polrmt* in the heart, TFAM remains stable despite the depletion of mtDNA and upregulation of LONP1 protease (Fig. 5A, Table S3, [34]). To identify additional factors that contribute to the stability of TFAM, we generated heart- and skeletal muscle-specific *Polrmt* knockout mice also containing the *Tfam-FLAG*<sup>BAC</sup> transgene (*Polrmt*<sup>LoxP/LoxP</sup>, +/Ckmm-cre, *Tfam*-
<figure><img src="image_8.png" /><figcaption>**Fig. 3.** Determination of the mt-nucleoid composition in mouse.</figcaption></figure>

(A, B) Linear density glycerol gradients performed in liver mitochondria of control animals (Tfam<sup>+/+</sup>). The mtDNA was amplified in each fraction with specific primers located in Nd1. Proteins were analyzed by western blotting. (A) Gradient with 20 mM NaCl. (B) Gradient with 100 mM NaCl. (C) Fraction of mitochondrial and non-mitochondrial proteins found by TFAM-FLAG affinity enrichment experiments. (D, E) Dot plot illustrating the fold-change enrichment of proteins found by TFAM-FLAG affinity enrichment experiments in the liver, heart, and kidney from Tfam-FLAG rescue animals. The mitochondrial and non-mitochondrial proteins are indicated. The circle size corresponds to the log2 fold-change (LFC), the color shows the -log10 adjusted P value (–Log10 adj.P), and the red circumference indicates an adj.P < 0.05 (Significant). n = 4 biological replicates. Samples genotype = Tfam<sup>-/-</sup>, Tfam-FLAG<sup>BAC</sup>. Controls genotype = Tfam<sup>+/+</sup> (D) Proteins found in 20 mM NaCl samples. (E) Core mt-nucleoid proteins are enriched in at least five of the six sample groups analyzed (three tissues, two NaCl concentrations). (F) Protein network interaction of the core mt-nucleoid proteins obtained from the STRING database and modified in Cytoscape v3.10.1. Green nodes indicate mitochondrial proteins while gray nodes correspond to non-mitochondrial proteins. Cyan circumference indicates proteins annotated as mt-nucleoid components. Gray circumference indicates proteins from the cytosolic ribosome. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A

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<caption>**Fig. 4.** Downregulation of *Mrps38* leads to the activation of mtDNA expression.</caption>

(A) Western blot analysis of TFAM, HSP60, and MRPS10 in cells treated with CAP or transfected with indicated siRNAs. Vinculin was used as a loading control (n = 4–5 replicates). Blot images from MRPS10, TFAM, HSP60, and Vinculin are the same as shown in Fig. S7G. (B) Quantification of (A). TFAM protein levels relative to siRNA Neg-1 control. (C) Steady-state mtDNA levels determined by qPCR of cells treated with CAP or the indicated siRNAs. Probes for 12S, Cox1, and Atp6 were used to measure mtDNA content, and normalized to the 18S probe nuclear DNA reference. The data is presented as a fold change from negative control (Neg-1). The boxplots show the median (horizontal line), the 25 to 75 percentile interquartile range (box), the maximum and minimum observations within 1.5 interquartile range (vertical lines), and outliers (filled dots). N = 6–9 independent replicates, *p* < 0.01 = ***, *p* < 0.05 = *, Two-tailed unpaired Student's t-test against the negative control (Neg-1). (D) Southern blot of mtDNA and 7S DNA from cells treated with CAP, *Mrps38-1* siRNA, and negative control siRNA (*Neg-1*). (E) Quantification of (D). The data is presented as a fold change from negative controls (Neg-1). N = 5 independent replicates, *p* < 0.001 = ***, two-tailed unpaired Student's t-test against the negative control (Neg-1). (F) Levels of *de novo* mtDNA synthesis in mitochondria isolated from cells treated with CAP or transfected with the indicated siRNA. Western blot analysis of MRPS38, HSP60, VDAC1, and VDAC3 (n = 3 independent replicates). (G) Levels of *de novo* transcription in mitochondria isolated from cells treated with CAP or transfected with the indicated siRNA. Western blot analysis of MRPS38, HSP60, VDAC1, and VDAC3 (n = 3 independent replicates). (H) Representative microscopy images of induced HEK cells showing single channels for DNA and FLAG, and merged channels. Fluorescence intensity profiles of DNA and FLAG are shown to the right of the images.

FLAG<sup>BAC</sup>, hereafter denoted *Polrmt*<sup>CKO</sup> mice) and compared with controls (*Polrmt*<sup>LoxP/LoxP</sup>, *Tfam-FLAG*<sup>BAC</sup>). After that, we analyzed the TFAM-FLAG interactors at both ionic strength conditions in the absence of POLRMT in heart mitochondria compared to control animals. In addition to C1QBP, we found 18 new mitochondrial TFAM-FLAG interactors upon depletion of POLRMT in the heart (Fig. 5B, Table S3). We determined that 15 of the mitochondrial proteins were specifically enriched in *Polrmt*<sup>CKO</sup> animals by comparing them with heart interactors from the *Tfam-FLAG* rescue mouse (Fig. 5B). The enrichment of the TFAM-FLAG interactors correlated with its upregulation in the total proteome (LFC > 1.5) from the heart of the *Polrmt*<sup>CKO</sup> mouse (Fig. 5C). Notably, we detected a strong interaction of LONP1 with TFAM-FLAG, yet the TFAM protein is not degraded (Fig. 5A). In addition, we identified ten specific TFAM-FLAG interactors that are not upregulated due to the absence of POLRMT in heart mitochondria (LFC < 1.5 in total proteome, Fig. 5D, and E). Among these, C1QBP is an interactor in *Tfam-FLAG* rescue animals while ACAD9 [63] is present only in the heart mt-nucleoids at low ionic strength (Fig. 5B). The complex III subunit UQCRH and the MICOS subunit CHCHD6 face the intermembrane space [64,65] while HSPA9 and GRPEL1 are chaperones that support the import of proteins into the mitochondrial matrix [66–68]. ATP5MF, CYC1, and UQCR10 are OXPHOS subunits [65,69], and threonine aldolase 1 (THA1), which is expressed in mice but not in humans [70], CHCHD6, CYC1, UQCRH, and UQCR10 have never been reported as mt-nucleoid proteins (Table S1). Finally, we checked the proteins enriched in control mitochondria that were missing in the *Polrmt*<sup>CKO</sup> samples. We observed that depletion of these proteins correlates with the downregulation of total protein abundance observed in heart mitochondria of *Polrmt*<sup>CKO</sup> mice (Fig. S9A-C), except for SSBP1, which was depleted specifically as a TFAM-FLAG interactor (Fig. S9A).

In summary, TFAM-FLAG interacts with LONP1 but is not degraded efficiently upon depletion of POLRMT in the heart. Our results suggest that the interaction of TFAM-FLAG with proteins upregulated in the absence of POLRMT prevents its degradation.

### 3.5. Analysis of the mt-nucleoid in the CAG-TFAM mouse model

The TFAM-to-mtDNA ratio determines the compaction of the mt-nucleoid that influences the extent of mtDNA transcription [15,16,19]. Therefore, we investigated the mt-nucleoid composition in a mouse model with high levels of FLAG-tagged TFAM protein expressed from the ROSA26 locus under the control of the CAG-promoter, hereafter denoted CAG-TFAM mice [16]. We previously showed that CAG-TFAM mice respond to the very high increase of TFAM in a tissue-specific fashion (the molecular genotype is recapitulated in Table 1). In the heart, the CAG-TFAM mice have high levels of TFAM protein and mtDNA resulting in a near-normal TFAM-to-mtDNA ratio and maintained mtDNA gene expression. In the liver, the CAG-TFAM mice have high TFAM protein levels but no increase in mtDNA levels. The CAG-TFAM mice maintain mtDNA gene expression in the liver despite the high TFAM to mtDNA ratio likely because POLRMT and the LONP1 protease are upregulated.

In sharp contrast to the situation in these two tissues, the skeletal muscle tissue of CAG-TFAM mice has very high TFAM levels that do not lead to an increase in mtDNA levels. The very high ratio of TFAM to mtDNA in the skeletal muscle of CAG-TFAM mice leads to a drastic reduction in levels of mtDNA transcripts and a severe respiratory chain deficiency [16]. The CAG-TFAM mice thus provide an interesting model for comparison of the mt-nucleoid composition in different tissues, and we, therefore, isolated mitochondria from liver, heart, and skeletal muscle from 16 to 18 days CAG-TFAM animals to analyze TFAM-FLAG interactors (Table S3). For the analysis of the mt-nucleoid in CAG-TFAM mouse tissues, we only included the low ionic strength samples because the high ionic strength samples enriched mainly non-mitochondrial proteins (Fig. 6A). Interestingly, TFAM-FLAG interactors (LFC < 1.5, adj.P value 0.1) in the liver and heart from CAG-TFAM mice include seven of the core mt-nucleoid components like C1QBP, POLG2, POLRMT, SSBP1, TEFM, TWNK, and YME1L1. In contrast, we only found C1QBP and SSBP1 in skeletal muscle (Fig. 6B). When we analyzed the shared enriched mitochondrial proteins in all analyzed tissues from the CAG-TFAM mouse (Fig. 6B), we detected C1QBP, PHB, PHB2, and SSBP1, all of which are TFAM-FLAG interactors in the low ionic strength conditions in the *Tfam-FLAG* rescue mouse (Fig. 3D). Additional interactors were the uncharacterized protein ACAD12, the intermembrane space chaperone CLPB [71], the mitochondrial calcium uniporter MCU [72,73], and four non-mitochondrial proteins. The interaction of TFAM-FLAG with these seven proteins thus does not require normal mtDNA transcription since they are also present in the skeletal muscle of the CAG-TFAM mouse.

We compared the TFAM-FLAG interactors between the CAG-TFAM and the *Tfam-FLAG* rescue mice to define specific proteins enriched in the heart and liver from the CAG-TFAM samples. We observed seven shared mitochondrial TFAM-FLAG interactors in the heart and liver in both mouse models: C1QBP, PHB, PHB2, POLG2, POLRMT, SSBP1, and YME1L1. Additionally, eight mitochondrial TFAM-FLAG interactors were only present in the CAG-TFAM samples (Fig. 6C). DHX30 is a mitoribosome assembly factor previously reported as a mt-nucleoid component [29]. AFG3L1 is a component of the m-AAA protease facing the mitochondrial matrix [74], and PTRH2 is a peptidyl-tRNA hydrolase with multiple subcellular localizations, including mitochondria [75]. Both proteins are new TFAM-FLAG interactors. Strikingly, we found five proteins involved in the fatty acid catabolic process, ACOT2, CPT1B, ECI2, ECH1, and HADHB [76]. CPT1B is an outer mitochondria membrane protein [76], and it does not interact with TFAM in the mt-nucleoid context, while ACOT2, ECI2, and HADHB have previously been reported as mt-nucleoid interactors (Table S1). In addition, seven non-mitochondrial TFAM-FLAG interactors were enriched only in CAG-TFAM samples. The heart and liver proteome of the CAG-TFAM mouse is widely affected by the very high levels of TFAM-FLAG [16]. Therefore, it is probable that some TFAM-FLAG interactors in the heart and liver of the CAG-TFAM mouse reflected total protein abundance changes. However, we did not find a correlation between total protein abundance and the enrichment of the TFAM-FLAG interactors in the heart of the
A

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

B

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

C

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

D

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

E

<figure><img src="image_22.png" /><figcaption>**Fig. 5.** Determination of TFAM-FLAG interactors upon ablation of *Polrmt* in heart mitochondria.</figcaption></figure>

(A) Western blot analysis of TFAM, LONP1, and POLRMT on heart mitochondrial lysates from *Polrmt*<sup>CKO</sup> and control animals. VDAC and HSP60 were used as loading control (n = 3 biological replicates). (B) Dot plot illustrating the fold-change enrichment of proteins found by TFAM-FLAG affinity enrichment experiments in the heart of *Polrmt*<sup>CKO</sup> and control animals. The mitochondrial proteins are indicated. The size of the circle represents the log2 fold-change (LFC), the color shows the -Log10 adjusted P value (−Log10 adj.P), and the red circumference indicates an adj.P < 0.05 (Significant). n = 4 biological replicates. Samples genotype = *Polrmt*<sup>L/L</sup>, +/Cre, *Tfam-FLAG<sup>BAC</sup>*. Controls genotype = *Polrmt*<sup>L/L</sup>, *Tfam-FLAG<sup>BAC</sup>* (C) Hierarchical clustering heatmap showing the Person correlation of TFAM-FLAG enriched proteins and total proteome changes in the heart of *Polrmt*<sup>CKO</sup> animals. (D, E) Scatter plot showing the log2 fold change (LFC) correlation between total proteome changes and the TFAM-FLAG enriched proteins in heart mitochondria of *Polrmt*<sup>CKO</sup> animals. Proteins with a positive correlation are indicated by gray dots, except LONP1 (orange dot). Blue dots indicate proteins enriched only in TFAM-FLAG immunoprecipitation. (D) Samples with 20 mM NaCl. (E) Samples with 100 mM NaCl. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
<table><caption>**Table 1**
The molecular genotype of the CAG-TFAM mouse model.</caption><thead><tr><th>Tissue</th><th>TFAM levels<sup>a</sup></th><th>mtDNA content<sup>a</sup></th><th>Transcript levels<sup>b</sup></th></tr></thead><tbody><tr><td>Heart</td><td>4.46 ± 0.24</td><td>6.35 ± 0.66</td><td>0.67 ± 0.13</td></tr><tr><td>Liver</td><td>2.1 ± 0.23</td><td>0.87 ± 0.09</td><td>0.89 ± 0.24</td></tr><tr><td>Skeletal muscle</td><td>3.84 ± 0.52</td><td>0.96 ± 0.08</td><td>0.09 ± 0.06</td></tr></tbody></table>

<sup>a</sup> Mean fold change ± SEM compared to control animals (n = 9–12 biological replicates for heart and liver; n = 8 for skeletal muscle).

<sup>b</sup> Mean fold change ± SEM of 12s, Nd1, Cox1 and Cytb mitochondrial transcripts compared to control animals (n = 4–5 biological replicates). Data was obtained from [16].

CAG-TFAM mouse (Fig. 6D). In the liver, ACOT2 and CPT1B were upregulated and correlated with their enrichment as TFAM-FLAG interactors, but that was not the case for the rest of the proteins (Fig. 6E, F). Hence, an upregulation of the total protein levels cannot explain the specific TFAM-FLAG interactions observed in the CAG-TFAM mouse.

In summary, we show that the mt-nucleoid in the liver and heart of the CAG-TFAM mouse contains most of the core components. Additionally, we found eight TFAM-FLAG interactors that might participate in the compensatory mechanisms to very high TFAM levels in the heart and liver of the CAG-TFAM mouse. Notably, these proteins are absent in the skeletal muscle of the CAG-TFAM mouse, where mitochondrial transcription is inhibited.

## 4. Discussion

In this work, we introduce a novel mouse strain, *Tfam-FLAG BAC* transgenic mice, as a valuable tool for investigating the mt-nucleoid composition *in vivo*. Importantly, we demonstrate that the TFAM-FLAG protein is functional as it can complement the absence of the wild-type TFAM protein in *Tfam-FLAG* rescue mice. Hence, we ensure that the results obtained by the proteomics analysis reflect physiological conditions. A plethora of previous studies in cell lines have relied on the addition of purification tags like FLAG, BirA, and APEX to study the composition of the mt-nucleoid [22,24–27]. However, in most cases, the functionality of the construct was not well characterized, and high expression levels that do not reflect physiological conditions were used. The importance of these experimental factors is well exemplified by modifications made to TFAM. For instance, high expression levels of TFAM are not well tolerated *in vivo*, as exemplified by the CAG-TFAM mice [16]. Previous attempts in our lab to generate transgenic mice with a mKATE2 fluorescence tag on TFAM resulted in unstable chimeric proteins even at low expression levels (Fig. S10A, B). Also, high expression levels of TFAM-mKATE2 were required to detect fluorescence in studied tissues (Fig. S10C).

The previous investigation in cell lines has been designed to document possible components of the mt-nucleoid. However, the mt-nucleoid composition varies considerably among different studies and this circumstance hampers efforts to define the core components [20] (Table S1). The diverse purification strategies and the incomplete functional characterization of the modified proteins likely contribute to these discrepancies [20]. The *Tfam-FLAG*<sup>BAC</sup> mouse addresses these two potential pitfalls, and we show that the TFAM-FLAG protein is functional and allows the purification of the mt-nucleoid components by affinity enrichment from differentiated mouse tissues.

The proteomic analysis from the *Tfam-FLAG* rescue mouse in three tissues allowed us to define 12 core mt-nucleoid components. Our results agree with a layered structure of the mt-nucleoid [21] where the replication (POLG2, POLG, SSBP1, TWNK) and transcription (TFAM, TEFM, POLRMT) factors are central components (Fig. 3). Additionally, we identified the i-AAA protease YME1L1. As its active site faces the intermembrane space [49], we ruled out a direct remodeling of the mt-nucleoid by proteolytic activity. YME1L1 has been implicated in the

control of mtDNA metabolism by maintaining pyrimidine cellular pools, and its deletion causes an increase in mtDNA copy number [50]. It is tempting to speculate that YME1L1 could act as a sensor of mtDNA copy number through direct interaction with the mt-nucleoid or TFAM. However, the deletion of YME1L1 causes a broad remodeling of the mitochondrial proteome and accumulation of several proteins that might affect mtDNA maintenance and expression in multiple ways [50].

Surprisingly, the mitoribosome subunits MRPS10 and MRPS38, along with the assembly factors DDX28 and C1QBP, were identified as core components of the mt-nucleoid. Both the synthesis of mitochondrial RNAs and the assembly of the mitoribosome occur near the mt-nucleoid and involve the mitochondrial RNA granules [29–31,51]. It has been proposed that active transcription within the mt-nucleoid may trigger a phase separation process, similar to that observed in regions of active transcription in the nucleus [77]. Our imaging results for C1QBP and MRPS38-FLAG suggest these proteins interact with TFAM, given their localization near the mt-nucleoid and highly reminiscent of that described for mitochondrial RNA granules [29,31,51]. However, we did not observe a consistent enrichment of other RNA processing enzymes as core components of the mt-nucleoid. Collectively, these observations support the conclusion that the presence of MRPS10, MRPS38, DDX28, and C1QBP in the mt-nucleoid is not solely due to their proximity to the mitochondrial RNA granules.

Additional investigations in MEFs revealed that depletion of MRPS38 leads to the upregulation of *de novo* mtDNA replication and transcription. MRPS38 makes specific contact with the large subunit in the monosome interphase [78], and MRPS38 in yeast is required for efficient mitochondrial translation [79]. Our findings support the idea of a feedback mechanism that upregulates mtDNA transcription/replication when mitochondrial translation is inhibited. In agreement with this model, deletion of the mitoribosome assembly factors MTERF3, MTERF4, or TFB1M in animal models leads to an increase in mtDNA copy number and mitochondrial transcript levels [80–83]. Moreover, MTERF3 has been suggested as a negative regulator of mtDNA transcription [81], but its levels remain stable upon MRPS38 depletion or in CAP-treated cells (Fig. S7G).

Our data also suggest that a less compacted mt-nucleoid may contribute to the upregulation of mtDNA expression since TFAM levels do not increase in correspondence to mtDNA copy number after depletion of MRPS38 or in CAP-treated cells. Additionally, we observed an upregulation of *de novo* mtDNA replication, but not the 7S DNA, indicating that inhibition of mitochondrial translation reduces abortive mtDNA replication or the stability of the 7S DNA. It is yet to be determined whether other factors are involved in the feedback mechanism, but some possible candidates are DDX28, C1QBP, and MRPS10, which have been identified as core mt-nucleoid components. Although depletion of DDX28 or C1QBP has been shown to cause a mild increase in mitochondrial transcripts but not mtDNA [29,57,58], depletion of MRPS10 did not affect mtDNA copy number or transcript levels. Further studies are needed to elucidate whether DDX28, C1QBP, and MRPS10 participate in the upregulation of *de novo* mtDNA replication and transcription upon inhibition of the mitochondrial translation.

Previous studies on the composition of the mt-nucleoid have highlighted its dynamic nature (Table S1), suggesting that it may have a tissue-specific composition. In line with this, mutations in proteins involved in mtDNA expression are often more detrimental in specific tissues [4,13]. The *Tfam-FLAG* rescue mouse serves as an excellent model for identifying tissue-specific components of the mt-nucleoid under physiological conditions. Here, we provide a list of candidate tissue-specific mt-nucleoid proteins for the liver, kidney, and heart, which can be further validated in future studies.

We demonstrate that the *Tfam-FLAG* transgene can be used to identify mt-nucleoid components. Therefore, we proceed to characterize TFAM-FLAG interactors in two mouse models of mitochondrial dysfunction. We generated mice with disrupted *Polrmt* expression in the heart that also contain the *Tfam-FLAG* transgene to unveil TFAM
<figure><img src="image_23.png" /><figcaption>Fig. 6. Composition of the mt-nucleoid in animals with high overexpression of TFAM-FLAG.</figcaption></figure>

(A) Fraction of mitochondria and non-mitochondria proteins found by TFAM-FLAG affinity enrichment experiments in the liver, heart, and skeletal muscle from CAG-TFAM-FLAG animals. (B, C) Dot plot illustrating the fold-change enrichment of proteins found by TFAM-FLAG affinity enrichment. The mitochondria and non-mitochondrial proteins are indicated. The circle size corresponds to the log2 fold-change (LFC), the color shows the -log10 adjusted P value (–Log10 adj.P), and the red circumference indicates an adj.P < 0.1 (Significant). N = 4 biological replicates. Samples genotype = CAG-Tfam-FLAG. Controls genotype = Tfam+/+ (B) Enriched proteins in the liver, heart, and skeletal muscle of CAG-TFAM animals. (C) Enriched proteins in the liver and heart of CAG-TFAM and Tfam-FLAG rescue animals. (D) Hierarchical clustering heatmap showing the Pearson correlation of TFAM-FLAG enriched proteins and total proteome changes in the liver and heart of CAG-TFAM animals. (E, F) Scatter plot showing the log2 fold change (LFC) correlation between total proteome changes and the TFAM-FLAG enriched proteins in CAG-TFAM animals. Orange dots indicate mitochondrial and gray dots indicate non-mitochondrial proteins. (E) Correlation in the liver. (F) Correlation in the heart. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
interactors that contribute to TFAM stability despite the depletion of mtDNA [34]. Notably, we found that TFAM-FLAG interacts with LONP1 in PolrmtCKO animals but it is not efficiently degraded. LONP1 is upregulated as a compensatory response to depletion of POLRMT in the heart [34], and our results suggest that global mitochondrial protein rewiring upon depletion of POLRMT contributes to TFAM stabilization. Similar proteomic changes can be observed upon ablation of TWNK in the heart (Fig. S9C). However, in this mouse model, the depletion of mtDNA results in the degradation of TFAM [84,85]. The TFAM-FLAG interactors do not prevent its interaction with LONP1 in the PolrmtCKO animals, and it remains to be investigated if they can decrease LONP1 catalytic activity to protect TFAM from degradation. HSP9A and GRPEL1 are chaperones that could impair the LONP1-mediated degradation of TFAM-FLAG [66–68]. As an alternative explanation, the import of TFAM-FLAG could be less efficient and promote the interaction with these chaperones, also, it would explain the strong interaction with CHCHD6 and UQCRH, two proteins found in the mitochondrial intermembrane space [64,65].

The compaction of the mt-nucleoid by TFAM has emerged as an important regulatory point of mtDNA expression. Therefore, we studied the mt-nucleoid composition in the CAG-TFAM mouse. The mt-nucleoid composition in the CAG-TFAM mouse correlates with the tissue-specific response to very high levels of TFAM [16]. In the liver and heart, mtDNA transcription is active, and the mt-nucleoid composition from the CAG-TFAM mouse resembles that observed in the Tfam-FLAG rescue mouse. In contrast, in skeletal muscle of the CAG-TFAM mouse, mtDNA transcription is drastically decreased and the mt-nucleoid lacks most of its core components (POLRMT, POLG2, TEFM, TWNK, and YME1L1). We found eight TFAM-FLAG interactors that might participate in the compensatory mechanisms to counteract negative effects of the very high TFAM levels in the heart and liver of the CAG-TFAM mouse, as exemplified by ACOT2, ECI2, ECH1, and HADHB that participate in fatty acid catabolism [76]. Still, it is unclear how these enzymes might contribute to maintaining mtDNA transcription active when TFAM levels are high. One alternative is that ACOT2, ECI2, ECH1, and HADHB hamper the interaction of TFAM with mtDNA to maintain a physiological TFAM-to-mtDNA ratio. However, as a side effect, lipid metabolism might be affected and lead to the development of steatosis in the CAG-TFAM mouse [16]. Another of the TFAM-FLAG interactors in the CAG-TFAM mouse was AFG3L1. This protein assembles as homohexamers or heterohexamers with AFG3L2 or paraplegin to form the m-AAA protease complex in the mitochondrial inner membrane [86]. The m-AAA protease is essential for mitochondrial quality control, and mutations in AFG3L2 and paraplegin cause diseases with neurological manifestations [49]. Deletion of AFG3L1 does not cause evident phenotypes in mice, while in humans, it is not expressed [74,87]. LONP1 has been proposed as the main protease that controls TFAM levels [17,62], but our data suggest that AFG3L1 might also be a regulatory protease when the TFAM levels are very high. The mitoribosome assembly factor DHX30 [29] was also enriched in the CAG-TFAM mouse, again suggesting crosstalk between mitochondrial translation and regulation of mtDNA transcription. Notably, these proteins are absent from the mt-nucleoid in the skeletal muscle of the CAG-TFAM mouse, which has severely decreased mitochondrial transcription [16].

In summary, we demonstrate that the Tfam-FLAG transgenic mouse is a powerful model for studying the mt-nucleoid composition under physiological conditions. We have defined the core components of the mt-nucleoid in vivo and our data support that there is a crosstalk between mitochondrial translation and mtDNA transcription/replication. Moreover, the Tfam-FLAG transgene can be used in models of mitochondrial dysfunction to uncover factors that might influence mtDNA gene expression for further investigation.

# 5. Study limitations

Our approach to defining mt-nucleoid composition in mouse tissues is focused on identifying reproducible components, which may mean that transient or weakly associated factors are overlooked. For example, the mitochondrial transcription initiation factor TFB2M [88] was only detected in the 100 mM NaCl heart and 20 mM NaCl liver samples (Table S2). The low reproducibility of TFB2M detection in our datasets may be attributed to its transient interaction with TFAM, as well as the relatively low proportion of transcriptionally active mt-nucleoids. Similarly, despite its critical role in mitochondrial transcription termination, MTERF1 [89] is absent from all our datasets. We find it puzzling that MTERF1 is not detected as a mt-nucleoid component, and we cannot offer a clear explanation for this observation. Additionally, our finding that C1QBP, DDX28, MRPS10, MRPS38, and YME1L1 may be core mt-nucleoid components needs further validation.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbamcr.2025.119955.

## CRediT authorship contribution statement

**Rodolfo García-Villegas:** Writing – review & editing, Writing – original draft, Visualization, Validation, Project administration, Methodology, Investigation, Data curation, Conceptualization. **Franka Odenthal:** Methodology, Investigation, Data curation. **Yvonne Gian-noula:** Visualization, Investigation, Data curation. **Nina A. Bonekamp:** Visualization, Methodology, Investigation, Conceptualization. **Inge Kühl:** Methodology, Investigation, Data curation. **Chan Bae Park:** Methodology. **Henrik Spåhr:** Methodology, Investigation. **Elisa Motori:** Supervision, Methodology, Funding acquisition. **Fredrik Levander:** Software, Formal analysis, Data curation. **Nils-Göran Larsson:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Funding acquisition, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nils-Göran Larsson reports a relationship with Pretzel Therapeutics, Inc. that includes: consulting or advisory and equity or stocks. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

N.-G.L. was supported by the Swedish Research Council (2015-00418), the Swedish Cancer Foundation (243520Pj), the Knut and Alice Wallenberg Foundation (2023.0224 and 2024.0081), the European Research Council (Advanced Grant 101141290), the Novo Nordisk Foundation (NNF22OC0078444), and grants from the Swedish state under the agreement between the Swedish government and the county councils (FOUI-1001906) and the Max Planck Society. E.M. was funded by the Deutsche Forschungsgemeinschaft (SFB 1218—269925409 and EXC 2030—390661388). I.K. was supported by the French Muscular Dystrophy Association (AFM-Téléthon #23294) and Agence nationale de la recherche (ANR-20-CE12-0011).

We thank the Proteomics Biomedicum Core Facility, Karolinska Institutet for mass spectrometry data acquisition (https://ki.se/en/research/proteomics-biomedicum-core-facility), and the CECAD Imaging Facility for the use of microscopes. We thank Maria Miranda for her help with mitochondrial isolations and tissue collection.
## Data availability

The mass spectrometry data from this publication have been deposited to the ProteomeXchange Consortium via the PRIDE [90] partner repository with the dataset identifier PXD047950. Source data are provided with the publication. Further requests should be directed to the corresponding authors.

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