Pathogenic roles of the IMNEPD-associated PTRH2 mutants in aggravating mitochondrial dynamics and its rescue

Abstract

Background

Mitochondrial dynamics, orchestrated by a finely tuned balance between fusion and fission, are critical for cellular homeostasis and development. Dysregulation of these processes has been increasingly implicated in various diseases, including developmental disorders. Although core components, such as mitofusin 1 and 2 (MFN1/2), have been characterized, the broader regulatory network remains incompletely defined.

Methods

Gain-of-function screening of mitochondrial proteins was performed in human cell lines to identify regulators of mitochondrial morphology. Deletion mutants of PTRH2, MFN1/2, and FKBP8, as well as disease-related PTRH2 truncation mutants (A90fs, W108*), were generated by site-directed mutagenesis. Protein–protein interactions were examined by co-immunoprecipitation and TurboID-based proximity labeling, while mitochondrial morphology was assessed by confocal microscopy. Functional consequences of PTRH2 modulation were evaluated using CRISPR/Cas9 knockout and overexpression models. Mitochondrial function was analyzed by assays of ATP production, membrane potential, and reactive oxygen species, and mitophagy activity was monitored using mito-Keima–based fluorescence imaging.

Results

Here, we identify peptidyl-tRNA hydrolase 2 (PTRH2) as a novel modulator of mitochondrial dynamics through a gain-of-function screening approach. PTRH2 interacts with MFN1/2, interfering with MFN dimerization and thereby suppressing mitochondrial fusion. Notably, the PTRH2 truncation mutants A90fs and W108*, implicated in infantile multisystem neurologic, endocrine, and pancreatic disease (IMNEPD), exhibit an enhanced binding to MFN1/2, leading to mitochondrial fragmentation. This inhibitory interaction is alleviated under amino acid deprivation, a condition known to promote mitochondrial fusion. Furthermore, sustained PTRH2-induced mitochondrial fragmentation results in perinuclear aggregation of the mitochondria via recruiting FK506-binding protein 8 (FKBP8) and impairing mitophagy (Fig. 1).

Conclusions

These findings establish PTRH2 as a crucial negative regulator of mitochondrial fusion, providing new insights into mitochondrial dynamics and the molecular pathology of IMNEPD.

Supplementary Information

The online version contains supplementary material available at 10.1186/s10020-026-01452-1.

Introduction

Mitochondria are dynamic organelles essential for energy metabolism, apoptosis, and the regulation of reactive oxygen species (ROS) (Disha et al., 2024). Their function is tightly coupled to continuous morphological remodeling via the fusion and fission processes, which are responsive to cellular conditions. Under low-energy states, fusion is promoted to optimize ATP production (Gao and Hu 2021; Nabi et al., 2022), while stress conditions favor fission to segregate damaged mitochondria or increase mitochondrial abundance (Westermann 2010). Central to both processes are the large dynamin-related GTPases—mitofusin 1 and 2 (MFN1/2), optic atrophy 1 (OPA1), and dynamin-related protein 1 (DRP1)—which orchestrate membrane remodeling through GTP binding and hydrolysis. MFN1/2 and OPA1 mediate outer and inner membrane fusion, respectively, while DRP1 assembles at the outer membrane to drive fission via the GTPase-dependent constriction (Westermann 2010; Quiles and Gustafsson 2022). Knockout of Mfn1, Opa1, or Drp1 in mice results in embryonic lethality, highlighting their essential roles in development and cellular viability. Given the centrality of GTPase activity in regulating mitochondrial dynamics, understanding how the activity of these GTPases is modulated—through post-translational modifications, interacting partners, or cellular signaling pathways—has become a subject of intense investigation. Nevertheless, the full spectrum of regulatory mechanisms that fine-tune mitochondrial dynamics remains incompletely understood.

Over 40% of mitochondrial proteins are linked to human diseases (Morgenstern et al., 2021), including metabolic disorders, neurodegenerative diseases, and cardiovascular diseases (Nunnari and Suomalainen 2012). Especially, given development is a highly energy-demanding process supported by mitochondria (Spinelli and Haigis 2018), the connection between mitochondrial dysfunction and developmental diseases has become an area of growing interest. Accordingly, defects in mitochondrial dynamics can disrupt these developmental processes, leading to severe congenital disorders, such as Leigh syndrome (Bakare et al., 2021), Pearson syndrome (Yoshimi et al., 2022), and Alpers-Huttenlocher syndrome (Gorman et al., 2016; Saneto et al., 2013) etc., which deserves for intense study to better understand the pathophysiology.

Peptidyl-tRNA hydrolase 2 (PTRH2), also known as Bcl-2 inhibitor of transcription 1 (BIT1), is localized to multiple cellular compartments, including the mitochondrial outer membrane (Jan et al., 2004), endoplasmic reticulum (ER), and Golgi apparatus (GA) (Corpuz et al., 2020). PTRH2, previously known for its role in hydrolyzing aberrant peptidyl-tRNA (Sharma et al., 2014), is also involved in diverse cellular processes, such as apoptosis (Jan et al., 2004), survival, and signaling pathways like PI3K/AKT and mTOR (Griffiths et al., 2011; Kairouz-Wahbe et al., 2008; Hu et al., 2014). In addition, PTRH2 influences myogenic differentiation and epithelial-to-mesenchymal transition during neural development (Griffiths et al., 2015; Wu et al., 2016), reflecting its role in cellular plasticity and developmental processes. Recently, mutations in PTRH2 gene have been identified as the cause of Infantile-Onset Multisystem Neurologic, Endocrine, and Pancreatic Disease (IMNEPD), a rare disorder marked by developmental delay, neurodegeneration, and endocrine dysfunction (Hu et al., 2014; Picker-Minh et al., 2016). These include missense and nonsense mutations that generate truncated PTRH2 proteins (Hu et al., 2014; Sharkia et al., 2017). Despite these functions, the mechanistic consequences of disease-associated truncated PTRH2 forms remain poorly understood.

With genome-wide functional screening, we identified a novel function of PTRH2 in regulating mitochondrial dynamics. PTRH2 regulates mitochondrial fusion and subsequently their aggregation through its interaction with MFNs and FKBP8. Intriguingly, IMNEPD-associated PTRH2 mutants exaggerate mitochondrial aggregation, disrupting mitochondrial dynamics, which is reversed by amino acid signaling, illustrating a crucial role of PTRH2 in balancing mitochondrial dynamics and pathophysiology, such as IMNEPD (Fig. 1).

Fig. 1.

Schematic model of PTRH2–mediated mitochondrial dynamics. PTRH2 regulates mitochondrial fusion through interaction with MFN. IMNEPD mutants exaggerate the interaction, leading to mitochondrial fragmentation and aggregation through the MFN–PTRH2–FKBP8 complex, reversed by amino acids deprivation. The illustration was created with BioRender.com

Result

PTRH2 affects mitochondrial dynamics via catalytic domain

In our previous gain-of-function screening using 517 cDNAs encoding mitochondrial proteins, a list of clones that induce mitochondrial fragmentation upon overexpression, including FKBP8 (Yoo et al., 2020), was identified (Supplementary Fig. S1a). Among them, we confirmed that overexpressed PTRH2 was potent to induce mitochondrial fragmentation in HeLa cells (Supplementary Fig. S1b). We thus decided to characterize the role of PTRH2 in mitochondrial dynamics. Given PTRH2 exhibits pathological phenotypes in liver cancer and IMNEPD (Corpuz et al., 2020), we have characterized its function in liver cells. As seen in Hela cells, PTRH2-GFP overexpression in human hepatocellular carcinoma Hep3B cells increased numbers of the fragmentated mitochondria compared to control cells (Fig. 2a, b). Intriguingly, we also observed aggregated and huge forms of mitochondria along with the fragmentated mitochondria (Fig. 2a, b). Using Mito-RFP, TIM23 immunostaining, and mitotracker assays, we confirmed the aggregated mitochondria (Supplementary Fig. S1c-e). The mitochondria aggregation triggered by PTRH2 expression was observed in other human hepatocellular carcinoma Huh-7 and HLE cells, and human pancreatic epithelioid carcinoma PANC1 cells (Supplementary Fig. S1f-h). This alteration in mitochondrial morphology progressed from the initial fragmentation to the aggregation at late time (Supplementary Fig. S1i), showing two consecutive phases.

Fig. 2.

Ectopic expression of PTRH2 triggers mitochondrial fragmentation and aggregation. a Hep3B cells were transfected with EGFP-N1 or PTRH2-GFP for 48 h, immunostained with anti-TOM20 antibody and observed under confocal microscope. Bar; 10 µm. b Hep3B cells were co-transfected with Mito-RFP and EGFP-N1 or PTRH2-GFP for 48 h and observed under fluorescence microscope. The percentages of cells showing abnormal morphologies of mitochondria were determined. n > 150 cells. c Schematic representation of PTRH2 WT and deletion (△) mutants (left). Their subcellular localization and effects on mitochondrial morphologies are summarized (right). Mito, mitochondria; ER, endoplasmic reticulum; +, mitochondrial fragmentation or aggregation; -, no mitochondrial fragmentation or aggregation. d Hep3B cells were transfected with EGFP-N1 or PTRH2-GFP (WT or deletion forms) for 48 h, immunostained with anti-TOM20 antibody and observed under confocal microscope. Bar; 10 µm. e Hep3B cells were co-transfected with Mito-RFP and EGFP-N1 or PTRH2-GFP (WT or deletion forms) for 48 h and observed under fluorescence microscope. The percentages of cells showing abnormal morphologies of mitochondria were determined. n > 150 cells. f, g Hep3B/sgCtrl and Hep3B/sgPTRH2 cells were transfected with Mito-RFP for 24 h and observed under fluorescence microscope. Bar; 20 µm (f). The length of mitochondrial branches in Fig. 2F was quantified using ImageJ (g). h Hep3B/sgCtrl and Hep3B/sgPTRH2 cells were incubated with serum-free media (ATP^Total) or 5 mM 2-Deoxy-D-glucose (2-DG) and sodium pyruvate in glucose/serum-free media (ATP^OxPhos) for 2 h. Cellular ATP levels were measured using Cell-Titer Glo assay and multi-plate reader Tristar 5, respectively. i Hep3B/sgCtrl and Hep3B/sgPTRH2 cells were stained with MitoSOX (2.5 μM) for 30 min, and the fluorescent signal were analyzed using FACS Canto II. As a reference for ROS induction, Hep3B/sgCtrl cells were treated with rotenone (5 μM) for 12 h prior to MitoSOX staining. j Hep3B/sgCtrl and Hep3B/sgPTRH2 cells were stained with JC-1 (20 µM) for 20 min, and the fluorescent signal were analyzed using FACS Canto II. To assess the response to mitochondrial perturbation, Hep3B/sgCtrl cells were treated with oligomycin (1 μg/ml) for 4 h prior to JC-1 staining. Bars represent mean ± s.e.m. (three independent experiments) (b, e, h, i). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test (g, h, i)

To address how PTRH2 affects mitochondrial dynamics, we first determined the PTRH2 domain responsible for mitochondrial fragmentation. We generated serial deletion mutants (∆) lacking each domain; a mitochondrial localization domain (residues 1–65) in which a single transmembrane (TM) domain is located (residues 15–40) and a catalytic hydrolase domain (residues 65–179) (Corpuz et al., 2020) (Fig. 2c). Deletion of TM domain (∆15–40) resulted in cytosolic localization of PTRH2 and showed no apparent changes in mitochondrial dynamics. Removal of mitochondrial localization sequences (∆2–14, ∆40–65) led to partial localization of PTRH2 in the ER as well as mitochondria and had a milder impact on mitochondrial morphology than WT (Fig. 2d, e). In contrast, deletion of catalytic domain (∆66–179) induced more pronounced changes in mitochondria morphology than WT (Fig. 2d, e). While overexpressed PTRH2 WT induced a roughly 1:1 ratio of fragmentation to aggregation, PTRH2 (∆66–179) mutant increased the fragmentation and decreased the aggregation, showing approximately a 3:1 ratio (Fig. 2e). PTRH2 also localizes in GA and ER (Yi et al., 2010) but did not induce any alteration in these organelles (Supplementary Fig. S1j).

To characterize mitochondrial features, we generated PTRH2 knockout (KO) Hep3B cells using sgRNA (Supplementary Fig. S2a). Western blotting revealed no significant differences in the levels of mitochondrial membrane proteins TOM20 and TIM23 between control and PTRH2 KO cells (Supplementary Fig. S2b, c). Intriguingly, we found that the length of mitochondrial branches significantly increased in PTRH2 KO cells (Fig. 2f, g). Thus, the increases in length of mitochondrial branches without alteration in mitochondria proteins are likely due to enhanced fusion rather than an increase in mitochondrial mass. Upon investigation of the expression of key proteins involved in mitochondrial dynamics, we found no significant changes of MFN1, MFN2, FIS1, OPA1, and DRP1 in PTRH2 KO cells (Supplementary Fig. S2b, c). Instead, we observed an increase in the phosphorylation of DRP1 serine 616 but not serine 637 (Supplementary Fig. S2b, c), which is known to promote mitochondrial fission (Xie et al., 2020), implying a possible adaptive response to the elongated mitochondrial branches in PTRH2 KO cells. We further examined the functional consequences of PTRH2 deficiency and observed increased mitochondrial membrane potential and mitochondrial ROS levels, accompanied by unchanged or modestly reduced mitochondrial ATP production (Fig. 2h–j). We believe that these functional alterations are interpreted as downstream consequences of altered mitochondrial morphology and homeostasis. Notably, despite the increased mitochondrial branch length, mitochondrial function—particularly ATP production—was not enhanced, suggesting that the elongated mitochondrial morphology reflects dysregulated mitochondrial dynamics and potentially excessive or abnormal fusion rather than a physiologically beneficial adaptation. Accordingly, these findings suggest that PTRH2 may act as a negative regulator of mitochondrial fusion.

PTRH2 interacts with MFNs to inhibit mitochondrial fusion

The notion that PTRH2 may negatively regulate mitochondrial fusion led us to identify the mitochondrial dynamics regulator. We first assessed whether PTRH2 interacts with the proteins involved in the mitochondrial dynamics. The immunoprecipitation (IP) assays revealed that PTRH2 interacts with MFN1/2, but not with FIS1, OPA1 or DRP1, in Hep3B cells (Fig. 3a). Similar to PTRH2, overexpression of MFN2 or a truncated GTPase form of MFN1 was previously shown to induce mitochondrial fragmentation and clustering (Huang et al., 2007; Koshiba et al. 2004). We confirmed that overexpression of MFN2 WT or mutants lacking GTPase (∆G), HR1 (∆H1), or HR2 (∆H2) domain induced mitochondrial clustering except for MFN2 mutant lacking TM domain (∆T) (Fig. 3b). We then assessed if the alteration of mitochondrial dynamics by PTRH2 is related to the mitochondrial clustering. With an IP assay, we found that PTRH2 binds to MFN2-WT, ∆H1, ∆H2, and ∆T, but not to MFN2-∆G (Fig. 3c). PTRH2 also failed to bind to MFN1-∆G (Supplementary Fig. S3a). Thus, PTRH2 interacts with MFNs through MFN GTPase domain and there is no close association between the mitochondrial clustering and the binding pattern of PTRH2 to MFNs (Fig. 3b). In addition, the mitochondrial clustering is mediated via homo- or hetero-dimerization via HR domain (Koshiba et al., 2004), whereas PTRH2 inhibited the formation of MFN1 homodimer (Fig. 3d), suggesting that the mitochondria morphological alteration induced by PTRH2 is distinct from the MFN-dependent clustering and is hereafter referred to as mitochondrial aggregation.

Fig. 3.

PTRH2 interacts with mitofusins to regulate mitochondrial dynamics. a Hep3B cell lysates were subjected to the immunoprecipitation (IP) assay using anti-PTRH2 antibody. The immunoprecipitates and cell lysates were analyzed by western blotting. b, c Schematic representation of MFN2 WT and deletion (△) mutants (left). Their binding to PTRH2 and effects on mitochondrial clustering are noted. +, mitochondrial clustering or interaction; -, no mitochondrial clustering or no interaction (right) (b). HEK293T cells were co-transfected with PTRH2-FLAG and MFN2-GFP (WT or deletion forms) for 24 h and subjected to IP assay using anti-FLAG antibody (c). d HEK293T cells were co-transfected with MFN1-HA, MFN1-FLAG and PTRH2-RFP for 24 h and subjected to IP assay using anti-FLAG antibody. e HEK293T cells were transfected with PTRH2-FLAG for 24 h and then incubated under the indicated conditions for 4 h. Full, DMEM with 10% FBS; -FBS, DMEM without FBS; EBSS, EBSS only; EBSS + FBS, EBSS with 10% FBS; EBSS + QLR, EBSS with 4 mM glutamine, 0.8 mM leucine and 0.4 mM arginine. Cell lysates were analyzed with IP assay using anti-FLAG antibody. f HEK293T cells were co-transfected with MFN2-FLAG and PTRH2-GFP (WT or deletion forms) for 24 h and subjected to IP assay using anti-FLAG antibody

Given PTRH2 disrupted the interactions between MFNs, we hypothesized that PTRH2 negatively regulates mitochondrial fusion to induce mitochondrial fragmentation. To explore this possibility, we sought to examine the interaction between PTRH2 and MFNs under the conditions driving mitochondrial fusion. Under EBSS conditions, it is known that the GTPase activity of MFN and OPA1 increases, leading to enhanced mitochondrial fusion, and this event is further amplified by the addition of glutamine (Q), leucine (L), and arginine (R) (Abdullah et al., 2022). As expected, the interaction between MFN1 and PTRH2 was reduced by the treatment with EBSS or EBSS plus QLR (Fig. 3e). Further analysis of the interaction revealed that deletion of the PTRH2 TM domain abolished the interaction of PTRH2 with MFN, whereas deletion of the PTRH2 catalytic domain enhanced it (Fig. 3f; Supplementary Fig. S3b), consistent with the effects of PTRH2 deletion mutants on mitochondrial fragmentation. Thus, the binding PTRH2 to MFNs suppresses mitochondria fusion via inhibiting the interaction between MFNs, balancing overall mitochondrial dynamics to fragmentation.

IMNEPD-associated PTRH2 mutants exacerbate mitochondrial aggregation

As mutations of PTRH2 cause the genetic disorder known as IMNEPD (Hu et al., 2014), we investigated whether these mutations in PTRH2 impact on mitochondrial dynamics. Among several IMNEPD-related PTRH2 forms, two deletion forms, CT deletion at positions 269 and 270 (A90fs) and a nonsense mutation (c.324G > A) at residue 108 (W108*), showed higher mitochondrial aggregation than PTRH2 WT in Hep3B cells (Fig. 4a, b; Supplementary Fig. S4a). Compared to PTRH2 WT, mitochondria aggregation triggered by A90fs and W108* was already high at 24 h and further increased at 48 h (Fig. 4c). Furthermore, we observed that MFN1 consistently bound better to A90fs or W108* than PTRH2 WT (Fig. 4d). We also examined whether this interaction is dependent on MFN GTPase activity. Based on the MFN1 mutants showing reduced GTPase activity (Cao et al., 2017; Lee et al., 2014; Ryu et al., 2012), we generated triple mutant (3 M: K88T, K222Q, and W239A) in GTPase domain. Compared to MFN1 WT, MFN1 3 M mutant showed enhanced binding to PTRH2 WT, A90fs, and W108* (Supplementary Fig. S4b). Together with the observation that PTRH2-MFN interaction is reduced under amino acid deprivation (Fig. 3e), these results suggest that PTRH2 WT, A90fs, and W108* interact with MFNs in a GTPase activity–dependent manner.

Fig. 4.

IMNEPD-associated PTRH2 mutants exacerbate mitochondrial aggregation. a Hep3B cells were transfected with Mito-RFP and PTRH2-GFP (WT or IMNEPD forms) for 48 h and the percentages of cells showing mitochondria aggregation were determined. Bars represent mean ± s.e.m. (n > 150 cells, three independent experiments). b Hep3B cells were transfected with PTRH2-GFP (WT or IMNEPD forms) for 24 h, immunostained with anti-TOM20 antibody and observed under confocal microscope. Bar; 10 µm. c Hep3B cells were co-transfected with Mito-RFP and EGFP-N1 or PTRH2-GFP (WT or IMNEPD forms) for 24 or 48 h. The percentages of cells showing abnormal morphology of mitochondria were determined. Bars represent mean ± s.e.m. (n > 150 cells, three independent experiments). d HEK293T cells were co-transfected with MFN1-HA and PTRH2-RFP (WT or IMNEPD forms) for 24 h and subjected to IP assay using anti-HA antibody (left). The signals of PTRH2-RFP on the blots were quantified by ImageJ and normalized by that of MFN1-HA in IP (right). e Hep3B cells were co-transfected with Mito-RFP and PTRH2-GFP (WT or IMNEPD forms) for 48 h. The mitochondrial branch length (RFP) and PTRH2 expression (GFP) of cells were measured using ImageJ and their relation was depicted by Pearson coefficient (R²). f, g Hep3B cells were transfected with EGFP-N1 or PTRH2-GFP (WT or IMNEPD forms) for 48 h and observed under a fluorescence microscope. Bar; 20 µm (f). Cell area was then quantified using ImageJ (g). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test (a, d, g)

We then analyzed the relationship between expression levels of PTRH2 WT, A90fs, and W108* and alteration of mitochondrial branches. Quantitative analysis showed that alteration of mitochondrial branch length was more sensitive to PTRH2 A90fs and W108* than WT while it increases following their expression levels (Fig. 4e; Supplementary Fig. S4c), suggesting that mutations in PTRH2 associated with IMNEPD disrupt mitochondrial dynamics. IMNEPD is a genetic disorder characterized by multi-organ atrophy, including the liver, which has been attributed to reduced mTOR activity leading to reduced cell size and cell loss (Hu et al., 2014). In our study, we also found that expressions of PTRH2 WT and IMNEPD forms (A90fs and W108*) significantly reduced cell size (Fig. 4f, g). However, we did not observe notable changes in mTOR activity in Hep3B cells (Supplementary Fig. S4d). Given previous reports linking mitochondrial network disruption to reduced cell size (Seel et al., 2023), our findings further support the role of PTRH2 in regulating mitochondrial dynamics, probably contributing to the atrophic phenotypes observed in IMNEPD.

FKBP8 binds to PTRH2 to mediate the IMNEPD-associated mitochondrial aggregation

To address the role of MFNs in the PTRH2-induced mitochondrial fragmentation, we generated MFN1/2 double knockout HCT116 cells (Fig. 5a). As reported (Filadi et al., 2018), we confirmed that mitochondria are highly fragmented in MFN1/2 double knockout HCT116 cells compared to control cells (Supplementary Fig. S5a). In these MFN1/2 knockout cells, ectopic expression of PTRH2 altered the proportional distribution of mitochondrial morphologies, resulting in a relative decrease in the fragmented mitochondria and a concomitant increase in the aggregated mitochondria (Fig. 5b; Supplementary Fig. S5a). It appears that MFN plays a role in the mitochondrial fragmentation but not much in the mitochondrial aggregation driven by PTRH2. We have thus searched for an additional factor which regulates the PTRH2-mediated mitochondrial aggregation. We established a cell-based assay showing the Mito-RFP-positive mitochondrial aggregation and performed gain-of-function screen using cDNAs expression library encoding 517 mitochondrial proteins (Fig. 5c). From the screen, we identified FKBP8, a member of the immunophilin protein family and mitophagy receptor located on the mitochondrial outer membrane, particularly independent of Parkin-mediated pathways (Yoo et al., 2020; Bhujabal et al., 2017), as a candidate (Fig. 5d).

Fig. 5.

FKBP8 binds to PTRH2 to mediate mitochondrial aggregation. a, b HCT116/sgCtrl and HCT116/sgMFN1/2 cells were generated (a) and co-transfected with Mito-RFP and EGFP-N1 or PTRH2-GFP for 48 h and observed under fluorescence microscope. The percentages of cells showing abnormal morphologies of mitochondria were determined (b). c, d Scheme of gain-of-function screen to identify a regulator of PTRH2-mdeiated mitochondrial dynamics (c). Hep3B cells were co-transfected with Mito-RFP, PTRH2-GFP (WT or IMNEPD forms) and cDNA expression library encoding mitochondria proteins for 48 h and cDNA clones affecting mitochondrial aggregation were determined (d). e After transfecting HEK293T cells with PTRH2-V5-TurboID (WT or IMNEPD forms) for 24 h, cells were further incubated with 50 µM biotin for 30 min, lysed and pulled-down by streptavidin. Cell lysates (Input) and the biotinylated proteins were analyzed by western blotting. f, g HeLa/sgCtrl or HeLa/sgFKBP8 cells were generated (f) and co-transfected with Mito-RFP and PTRH2-GFP (WT or IMNEPD forms) for 48 h and observed under fluorescence microscope. The percentages of cells showing abnormal morphologies of mitochondria were determined (g). h HEK293T cells were co-transfected with PTRH2-FLAG and HA-FKBP8 (WT or deletion forms) for 24 h and subjected with immunoprecipitation (IP) assay. i Hep3B/sgCtrl and Hep3B/sgFKBP8 cells were co-transfected with Mito-RFP, PTRH2-FLAG (WT or IMNEPD forms) and HA-FKBP8 (WT or deletion forms) for 48 h. The percentage of cells showing mitochondrial aggregation were determined. Bars represent mean ± s.e.m. (b, d, g, i) (n > 150 cells, three independent experiments). Statistical analysis was performed using two-way ANOVA with interaction, followed by Tukey’s (d) or Dunnett’s (i) multiple comparisons test

To assess its function in mitochondria dynamics we generated a PTRH2-V5-TurboID fusion protein for a TurboID assay (Fig. 5e), which allows detection of a temporal binding state of proximity proteins through the biotinylation (Cho et al., 2020). Biotinylation and IP assays revealed that FKBP8 also interacts with PTRH2 as well as MFNs, with this interaction being further enhanced in A90fs or W108* (Fig. 5e; Supplementary Fig. S5b). Moreover, as seen in control cells, mitochondrial fragmentation similarly occurred in FKBP8 knockout HeLa cells following the expression of PTRH2 WT or IMNEPD forms (Fig. 5f, g; Supplementary Fig. S5c). In contrast, the mitochondrial aggregation was significantly reduced in FKBP8 knockout HeLa cells (Fig. 5f, g; Supplementary Fig. S5c). With IP assay, we found that PTRH2 binds to FKBP8 but not to FKBP8 mutant lacking the C-terminal region (a.a. 391–413), including TM region (△TM, a.a. 391–410) and C-term end region (△C, a.a. 410–413) (Fig. 5h; Supplementary Fig. S5d). It appears that PTRH2 induces mitochondrial aggregation via binding to the C-terminus of FKBP8, which is distinct from the role of FKBP8 playing in the mitochondrial fragmentation via the LIR motif near its N-terminus (Bhujabal et al., 2017). In addition, unlike FKBP8 WT, re-expressions of PTRH2-binding-defective FKBP8-△TM or △C mutant in FKBP8 knockout Hep3B cells did not affect the mitochondrial aggregation induced by PTRH2 WT or IMNEPD forms (Fig. 5i; Supplementary Fig. S5e). Together these results suggest that FKBP8 collaborates with PTRH2 in the mitochondrial aggregation.

PTRH2 forms a ternary complex with FKBP8 and MFNs to regulate mitochondrial dynamics

We further investigated how PTRH2 interacts with MFN and FKBP8 to regulate mitochondrial dynamics. With IP assay using PTRH2 deletion mutants, we found that PTRH2 interacts with FKBP8 through its catalytic domain (Fig. 6a), reflecting our results showing that PTRH2 mutant lacking catalytic domain induced mitochondrial fragmentation but not aggregation. On the other hand, deletion of PTRH2 catalytic domain well interacted with MFN (Fig. 6b). Interestingly, PTRH2 potentially formed a tripartite protein complex with MFN and FKBP8. Notably, FKBP8 showed stronger association with the PTRH2–MFN complex than with PTRH2 alone in the transfected cells (Fig. 6c), suggesting that FKBP8 is recruited to the MFN–PTRH2 complex. This aspect was further supported by TurboID-based proximity labeling assay, where FKBP8 biotinylation was increased by PTRH2 IMNEPD forms that interact more strongly with MFNs than PTRH2 WT (Fig. 5e).

Fig. 6.

Enforced interaction of PTRH2 with MFN/FKBP8 disrupts mitochondrial dynamics and contributes to pathological atrophy in IMNEPD. a HEK293T cells expressing HA-FKBP8 and PTRH2-FLAG (WT or deletion forms) were analyzed with immunoprecipitation (IP) assay using anti-FLAG antibody. b Summary of the interaction between PTRH2 (WT or deletion forms) and either MFN or FKBP8. +, interaction; -, no interaction. c HEK293T cells were co-transfected with MFN2-GFP, HA-FKBP8 and PTRH2-FLAG for 24 h and subjected to IP assay using anti-FLAG antibody. d HEK293T cells were co-transfected with V5-TurboID-MFN2 and PTRH2-GFP (WT or phosphorylation mutants) for 24 h and subjected to IP assay using anti-GFP antibody. e Hep3B cells were co-transfected with Mito-RFP and PTRH2-GFP (WT or phosphorylation mutants) for 48 h and observed under fluorescence microscope. The percentages of cells showing abnormal morphologies of mitochondria were determined. Bars represent mean ± s.e.m. (n > 150 cells, three independent experiments). f Hep3B cells were co-transfected with mt-Keima and CMV14 or PTRH2-FLAG (WT or IMNEPD forms) for 48 h and the signals of 550 nm and 440 nm were quantified using ImageJ. Bars represent mean ± s.e.m. Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test. (n = 47 ~ 89 cells). g Hep3B cells expressing RFP-N1 or PTRH2-RFP were immunostained with anti-p62 antibody and observed under confocal microscope. Bar; 20 µm. h, i HEK293T cells expressing PTRH2-FLAG (h) or PTRH2-A90fs-FLAG (i) were left untreated (-) or treated with EBSS + QLR (4 mM glutamine, 0.8 mM leucine and 0.4 mM arginine, 4 h), palmitate (200 µM, 8 h) (h, i) and hypoxia (1% O₂, 4 h) (i). Cell lysates were subjected to IP assay using anti-FLAG antibody. j Hep3B cells expressing Mito-RFP and PTRH2-GFP (WT or IMNEPD forms) were left untreated (Ctrl) or treated with palmitate (200 µM, 8 h), EBSS + QLR (4 mM glutamine, 0.8 mM leucine and 0.4 mM arginine, 4 h) or H₂O₂ (100 µM, 4 h). The percentages of cells showing mitochondrial aggregation were determined. Bars represent mean ± s.e.m. Statistical analysis was performed using two-way ANOVA with interaction, followed by Dunnett’s multiple comparisons test. (n > 150 cells, four independent experiments)

We then addressed an important question on how the interaction among these proteins is regulated. With the predictions from PhosphoSitePlus, we presumed the possible post-translational modifications of PTRH2 and generated point mutants of candidate residues within the N-terminal region (1–89 aa), including serine 5 which was previously reported to regulate subcellular localization via phosphorylation (Biliran et al., 2008). Among these mutants, the phosphorylation at PTRH2 Ser54 was found to potentially modulate its interaction with FKBP8 (Supplementary Fig. S6a). To further explore this, we generated a phosphorylation-mimic mutant (S54D). Compared to PTRH2 WT, PTRH2 S54D showed reduced interaction with MFNs (Fig. 6d) and induced less mitochondria aggregation (Fig. 6e). On the contrary, the phosphorylation-defective mutant (S54A) showed increased interaction with FKBP8 and a trend toward enhanced mitochondrial aggregation. It appears that the phosphorylation at Ser54 modulates PTRH2’s binding ability to its partners, thereby influencing the formation of the PTRH2–MFN–FKBP8 complex and regulating mitochondrial dynamics.

Disrupting IMNEPD PTRH2-MFNs complex rescues the impaired mitochondrial dynamics

Since it was reported that mitochondrial aggregation occurs near the perinucleus during mitophagy (Itakura et al., 2012), we determined whether the PTRH2-induced mitochondrial aggregation occurs during mitophagy using the mt-Keima assay (Sun et al., 2017). As reported (Yoo et al., 2020; Bhujabal et al., 2017), FKBP8 overexpression increased fluorescence at 550 nm in mitochondrial aggregates. However, there was little change in the mitophagy of mitochondrial aggregates upon PTRH2-WT, A90fs or W108* overexpression (Fig. 6f; Supplementary Fig. S6c). In addition, LC3, an autophagy marker, was recruited to mitochondrial aggregates upon FKBP8 expression but not by PTRH2 expression (Supplementary Fig. S6d). Thus, the mitochondrial aggregation induced by PTRH2 expression is not related to mitophagy. Further, the FKBP8-induced LC3 recruitment to mitochondrial aggregates was impaired by co-expression of PTRH2 (Supplementary Fig. S6e). Interestingly, the PTRH2-induced mitochondrial aggregation was accompanied by p62 accumulation without subsequent recruitment to lysosomes (Fig. 6g; Supplementary Fig. S6f), showing a blockade in the mitophagy pathway. While FKBP8 functions as a mitophagy receptor interacting with LC3 (Yoo et al., 2020; Bhujabal et al., 2017), this interaction was not affected by PTRH2 (Supplementary Fig. S6g). Thus, the mitochondrial aggregation mediated by the PTRH2-FKBP8 interaction accompanies a blockade of mitophagy.

Since the PTRH2-MFNs interaction is diminished upon promoting mitochondrial fusion, we examined if the signals influence the PTRH2-FKBP8 interaction. As seen in the PTRH2-MFNs interaction, the PTRH2-FKBP8 interaction was reduced under EBSS + QLR treatment (Fig. 6h). In contrast, treatment with palmitate, which promotes mitochondrial fission (Li et al., 2017), led to an increase in the interaction of PTRH2 with MFN or FKBP8 (Fig. 6h). Furthermore, we found that the interactions of A90fs or W108* with MFNs were all reduced in cells exposed to EBSS + QLR (Fig. 6i; Supplementary Fig. S6h). At the same time, these interactions all increased in cells treated with palmitate or hypoxia (1% O₂) which also stimulates mitochondrial fission (Han et al., 2019). Accordingly, the mitochondrial aggregation was reduced in cells exposed to EBSS + QLR (Fig. 6j), consistent to the interaction patterns of PTRH2 with MFNs or FKBP8. These results suggest that alterations in mitochondrial dynamics driven by A90fs and W108* could be rescued by a condition that reduces their interaction with MFNs. Collectively, the role of PTRH2 in regulating mitochondrial dynamics might underlie the pathogenesis of IMNEPD disorder, particularly in PTRH2 A90fs and W108* mutants.

Discussion

Here, we demonstrated that PTRH2 regulates mitochondrial dynamics through its sequential interaction with MFNs and FKBP8. In this process, PTRH2 counteracts MFN activity by binding to it. Notably, PTRH2 interacts with the GTPase domain of MFNs—a critical component shared by mitochondrial dynamics regulators, such as OPA1 and DRP1—which control membrane fusion and fission through their intrinsic GTPase activity. Regulation of this domain has been implicated in diseases, including cancer. Recently, FUNDC2, another regulator of mitochondrial dynamics, binds to this domain of MFN1 and promotes liver tumorigenesis (Li et al., 2022a). The interactions of MFNs with PTRH2 or FUNDC2 likely require fine modulation to balance fusion and fission under pathophysiological conditions. Intriguingly, our findings provide some clues to this regulation, including a potential phosphorylation of PTRH2 and amino acid starvation signaling. However, further investigations on the detailed interaction mode between MFNs and PTRH2, as well as the broader regulation of the MFN GTPase domain, are warranted for better understanding of mitochondrial dynamics.

Following the mitochondrial fragmentation due to enhanced fission, mitochondrial aggregation, clumping, or gathering of mitochondria has occasionally been reported under a few cellular conditions, such as apoptosis (Martinou and Youle 2011), mitophagy (Narendra et al., 2008), and chemical treatments (e.g., cytochalasin B). In apoptosis, mitochondrial aggregation precedes cytochrome c release from mitochondria (Haga et al., 2003). Consistent with this, cells harboring PTRH2-induced mitochondrial aggregates become susceptible to cell death (data not shown). Mitochondrial aggregation can also be triggered by DNA nanodevices (Zhu et al., 2023) or chemicals like cytochalasin B to compromise the efficiency and safety of embryo (Li et al., 2022b). It may also facilitate mitophagy by segregating damaged mitochondria for autophagic removal (Ma et al., 2020). In such cases, impaired mitophagy and cellular stress contribute to the accumulation of damaged mitochondria.

With this perspective, we found that MFNs and FKBP8 bind to distinct PTRH2 regions, supporting the formation of an MFN–PTRH2–FKBP8 complex that may mediate mitochondrial aggregation in response to damages. Unlike MFN, we failed to detect the endogenous FKBP8–PTRH2 complex via IP assay under normal conditions, suggesting that FKBP8 might be recruited to the complex under pathological condition. A possible explanation for the mitochondrial damages associated with this complex could be the alteration of mitochondrial dynamics and mitochondria–ER contacts. Indeed, PTRH2, FKBP8, and MFN2—including its isoforms—are localized to both mitochondria and ER, where they are known to regulate the contact sites, potentially coordinating mitochondrial dynamics and inter-organelle communication (Nakamura et al., 2025; Naón et al., 2023). Moreover, the PTRH2–FKBP8 interaction appears to suppress FKBP8-mediated mitophagy, which we previously demonstrated to occur via FKBP8's binding to LC3 (Yoo et al., 2020), probably by preventing LC3 recruitment. To our knowledge, this is the first demonstration showing the MFN-PTRH2-FKBP8 axis as a regulator of mitochondrial dynamics, providing broader insights into the regulatory landscape of mitochondrial dynamics.

IMNEPD, caused by PTRH2 mutations, has been reported in the context of PTRH2 loss of function (Hu et al., 2014). PTRH2’s hydrolase activity is conserved, with catalytic residues well characterized in PTRH1 (Schmitt et al., 1997; Kuroha et al., 2018). In contrast, PTRH2’s active site remains unconfirmed and its structural divergence from PTRH1 complicates predictions (Pereda et al., 2004). IMNEPD mutations do not overlap with the known catalytic residues, although Q85P is predicted to impair enzymatic activity (Sharkia et al., 2023). While the relationship between PTRH2 activity and mitochondrial dynamics remains unclear, many IMNEPD-associated mutations reside within the hydrolase domain. Among several mutations, two deletion mutants lacking hydrolase activity, A90fs and W108*, aggravate the mitochondrial dynamics and point mutants in Q85P and T48E did not. Further investigation on the relationship between the hydrolase activity of PTRH2, mitochondrial dynamics, and IMNEPD pathogenesis may clarify their mechanistic links.

Given that regulation of mitochondrial dynamics is critical for development (Chen et al., 2003; He and Maheshwari 2023; He et al., 2023), our findings suggest that PTRH2’s role in this process may contribute to IMNEPD pathogenesis. Although alterations of mitochondrial morphologies were not directly examined in disease-relevant tissues or patient-derived cells in this study, we observed that altered PTRH2 expression leads to cell size reduction, a phenotype consistent with the previous reports (Hu et al., 2014), implicating a role of PTRH2 in cell size regulation and with the progressive atrophy characteristic of IMNEPD. In this context, our data raise the possibility that PTRH2-dependent dysregulation of mitochondrial dynamics may represent a cellular mechanism underlying disease pathology. Moreover, if mitochondrial dynamics are disrupted in IMNEPD patients, our study provides a potential therapeutic framework. Excitingly, we found that amino acid deprivation weakens the PTRH2–MFN interaction and partially rescues mitochondrial defects. While amino acid deprivation often induces aberrant mitochondrial hyperfusion (Abdullah et al., 2022) and is therefore unlikely to fully restore mitochondrial dynamics and functions, these findings raise a new possibility in that targeting nutrient signaling or the PTRH2–MFN interaction could offer a therapeutic avenue for alleviating symptoms in IMNEPD patients.

Conclusion

In conclusion, this study presents a role of the MFN-PTRH2-FKBP8 axis in regulating mitochondrial dynamics, such as mitochondrial fusion and aggregation. Understanding how PTRH2 and its mutant regulate mitochondrial dynamics expands our understanding of the IMNEPD pathogenesis and therapeutic opportunity.

Materials and methods

Antibodies and reagents

The following antibodies were used for western blotting, immunofluorescence, and immunoprecipitation assays. PTRH2 (LS-C482623) from LSBio; MFN1 (A21293) and MFN2 (A19678) from ABclonal; TOM20 (sc-17764), FIS1 (sc-37644) GFP (sc-9996), HA (sc-7392), Actin (sc-47778), and TUBA (sc-23948) from Santa Cruz Biotechnology; FLAG M2 (F3165) and FLAG M2 affinity gel (A2220) from Sigma-Aldrich; RFP (PM005) from MBL Life Science; TIM23 (611,222) and OPA1 (612,607) from BD Biosciences; DRP1 (8570), pDRP1-S616 (4494), pDRP1-S637 (6319), S6 (2317), and pS6 (5364) from Cell Signaling Technology; FKBP8 (MAB3580) from R&D systems; LC3B (NB100-2220), LAMP2 (NBP2-22,217), and p62 (NBP1-48,320) from Novus Biologicals; V5 (ab27671) from Abcam; Streptavidin-Agarose (16–126) from Millipore; Protein G Sepharose 4 Fast Flow (17–0618–05) from GE Healthcare. Oligomycin (75,351), L-Glutamine (G7513), L-Leucine (L1512), L-Arginine (A5006), and Rotenone (R8875) were purchased from Sigma-Aldrich.

Cell culture and transfection

To investigate the role of PTRH2 and its disease-associated mutants in regulating mitochondrial dynamics, a series of human cell lines were used for overexpression, knockout, and interaction analyses. Hep3B, Huh-7, HLE, PANC1, HeLa, HCT-116, and HEK293T cells were cultured in Dulbecco’s modified Eagle medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; Gibco) and 100 µg/ml penicillin–streptomycin (Gibco). Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. HeLa/sgCtrl and HeLa/sgFKBP8 cells were previously established in our study (Yoo et al., 2020) and maintained under same culture conditions. Transient transfections of plasmids encoding PTRH2, its mutants, or other indicated constructs were performed using Lipofector-pMax (AptaBio) according to the manufacturer’s instructions.

DNA construction

To examine the role of PTRH2 and its interaction partners in mitochondrial dynamics, expression constructs for PTRH2, MFN1, MFN2, and related mutants were generated. Human PTRH2, MFN1, and MFN2 cDNAs were amplified by PCR and inserted into pEGFP-N1, p3xFLAG-CMV14, pcDNA3-HA, pRFP-N1, or pV5-TurboID-Sec61b vectors. Mutations or deletions of PTRH2, MFN1, and MFN2 were generated by site-directed mutagenesis or PCR-based cloning. All FKBP8 constructs were previously described (Yoo et al., 2020). Restriction enzyme sites and primer sequences are listed in Supplementary Table S2. All constructs were verified by DNA sequencing.

Generation of knockout cell lines

To investigate the functional contribution of PTRH2, FKBP8, MFN1, and MFN2 to mitochondrial morphology and dynamics, knockout cell lines were generated using a CRISPR–Cas9 system. Hep3B or HCT116 cells were transfected with LentiCRISPR v2 vectors containing sgRNAs targeting PTRH2, FKBP8, MFN1, or MFN2 for 48 h. Cells were subsequently selected with 5 µg/ml puromycin for 72 h, and monoclonal populations were established by limited dilution. Expression level of PTRH2 or FKBP8 was analyzed by western blotting. PTRH2, MFN1 and MFN2 sgRNA sequences are listed in Supplementary Table S2 and FKBP8 sgRNA constructs were previously described (Yoo et al., 2020).

Imaging and immunofluorescence

Immunofluorescence microscopy was performed to analyze mitochondrial morphology and subcellular localization of target proteins under the indicated experimental conditions. Cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) in PBS for 20 min. Then, cells were permeabilized with 0.005% digitonin for 20 min and blocked with 3% BSA in PBS for 1 h at room temperature. Cells were incubated with primary antibodies (1:200) in PBS containing 1% BSA overnight, washed with PBS three times, and incubated with secondary antibodies (1:500) in PBS containing 1% BSA for 1 h at room temperature. Samples were captured by a confocal microscope LSM700 (Zeiss).

Gain-of-function screening

To identify mitochondrial proteins that influence mitochondrial morphology, a gain-of-function screening was performed. Among ~ 1,600 known mitochondrial proteins, 517 cDNAs encoding mitochondrial proteins were collected (Supplementary Table S1). HeLa or Hep3B cells were co-transfected with each of mitochondrial cDNAs and Mito-RFP using Lipofector-pMax (AptaBio) and incubated for 24–48 h. Then, mitochondrial morphology was observed under a fluorescence microscope IX71 (Olympus).

Measurement of cellular ATP levels

Cellular ATP levels were measured to assess mitochondrial functional consequences associated with PTRH2 deficiency. Hep3B/sgCtrl and Hep3B/sgPTRH2 cells were washed with PBS and incubated either in serum-free DMEM (ATP^Total) or in glucose- and serum-free medium supplemented with 5 mM 2-deoxy-D-glucose and sodium pyruvate (ATP^OxPhos) for 2 h. ATP levels were quantified using the CellTiter-Glo® Luminescent Cell Viability Assay (G7570; Promega) according to the manufacturer’s instructions. An equal volume of CellTiter-Glo reagent was added to each well, and the plate was shaken for 2 min to induce cell lysis, followed by incubation at room temperature for 10 min to stabilize the luminescent signal. Luminescence was measured using a Tristar 5 multimode plate reader (Berthold Technologies).

Mitochondrial ROS by flow cytometry

To assess mitochondrial reactive oxygen species (ROS) levels associated with PTRH2 deficiency, Hep3B/sgCtrl and Hep3B/sgPTRH2 knockout cells were treated with DMSO or 5 μM rotenone for 12 h. Cells were incubated with 2.5 μM MitoSOX (M36008; Thermo Fisher Scientific) for 30 min at 37 °C and then washed twice with PBS. Cells were detached using trypsin–EDTA, transferred to new tubes, and analyzed by flow cytometry using a FACS Canto II (BD Biosciences).

Mitochondrial membrane potential by flow cytometry

To evaluate changes in mitochondrial membrane potential upon PTRH2 depletion, Hep3B/sgCtrl and Hep3B/sgPTRH2 knockout cells were treated with DMSO or 1 μg/ml oligomycin for 4 h. Cells were incubated with 20 μM JC-1 (HY-15534; MedChemExpress) for 20 min at 37 °C and washed twice with PBS. Cells were transferred to new tubes and analyzed by flow cytometry using a FACS Canto II (BD Biosciences).

Western blotting

Cells were lysed with radioimmunoprecipitation assay (RIPA) Buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 50 mM Tris–Cl pH 7.5, and 0.1% SDS) containing protease inhibitor cocktail (Quartett) and 1 mM PMSF at 4 °C. After centrifugation at 12,000 rpm for 20 min, protein concentration of supernatants was measured by Bradford protein assay (Bio-Rad Laboratories). Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane using semi-dry transfer system WSE-4025 HorizeBLOT 2 M-R (ATTO). The membranes were blocked with 5% BSA (GenDEPOT) at room temperature for 1 h and incubated with primary antibodies overnight at 4 °C. Subsequently, the membrane was incubated with a secondary antibody and then incubated with ECL for detection. Signals were visualized using X-ray films.

Immunoprecipitation assay

To analyze protein–protein interactions, cells were lysed with lysis buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 50 mM Tris–Cl pH 7.5) containing protease inhibitor cocktail (Quartett) and 1 mM PMSF at 4 °C. After centrifugation at 12,000 rpm for 20 min, supernatants were incubated with primary antibody in lysis buffer overnight at 4 °C. Samples were further incubated with Protein G Sepharose beads (GE Healthcare) or FLAG M2 beads (Sigma-Aldrich) for 4 h at 4 °C. Beads were washed four times with lysis buffer and analyzed by western blotting.

Quantification of cell size

Cell size was measured using ImageJ. Brightfield or fluorescence images were acquired using a fluorescence microscope IX71 (Olympus). Cell boundaries were manually traced using the polygon selection tool in ImageJ. The “Measure” function was used to calculate the area of each cell, expressed in square micrometers, after calibration with a scale bar. More than 100 cells were analyzed from randomly selected fields to ensure representative quantification.

TurboID-based proximity labeling assay

To identify proximal interactors of PTRH2, HEK293T cells were transfected with PTRH2-V5-TurboID (WT or IMNEPD forms) for 24 h and treated with 50 μM biotin (Sigma-Aldrich) for 30 min. Then, cells were washed with PBS and lysed with lysis buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 50 mM Tris–Cl pH 7.5) containing protease inhibitor cocktail (Quartett) and 1 mM PMSF at 4 °C. After centrifugation at 12,000 rpm for 20 min, supernatants were pull-downed using Streptavidin-Agarose bead slurry (16–126, EMD Millipore) in lysis buffer overnight at 4 °C. Beads were washed four times with lysis buffer and analyzed by western blotting.

Mitophagy assessment using mito-Keima

Cells were transfected with mitochondrial-targeted Keima plasmid (mito-Keima) using Lipofector-pMax (AptaBio). After 48 h, fluorescence images were acquired using a fluorescence microscope IX71 (Olympus) equipped with appropriate filter sets for dual excitation ratiometric imaging (Ex = 440 nm and 550 nm, Em = 620 nm). Image analysis was performed using ImageJ. Regions of interest (ROIs) were selected for individual cells, and the ratio of fluorescence intensity at 550 nm to that at 440 nm (550/440) was calculated to assess mitophagy activity. Background was subtracted from each channel before ratio analysis.

Quantification and statistical analysis

Fluorescence intensities were quantified using ImageJ. Mitochondrial branch length was analyzed using an ImageJ-based method (Valente et al., 2017). Statistical analyses were performed using GraphPad Prism 10 software. All data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Dunnett’s or Tukey’s multiple comparisons test.

Abbreviations

ROS

Reactive Oxygen Species

MFN1

Mitofusin 1

MFN2

Mitofusin 2

OPA1

Optic Atrophy 1

DRP1

Dynamin-Related Protein 1

FIS1

Mitochondrial Fission 1 Protein

PTRH2

Peptidyl-tRNA Hydrolase 2

BIT1

Bcl-2 Inhibitor of Transcription 1 (alias of PTRH2)

IMNEPD

Infantile-Onset Multisystem Neurologic, Endocrine, and Pancreatic Disease

ER

Endoplasmic Reticulum

GA

Golgi Apparatus

TM

Transmembrane (domain)

WT

Wild-Type

KO

Knockout

IP

Immunoprecipitation

EBSS

Earle’s Balanced Salt Solution

QLR

Glutamine, Leucine, and Arginine

mt-Keima

Mitochondria-Targeted Keima (fluorescent protein)

LC3

Microtubule-Associated Protein 1 Light Chain 3

ATP

Adenosine Triphosphate

Authors’ contributions

D.H.N.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Validation, Visualization, Writing – original draft. S.M.Y.: Conceptualization, Investigation, Methodology, Validation, Visualization. M.H.J.: Investigation, Validation. N.H.L.: Investigation. Y.W.K.: Investigation. Y.B.K.: Investigation. D.H.C.: Funding acquisition, Resources, Supervision. Y.K.J.: Conceptualization, Funding acquisition, Project administration, Supervision, Writing – review & editing.

Funding

This work was supported by the Bio-medical Technology Development Program (RS-2024–00439842) and (RS-2024–00338475), and a grant (RS-2025-00519823) of the National Research Foundation (NRF) funded by the Ministry of Science and ICT, Republic of Korea.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.