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Published in final edited form as: J Mol Biol. 2025 Sep 6;437(21):169433. doi: 10.1016/j.jmb.2025.169433

Phosphomimetic Experiments Do Not Support a Causal Role for TFAM Phosphorylation in mtDNA Elimination in Sperm

Natalya Kozhukhar 1, Yidong Bai 2,3, Mikhail F Alexeyev 1
PMCID: PMC12930458  NIHMSID: NIHMS2140199  PMID: 40921294

Abstract

In sexually reproducing eukaryotes—particularly mammals—mitochondrial DNA (mtDNA) is typically inherited from a single parent, making uniparental mtDNA inheritance a fundamental feature of eukaryotic biology. Recently, it has been suggested that spermatozoa contain no mtDNA because the matrix targeting sequence (MTS) of the mitochondrial transcription factor A (TFAM) becomes phosphorylated, which prevents the mitochondrial import of this protein essential for mtDNA replication. In this study, we used a combination of the GeneSwap technique and phosphomimetic mutations to investigate the impact of TFAM MTS phosphorylation on mtDNA maintenance in cultured cells. TFAM variants carrying phosphomimetic substitutions—S31D, S34D (TFAM-DD), and the double mutants S31D, P32D/S34D, F35D (TFAM-4D)—supported mtDNA maintenance in 143B cells, with their MTSs at least partially processed. This occurred despite the overall negative charge of the MTS in the TFAM-4D variant. Moreover, blocking the MTS processing by a combination of an overall negative charge and a mutation in the arginine residue critical for MTS cleavage did not prevent mtDNA maintenance. These observations led us to conclude that TFAM MTS phosphorylation alone is unlikely to explain mtDNA loss in human sperm during maturation.

Keywords: TFAM, phosphorylation, mtDNA loss, maternal mtDNA inheritance, mitochondrial import

Introduction

It has long been established that human spermatozoa contain the lowest copy number of mitochondrial DNA (mtDNA) among all human cell types, excluding erythrocytes, which lack mtDNA entirely [1]. Despite occasional reports of paternal mtDNA transmission (“leakage”) in animals [2-8] and human patients [9-11], such findings are often dismissed or contested on technical grounds [12-14], thereby reinforcing the long-standing dogma of strictly maternal mtDNA inheritance in humans.

No single mechanism accounts for uniparental mtDNA inheritance (UMI) across all animal species. In nematodes, for instance, the breakdown of the mitochondrial inner membrane in sperm allows CPS-6 (also known as endonuclease G or EndoG), typically located in the intermembrane space, to access and degrade paternal mtDNA [15]. In addition, sperm mitochondria are actively degraded within the zygote, eliminating any remaining mtDNA not degraded by EndoG [16-19]. In Drosophila, EndoG is similarly implicated; however, even in EndoG mutants, a seemingly redundant mechanism ensures the removal of paternal mtDNA [20]. Recent studies have shown that Poldip2 exonuclease functions in concert with EndoG in this organism to degrade mtDNA [21,22]. However, despite the presence of multiple mechanisms for paternal mtDNA elimination, emerging evidence suggests that paternal mtDNA leakage may represent an evolutionarily conserved and regulated process [23].

In monkeys and cows, sperm mitochondria are ubiquitinated during spermatogenesis, marking them—and their mtDNA—for degradation in the fertilized egg [24]. In fish, paternal mtDNA is eliminated in two phases: first, by reducing its copy number during spermatogenesis, and then through active destruction post-fertilization [25]. In mice, sperm mtDNA is eliminated before reaching the oviduct [26], a process found to be independent of both EndoG [27] and autophagy [28]. Notably, failure to remove mtDNA at this stage results in paternal transmission to offspring, indicating that autophagic degradation of sperm mitochondria does not play a central role in mouse UMI [26].

Generally, in short-lived model organisms such as Caenorhabditis elegans and Drosophila melanogaster, spermatogenesis is brief (6–7 h and 2 days, respectively [29,30]), necessitating active mtDNA degradation for timely completion. In contrast, human spermatogenesis spans approximately 74 days [31], a duration that may allow for the elimination of mtDNA through passive turnover upon cessation of its replication.

In humans, the exact mechanism of UMI remains unresolved and is currently under active debate [32,33]. The ‘TFAM sequestration” model proposed by Lee et al. suggests that sperm-specific phosphorylation of the mitochondrial targeting sequence (MTS) of mitochondrial transcription factor A (TFAM) reduces its net positive charge. This modification prevents mitochondrial import of TFAM, leading to its sequestration in the nucleus and/or cytosol, thereby rendering it unavailable for mtDNA replication and protection [32].

In contrast, the competing “mtDNA turnover” model emphasizes the absence of key mtDNA replication factors in sperm, such as the catalytic subunit of mitochondrial DNA polymerase (POLG) and mitochondrial RNA polymerase (POLRMT) [33]. Knockout of either protein in cultured cells results in mtDNA loss [34], supporting a model in which mtDNA is gradually lost through natural turnover during the extended process of sperm maturation.

Given that the TFAM sequestration model hinges on phosphorylation-driven reduction of the TFAM MTS net positive charge, we employed a combination of GeneSwap and phosphomimetic validation approaches to investigate the consequences of this modification on mtDNA maintenance. Our results demonstrate that phosphomimetic substitutions mimicking TFAM MTS phosphorylation, even when combined with MTS charge reversal and inhibition of MTS processing, do not fully block mtDNA maintenance. These findings suggest that TFAM phosphorylation alone is unlikely to be the sole determinant of UMI in humans.

Results

Single-substituted TFAM phosphomimetics support mtDNA maintenance

TFAM GeneSwap 143B#6 human osteosarcoma cells were described previously [34]. This cell line carries a biallelic CRISPR/Cas9-mediated inactivation of the TFAM gene rescued with retrovirally delivered wt “floxed” hTFAM cDNA, which can be removed with cre recombinase, rendering host cells TFAM−/− and inducing the loss of mtDNA unless a functional TFAM variant is co-introduced with cre recombinase.

In studies of phosphoregulation, it is common practice to mimic in vivo phosphorylation by substituting phosphorylatable serine and threonine residues with “phosphomimetic” aspartate and glutamate residues, respectively. These substitutions introduce negatively charged side chains at positions that are typically phosphorylated, thereby simulating constitutive phosphorylation [35].

Accordingly, we replaced serine residues at positions 31 and 34 with aspartates (S31D/S34D). As controls, we also generated alanine substitutions at the same positions (S31A/S34A). Both the alanine (TFAM-AA, 15/15 clones) and phosphomimetic (TFAM-DD, 21/22 clones) variants generally supported mtDNA maintenance (Figure 1C, D and Supplementary Figure S3C and D). These TFAM-AA and TFAM-DD constructs were delivered using retroviral vectors flanked by PhiC31 recombinase recognition sites (Figure 1, diagram E), allowing for selective excision of the transgenes. Excision of either TFAM-AA or TFAM-DD using PhiC31 recombinase resulted in mtDNA loss, confirming that both variants functionally supported mtDNA maintenance (Figure 1F).

Figure 1.

Figure 1.

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Single-substituted TFAM phosphomimetics support mtDNA maintenance. TFAMwt was swapped for TFAMwt, TFAM-AA, or TFAM-DD in 143B#6 cells, and TFAM expression and mtCN were examined in the resulting clones. C, TFAM-AA supports mtDNA maintenance. Diagrams a and b correspond to PCR diagnostic strategies in subpanels a and b. NTC, No template control. D, TFAM-DD supports mtDNA maintenance. Diagrams A and B correspond to PCR diagnostic strategies in subpanels a and b. F, mtDNA loss upon excision of TFAM-AA and TFAM-DD validates that these mutants support mtDNA maintenance. Diagram E corresponds to the PCR diagnostic strategy in subpanel e. G and H, TFAM expression and mtCN in GeneSwap clones. TFAM-DD#12 in panel D is in the process of mtDNA loss. It shows no TFAM expression in G or mtDNA in H. ΔTFAM, TFAMwt in 143B#6 cell line was excised with Cre recombinase without introducing a TFAM variant (TFAMvar, no GeneSwap control). mtDNA and TFAM expression are lost in ΔTFAM cells.

Among the 22 TFAM-DD, one (clone #12) exhibited weak mtDNA amplification during initial screening (Figure 1D) and eventually lost its mtDNA entirely (Figure 1H). Analysis of this clone revealed no detectable TFAM expression (Figure 1G), likely due to viral rearrangement, transcriptional silencing, or positional effects associated with vector integration.

Importantly, none of the three examined TFAM-DD phosphomimetic clones displayed defects in TFAM processing, which would be indicated by the presence of a higher molecular weight band (by ~5 kDa) corresponding to unprocessed TFAM (Figure 1G).

Clone #3 of TFAM-DD maintained a mitochondrial DNA copy number (mtCN) comparable to that of wild-type TFAM, although it exhibited higher expression levels, suggesting possible regulatory differences. Clones #16 and #23 of TFAM-DD expressed TFAM at levels similar to TFAM-AA in clone #8, and their mtCNs were also similar (Figure 1G, H). These results indicate that the phosphomimetic TFAM variants are functionally comparable to the alanine-substituted controls.

Similarly, TFAM expression levels and mtCNs were comparable among TFAM-AA clones #6 and #11 and TFAM-DD clone #3, further supporting the conclusion that the negative charge introduced by aspartate substitutions does not impair TFAM function in mtDNA maintenance.

Nevertheless, clones TFAM-AA#8 and TFAM-DD#16 and #23 with TFAM expression similar to that of wt TFAM had reduced mtCNs compared to wt TFAM, suggesting a possible generalized defect.

Double-substituted TFAM phosphomimetics support mtDNA maintenance

It has been proposed that phosphorylation introduces a greater negative charge (approximately −1.5) than aspartate substitution (−1). Consequently, substituting two adjacent amino acids with aspartates (cumulative charge −2) may more closely mimic the effects of in vivo phosphorylation at a single serine residue [35-37]. To test whether such double aspartate substitutions prevent the mitochondrial import of TFAM, we generated TFAM-4A (S31A, P32A/S34A, F35A) and TFAM-4D (S31D, P32D/S34D, F35D) variants. These constructs had previously been used to support the hypothesis that phosphorylation of the TFAM mitochondrial targeting sequence (MTS) hinders its mitochondrial import [32].

Following replacement of wild-type TFAM with these mutants in 143B#6 cells, we observed that both TFAM-4A and TFAM-4D supported mtDNA maintenance (Figure 2A, B, and Supplementary Figure S3E, F). Similar to our observations with TFAM-DD, one of the 23 randomly selected TFAM-4D clones (clone #13) lacked mtDNA (Figure 2B, D), and further analysis revealed an absence of TFAM expression in this clone (Figure 2E), likely due to vector-related issues. As expected, MT-CO2 expression in the remaining clones correlated with TFAM expression levels (Figure 2E). Notably, several clones expressing TFAM-4D, along with TFAM-4A clone #11, exhibited an additional higher molecular weight TFAM band, consistent with the presence of an unprocessed MTS.

Figure 2.

Figure 2.

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Double-substituted TFAM phosphomimetics support mtDNA maintenance. TFAMwt was swapped for TFAMwt, TFAM-4A, or TFAM-4D in 143B#6 cells, and TFAM expression and mtCN were examined in the resulting clones. A, TFAM-4A supports mtDNA maintenance. B, TFAM-4D supports mtDNA maintenance. C, mtDNA loss upon excision of TFAM-4A and TFAM-4D validates that these mutants support mtDNA maintenance. D and E, mtCN and TFAM expression in GeneSwap clones. TFAM-4D#13 in panel B lost its mtDNA. It shows no TFAM expression in E or mtDNA in D. ΔTFAM, TFAMwt in 143B#6 cell line was excised with Cre recombinase without introducing a TFAM variant (no GeneSwap control). mtDNA and TFAM expression are lost in ΔTFAM cells. Recombination schematics and PCR strategies in subpanels a, b, and e correspond to those in Figure 1. hTFAM short exposure and hTFAM long exposure correspond to short and long exposures of the same membrane probed with TFAM antibodies.

According to the “TFAM sequestration” model [32], reduced MTS positive charge resulting from phosphorylation would prevent mitochondrial import, leading to cytosolic retention and unprocessed MTS. To investigate whether the higher molecular weight TFAM isoform represents cytosolic TFAM, we tested its accessibility to proteinase K in crude mitochondrial preparations.

Surprisingly, this TFAM isoform was resistant to proteinase K digestion, similar to mitochondrial matrix proteins such as SOD2, SSBP1, and MT-ATP5, and in contrast to nuclear FEN1, outer mitochondrial membrane protein TOM70, and cytosolic GAPDH—all of which were digested. This result suggests that, contrary to predictions of the “TFAM sequestration” model, the higher molecular weight TFAM isoform is not cytosolic but is instead protected within mitochondria (Figure 3A).

Figure 3.

Figure 3.

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The high molecular weight TFAM 4D and TFAM S31D, P32D/S34D, F35D/R40E (TFAM-4D/E) isoforms are inaccessible to extramitochondrial proteinase K. A, Crude mitochondria were isolated from cells expressing wt TFAM, TFAM-4D, or TFAM-4D/E, were treated with or without proteinase K, and subjected to western blotting with the indicated antibodies. B, Sizing the high molecular weight TFAM band. Cruse mitochondrial preps from cells expressing either wt or mutant TFAM were run side-by-side with TFAM translated in vitro using an E. coli coupled in vitro transcription/translation system (IVT).

To properly size the high molecular weight TFAM band corresponding to TFAM-4D, we ran the mitochondrial lysate from cells expressing this construct side-by-side with TFAM produced using an Escherichia coli coupled in vitro transcription/translation system. Since E. coli lacks mitochondria, TFAM synthesized by this system retains its MTS and can serve as a reference for non-processed TFAM. Indeed, the two bands observed in proteinase K-treated crude mitochondrial preparations of cells expressing TFAM 4D correspond to TFAM with cleaved and retained MTS (Figure 3B).

TFAM MTS charge reversal, combined with the blockage of MTS processing, does not necessarily block mitochondrial import

According to the prevailing theory, both the net positive charge of the mitochondrial targeting sequence (MTS) and the presence of an arginine residue at the −2 position relative to the MTS cleavage site (the “R–2 rule”) are critical for proper MTS processing [38]. The absence of MTS processing in sperm TFAM is a key piece of evidence supporting the “TFAM sequestration” model [32]. To further investigate this model, we examined mtDNA maintenance in cells expressing a TFAM-4D mutant in which MTS processing was further impaired by substituting the arginine at position −2 with glutamate, generating the TFAM-4D/E variant.

This modification resulted in a predicted MTS net charge of −2.25 at pH 7 as calculated by VectorNTI 10.3.0 software, a reversal of the strongly positive +3.74 net charge seen in wild-type (wt) and alanine-substituted TFAM MTS sequences. Initial screening of cells GeneSwapped with TFAM-4D/E revealed that most clones retained mtDNA (Figure 4A). However, the majority showed reduced mtCN (Figure 4B), which correlated with decreased expression of both TFAM and the mtDNA-encoded gene MT-CO2 (Figure 4C). Notably, clones #6-II and #12 maintained mtCN levels within 50% of those seen in cells expressing wt TFAM, and mtDNA nucleoid staining was readily identifiable (Supplementary Figure S3G).

Figure 4.

Figure 4.

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TFAM 4D/E can support mtDNA maintenance. A, Most TFAM4D/E clones retain mtDNA on initial screening. Recombination schematics and PCR strategies in subpanels a, b, and e correspond to those in Figure 1. B, mtDNA in TFAM 4D/E clones #6-II and #12 is maintained at at least 50% of TFAM wt. C, Low mtCN in TFAM 4D/E clones correlates with low TFAM expression. MTS in TFAM 4D/E is not processed as suggested by higher molecular weight bands. TFAM short and TFAM long refer to exposure time. D, PhiC31-mediated excision of the TFAM 4D/E in clone #6-II results in the loss of mtDNA, suggesting that this mutant can support mtDNA maintenance.

Importantly, TFAM-4D/E migrated at a higher molecular weight, consistent with impaired MTS processing. To confirm its functional role in mtDNA maintenance, we performed PhiC31 recombinase-mediated excision of the TFAM-4D/E gene in clone #6-II. The removal of TFAM-4D/E led to the loss of mtDNA, confirming that this variant was indeed supporting mtDNA maintenance in that clone (Figure 4D).

To assess whether any of TFAM-4D/E was localized inside mitochondria, we performed proteinase K protection assays using crude mitochondrial preparations. Similar to wt TFAM and mitochondrial matrix proteins SSBP1 and SOD2, most of TFAM-4D/E was protected from proteinase K digestion, suggesting its localization within mitochondria (Figure 3A). In contrast, nuclear protein FEN1, cytosolic GAPDH, and the mitochondrial outer membrane protein TOM70—all located outside the mitochondrial outer membrane—were fully digested by proteinase K treatment.

Finally, we compared the size of TFAM-4D/E to wt TFAM extracted from the mitochondrial matrix, as well as to in vitro-translated TFAM synthesized using an E. coli transcription/translation system (which retains the unprocessed MTS). As with high molecular weight TFAM-4D, the TFAM-4D/E variant corresponded in size to the unprocessed form, confirming that it retains its MTS (Figure 3C).

Discussion

Despite its fundamental importance, the mechanism underlying maternal mtDNA inheritance in humans remains unresolved. Recently, significant debate has emerged around two competing models describing how mtDNA is lost in sperm. The “TFAM sequestration” model posits that mtDNA loss results from the extramitochondrial retention of TFAM, driven by phosphorylation of its mitochondrial targeting sequence (MTS). This modification is thought to prevent TFAM from entering mitochondria, rendering it unavailable for mtDNA replication and for shielding mtDNA from degradation by unidentified nucleases [32].

In contrast, the “mtDNA turnover” model argues that the idea of TFAM physically protecting mtDNA is inconsistent with experimental data. Instead, it emphasizes that several essential components of the mtDNA replication machinery—most notably POLG and POLRMT—are not expressed in sperm. Thus, mtDNA loss is attributed to the inability to replicate mtDNA during the ~74-day maturation process, coupled with its natural turnover [33].

The “TFAM sequestration” model, however, does not provide direct evidence for a causal relationship between TFAM MTS phosphorylation, MTS cleavage, and the inhibition of mitochondrial import. Rather, it demonstrates that in sperm, most TFAM retains its MTS, the MTS is phosphorylated, and TFAM is predominantly localized outside mitochondria [32]. Additionally, it shows that phosphomimetic TFAM-4D exhibits both nuclear and mitochondrial localization in HEK293 cells. Given that overexpression via transfection can lead to TFAM mislocalization [39,40], we aimed to investigate the role of TFAM MTS charge and processing in mtDNA maintenance under physiologically relevant conditions. This was achieved using the GeneSwap approach to deliver a single-copy TFAM gene, ensuring normal expression levels [34].

Our results clearly demonstrate that both TFAM-DD and TFAM-4D variants are capable of supporting mtDNA maintenance, and that approximately half of the TFAM-4D protein undergoes MTS processing. This occurs despite the reversed net charge of the TFAM-4D MTS compared to the phosphorylated TFAM MTS (−0.25 vs. +0.74, respectively). This discrepancy indicates that MTS net charge alone does not determine MTS processing or TFAM localization in situ.

Remarkably, even when MTS processing was inhibited by mutating the −2 arginine residue—thereby increasing the MTS net negative charge to −2.25 in the TFAM-4D/E variant—mtDNA maintenance was not completely abolished. Furthermore, the putatively unprocessed TFAM variants in both TFAM-4D and TFAM-4D/E were resistant to digestion by extramitochondrial proteinase K, suggesting their import into mitochondria.

Notably, we did not observe a massive relocalization of TFAM phosphomimetics to the nucleus, as predicted by the TFAM sequestration model (Supplementary Figure S4).

Taken together with their capacity to support mtDNA maintenance, these findings strongly suggest that TFAM-4D and TFAM-4D/E variants at least partially localize to the mitochondrial matrix. The ability of precursor proteins to reach the mitochondrial matrix without undergoing MTS cleavage is not without precedent. In line with our findings, reduction of the MTS positive charge in rat aldehyde dehydrogenase also prevented its processing but did not impair mitochondrial matrix localization or enzymatic activity of the precursor protein [41].

These observations directly challenge the “TFAM sequestration model,” which posits a causal link between unprocessed MTS and extramitochondrial localization. Instead, our data support a model in which MTS processing is not an absolute requirement for TFAM import or function within mitochondria.

Importantly, although some clones geneswapped with TFAM-DD or TFAM-4D—and the majority of those gene-swapped with TFAM-4D/E—exhibited either reduced mtDNA copy number (mtCN) or complete mtDNA loss, these clones also displayed little or no detectable TFAM expression. This finding contradicts the sequestration model, which predicts mislocalization of TFAM rather than reduced expression. Collectively, our results challenge the core assumptions of the TFAM sequestration hypothesis.

A second key conclusion of our study pertains to the roles of MTS net positive charge and the coupling of mitochondrial protein import to MTS cleavage. We show that TFAM-4D/E supports mtDNA maintenance in situ despite a reversed MTS charge (+3.74 to −2.25) and the absence of MTS cleavage. According to the current paradigm, both features are essential for mitochondrial TFAM import [42-45], which, in turn, is required for mtDNA maintenance. However, this paradigm is largely based on in vitro studies employing simplified, reconstituted systems, which may not fully replicate physiological conditions, and in vivo requirement for MTS positive charge and cleavage may be more flexible than previously thought. Alternatively, TFAM-4D and TFAM-4D/E may be imported via a non-canonical, presequence-independent pathway [46], which is consistent with previously suggested multiple subcellular localization signals present in this protein [47].

Materials and Methods

Cell lines and cultivation

Cells were routinely propagated in Dulbecco’s Modified Eagle Medium (DMEM, VWR cat# 45000-304) containing 4.5 g/L glucose, 10% Fetal Bovine Serum (FBS), 50 μg/ml gentamycin, 50 μg/ml uridine, and 1 mM sodium pyruvate in a humidified atmosphere containing 5% CO2 at 37 °C. This modification of the medium (+UP) is permissive for the growth of the ρ0 cells. After confirming the presence of mtDNA, ρ+ GeneSwapped cells expressing mutant TFAMs were maintained in the medium without uridine or pyruvate.

All cell lines were authenticated by STR analysis (LabCorp, Burlington, NC) and tested quarterly for mycoplasma contamination.

DNA constructs

Retroviral constructs used in this study are presented in Supplementary Figure S1. Plasmids were constructed according to standard recombinant DNA protocols [48]. Site-directed mutagenesis was performed by an overlap extension method as described previously [47]. For GeneSwap experiments, wt and mutant TFAM variants were cloned in retroviral vector pMA4659 (Addgene#184854), in which the polylinker is flanked by attP and attB sites for PhiC31 recombinase, allowing for the subsequent transgene excision with PhiC31. For the excision of TFAM variants, PhiC31 recombinase was delivered with the help of the retroviral vector pMA5136 (Addgene#184853).

Retrovirus production

Retroviral supernatants were produced by standard protocols [34]. Phoenix Ampho cells (ATCC CRL-3213) were plated in 60-mm dishes overnight at 2.5 * 106 cells per well to achieve ~80% confluency the next day. Cells were transfected with a mixture of 3.5 μg of retroviral vector plasmid plus 3.5 μg of GAG-Pol plasmid using linear polyethyleneimine (PEI) MW = 25,000 (Polysciences, Cat#23966-100, Warrington, PA, USA) at 1:3 (w:w) ratio of DNA to PEI. Retrovirus-containing supernatants were collected 48 h after transfection, filtered through 0.45 μm filters (Fisher Cat# 09-928-063, Thermofisher, Waltham, MA, USA), and stored at −80 °C.

Viral transduction

Viral transduction was performed as previously described [49]. Briefly, 143B#6 cells were seeded at 20–40% confluence in wells of a 6-well plate and allowed to attach for 4–24 h. Once cells were attached, the medium was replaced with 2 ml of a mixture consisting of fresh DMEM (1 ml) plus retroviral supernatant (1 ml) and supplemented with polybrene (10 μg/ml). After overnight incubation, the medium was replaced with 2 ml of fresh DMEM medium, and cells were grown in this medium for another 24 h, trypsinized, serially diluted, and plated into 150-mm dishes in DMEM medium supplemented with appropriate antibiotics. After colonies appeared, they were picked into 24-well plates, expanded, and analyzed by polymerase chain reaction (PCR).

GeneSwap approach

GeneSwap in 143B#6 cells was executed as described earlier [34]. To implement GeneSwap (see Supplementary Figure S2 for diagrams), 143B#6 cells were cotransduced with rv.3442 and a retrovirus carrying a hTFAM variant. This resulted in the simultaneous excision of the endogenous hTFAMwt and the introduction of either wt or mutant TFAM cDNA.

Genotyping of hTFAM excision in 143B#6 Cells. PCR diagnostics of hTFAM excision in 143B#6 cells was conducted using primers listed in Supplementary Table S1 according to Figure 1 scheme A.

Introduction of mutant TFAMs was verified by PCR using primers listed in Supplementary Table S1 according to Figure 1 scheme B. Subsequently, the identity of the mutants was confirmed in selected clones by PCR amplifying the proviral insert and sequencing the PCR product.

PhiC31-mediated excision of proviral inserts

TFAM variants were excised by transducing cells with rv.5136 (Supplementary Figure S1), which encodes a codon-optimized PhiC31 recombinase (PhiC31o) and hygromycin resistance. Successful excision was verified by PCR genotyping using primers listed in Supplementary Table S1 according to Figure 1 scheme E.

mtDNA detection

In the diagnostic screens of GeneSwapped clones, two duplex PCR primer mixes were used (Supplementary Table S1). Both mixes contained primers targeting the mtDNA control region. However, in Figure 2A and B, nDNA primers amplified a 618 bp fragment of the single-copy POLRMT gene, while in the remaining panels, a different mix containing nDNA primers amplified a 389 bp fragment of the multicopy 18S rRNA gene. Consequently, mtDNA amplification predominates over nDNA amplification in most clones shown in Figure 2A and B. nDNA amplification is only observed in clones with absent mtDNA or reduced mtCN.

Quantification of mtDNA

mtCN was determined as described previously [50]. Briefly, cells were collected by trypsinization, counted, and ~106-cell pellets were generated and frozen at −80 °C. Pellets were resuspended in PBS at ~10,000 cells/μL, and 10 μL aliquots were removed and mixed with 90 μL of solution containing 50 μg proteinase K, 40 μL of H2O, and 50 μL of the DirectPCR solution (Genprice Inc., San Jose, CA, USA Cat# 388-302-C). The mix was incubated at 50 °C for 30 min and then at 95 °C for another 30 min. The solution was adjusted to 500 μL with H2O, and 3 μL of the resulting solution was used as a template in 20 μL ddPCR to determine nuclear DNA (nDNA) content using the primers and probes listed in Supplementary Table S1. For mtDNA quantification, nDNA samples were diluted 500-fold, and 3 μL of the resulting dilution was used in 20 μL ddPCRs with the primers and probes listed in Supplementary Table S1. dddPCRs contained 0.9 μM of each forward and reverse primer, 0.25 μM probe, 10 μL of the 2× ddPCR Supermix for Probes (No dUTP, Bio-Rad, Hercules, CA Cat#1863023), 10 units of EcoRI HF restriction enzyme (New England Biolabs, Beverly, MA, USA, Cat# R3101S), and the balance of water. The cycling parameters were as follows: initial denaturation for 10 min at 95 °C, followed by 40 cycles of 20 s at 94 °C + 1 min at 60 °C, 10 min at 98 °C, and held at 4 °C. Each sample was measured in 2 technical replicates. To calculate mtCN per cell, the concentration of mtDNA targets was multiplied by the dilution factor and divided by the 0.5 × concentration of nDNA targets. Each mtDNA template concentration was combined with each nDNA template concentration, generating four mtCN values for each sample.

Preparation of the crude mitochondrial fractions and proteinase K treatment

5–150 million cells were lifted off four × 150-mm dishes with trypsin, washed twice with PBS, resuspended in 800 μL MSH buffer (210 mM Mannitol, 70 mM sucrose, 5 mM HEPES pH 7.5), and homogenized with a Kontes dounce homogenizer until ~80% were broken, judging by trypan blue staining. The suspension was centrifuged at 1,200g for 5 min at 4 °C, the supernatant was removed into a fresh tube, and the centrifugation was repeated. Mitochondria were pelleted at 14,000g for 5 min at 4 °C. The pellet was resuspended in the fresh MSH buffer, split into aliquots, and treated with 300 μg/ml proteinase K for 15 min on ice, after which proteinase K was inactivated with PMSF (1 mM final concentration) for 5 min on ice. Mitochondria were pelleted at 14,000g for 5 min at 4 °C. These final mitochondrial pellets were dissolved in 100 μL of 1× SDS-PAGE sample buffer, sonicated, boiled for 5 min, and separated by SDS-PAGE.

In vitro translation of TFAM

TFAM wt and its mutants were cloned into NEBExpress Control DHFR-His plasmid in place of the DHFR gene and used to generate in vitro translated products according to the manufacturer’s recommendations (New England Biolabs). In vitro-translated products were diluted and run side by side with mitochondrial lysates as a reference.

Western blotting

Western blotting was performed as described previously [51,52]. The antibodies were: α-Fen1, GeneTex GTX101777; α-GAPDH Cell Signaling Technology #2118; α-MT-ATP8, Abclonal A17890; α-SOD2, Santa Cruz sc-137254; α-SSBP1, Proteintech 12212-1-AP; α-TFAM, Abclonal A3173 and Proteintech 22586-1-AP; TOM70, Proteintech 14528-1-AP.

Microscopy

Live cells were grown on coverglass and stained in the growth media supplemented with 100 nM MitoTracker Red CMXRos (MTR, Fisher Scientific) and a 1:10,000 dilution of the commercial preparation of SYBR Gold (Fisher Scientific) for 1 h at 37 °C in a humidified atmosphere containing 5% CO2.

After staining, the medium was replaced with regular growth medium, and images were captured using a Zeiss LSM 980 laser scanning confocal microscope equipped with an AiryScan II array-based detector and a 63× oil-immersion objective (N.A. = 1.4). SybrGold dye was excited with a 488 nm laser, while Mitotracker CMX-ROS was excited using a 561 nm laser. A super-resolved AiryScan image at 3× confocal zoom was first acquired of a cell of interest, with separate channels for each wavelength. Subsequently, a reference image was taken using the standard confocal detector at 1× confocal zoom of the same location, including SybrGold and Mitotracker channels. All images were collected at room temperature to minimize mitochondrial movement.

For intracellular TFAM localization via immunofluorescence, cells were cultured and stained with MTR as described above. Following this, they were fixed with 4% formaldehyde in PBS for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for an additional 15 min, and incubated overnight at 4 °C with a 1:200 dilution of α-TFAM antibody (Proteintech 22586-1-AP) in PBS containing 10% goat serum.

After the overnight incubation, the primary antibody was removed, and cells were washed three times with PBS. They were then incubated with a 1:400 dilution of Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (Fisher Scientific A11008) for 1 h at room temperature in PBS supplemented with 10% goat serum. Following secondary antibody incubation, cells were washed again as described above and mounted using ProLong™ Gold Antifade Mountant (Fisher Scientific P10144).

Mounted slides were imaged as above, except without zoom.

Supplementary Material

Supplementary

Acknowledgements:

The authors wish to acknowledge Viktoriya Solodushko and Drs. Joel Andrews, Exing Wang, and Qing Zhou for their advice and assistance with immunofluorescence and confocal imaging.

Funding

The authors were supported by grants from the Baptist Health Foundation of San Antonio (YB), NIH 5P30CA054174 (YB), 1R01CA283840 (YB), 1R21AI171940 (YB), S10OD025089 (MA), NSF FAIN2419655 (MA), DoD 81XWH2110161 (MA), and W81XWH2110669 (MA).

Appendix A. Supplementary material

Supplementary material to this article can be found online at https://doi.org/10.1016/j.jmb.2025.169433.

Footnotes

CRediT authorship contribution statement

Natalya Kozhukhar: Writing – original draft, Investigation. Yidong Bai: Writing – review & editing, Writing – original draft, Formal analysis. Mikhail F. Alexeyev: Writing – review & editing, Writing – original draft, Project administration, Investigation, Funding acquisition.

DECLARATION OF COMPETING INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

DATA AVAILABILITY

No data was used for the research described in the article.

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