Abstract
Mitochondrial protein import through the outer and inner membranes is key to mitochondrial biogenesis. Recent studies have explored how cells respond when import is impaired by a variety of different insults. Here, we developed a mammalian import blocking system using dihydrofolate reductase fused to the N terminus of the inner membrane protein MIC60. While stabilization of the dihydrofolate reductase domain by methotrexate inhibited endogenous mitochondrial protein import, it neither activated the transcription factor ATF4, nor was affected by ATAD1 expression or by VCP/p97 inhibition. On the other hand, notably, plugging the channel of translocase of the outer membrane) induced YME1L1, an ATP-dependent protease, to eliminate translocase of the inner membrane (TIM23) channel components TIMM17A and TIMM23. The data suggest that unoccupied TIM23 complexes expose a C-terminal degron on TIMM17A to YME1L1 for degradation. Import plugging caused a cell growth defect and loss of YME1L1 exacerbated the growth inhibition, showing the protective effect of YME1L1 activity. YME1L1 seems to play a crucial role in mitochondrial quality control to counteract precursor stalling in the translocase of the outer membrane complex and unoccupied TIM23 channels.
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Data availability
Source data and mass spectrometry data are provided with this paper. All other data supporting the finding of this study are available from the corresponding authors on reasonable request. The MitoCarta 3.0 database (https://www.broadinstitute.org/mitocarta/mitocarta30-inventory-mammalian-mitochondrial-proteins-and-pathways) was used for proteomics data analysis. Proteomics from SILAC mass spectrometry has been deposited to the ProteomeXchange consortium with the dataset identifier PXD057163. Source data are provided with this paper.
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Acknowledgements
We thank D. Narendra for thoughtful reading of the manuscript and the Youle laboratory for feedback. We also thank the National Institute of Neurological Disorders and Stroke (NINDS) Light Imaging Facility, National Heart, Lung, and Blood Institute Flow Cytometry Core Facility and NINDS Protein/Peptide Sequencing Facility for technical assistance. We thank T. Langer for the YME1L1 KO HeLa cells and H. Takahashi for the pEU-E01-MCS(C1)-His vector. This work was supported by the NINDS intramural program, the National Taiwan University startup funding (grant number 111L7475, 2022) (to M.-C.H.), the National Science and Technology Council Grant (112-2320-B-002-061 to M.-C.H.), Nanken-Kyoten TMDU (2024-kokusai12) (to K.Y.) and the JSPS KAKENHI grants 22H02577 and 23H04923 (to K.Y.).
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M.-C.H., K.Y. and R.J.Y. designed the study. M.-C.H., H.K., L.L., R.K. and K.Y. performed the experiments. M.-C.H., H.K., L.L., R.K. and K.Y. analysed the data and/or its significance. M.-C.H., K.Y. and R.J.Y. wrote the paper with contributions from H.K. and L.L. M.-C.H., K.Y. and R.J.Y. acquired funding.
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Extended data
Extended Data Fig. 1 The immunofluorescent staining of IDF with anti-DHFR antibody.
HeLa6-TetOn-IDF cells prepared as in Fig. 1b were analysed by immunofluorescent staining with anti-DHFR antibody. All images are representative of at least two independent experiments and were shown as Z-projected results.
Extended Data Fig. 2 IDF-MTX complex does not dissipate the mitochondrial membrane potential.
(a) HeLa6-TetOn-IDF cells were treated with DMSO or Dox/MTX for 24 hours or further treated with CCCP for the last 1 hour. Mitochondrial membrane potential was visualized with TMRE staining. DIC, differential interference contrast. Bars, 50 μm. All images are representative of at least two independent experiments. (b) Quantification of TMRE signals in (a). The TMRE-positive area values per cell were shown as jittered dots. The data were obtained from two independent experiments (n=207 for DMSO, n=225 for Dox/MTX, n=206 for Dox/MTX+CCCP=206). Horizontal lines are displayed as the median. Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple comparisons test (ns, not significant; *** p<0.001). Source numerical data are available in source data.
Extended Data Fig. 3 VCP/p97 does not remove the mature IDF-MTX complex from mitochondria.
(a) HeLa6-TetOn-IDF cells were treated with Dox or Dox/MTX for the indicated times. The cell lysates were analysed by IB. p, precursor; m, mature form. All blots are representative of three independent experiments. (b) HeLa6-TetOn-IDF cells were treated with the indicated reagents and times. The cells (Total) were fractionated into mitochondrial (Mito) and cytosolic (Cyto) fractions, and analysed by IB. d, degraded intermediate of IDF. NMS-873, a VCP/p97 inhibitor. All blots are representative of two independent experiments. (c) HeLa6-TetOn-IDF cells were treated with the indicated reagents and times. The cell lysates were analysed by IB. All blots are representative of two independent experiments. Unprocessed blots are available in source data.
Extended Data Fig. 4 IDF plugging does not induce PINK1 stabilization.
HeLa6-WT and HeLa6-TetOn-IDF cells were treated with the indicated reagents and times. The cell lysates were analysed by IB. All blots are representative of two independent experiments. O/A, oligomycin and antimycin A; Epo, epoxomicin. Unprocessed blots are available in source data.
Extended Data Fig. 5 IDF plugging induces selective degradation of TIMM17A and TIMM23.
(a) The cell lysates prepared as in Fig. 5b were analysed by IB with antibodies against TOM subunits and TIMM50. All blots are representative of four independent experiments. (b) Volcano plot representation of the change in the putative YME1L1 substrates upon IDF plugging. The quantitative proteomic data was the same as in Fig. 5a but with different annotations. Class I proteins are the 29 putative YME1L1 substrates that are downregulated under hypoxic conditions; Class II proteins are the remaining 35 putative YME1L1 substrates that accumulated in YME1L1 KO MEF cells during normoxia. The annotation of YME1L1 substrates was retrieved from the previous report23. Unprocessed blots are available in source data.
Extended Data Fig. 6 Relationship between IDF-dependent TIMM17A degradation and mTOR activity or the imbalance of mitochondria DNA- and nuclear-encoded proteins.
(a) HeLa6-TetOn-IDF cells were treated with the indicated reagents for 24 hours. The cell lysates were analysed by IB. All blots are representative of three independent experiments. (b) Quantification of p-S6K (Ser371), TIMM17A and TIMM23 in (a) determined by densitometry. For p-S6K (Ser371), the blot densities were normalized first to total S6K and subsequently to DMSO treatment (lane 1). For TIMM17A and TIMM23, the blot densities were normalized first to HSP90 and subsequently to DMSO treatment (lane 1). The bars are displayed as mean ± SD from n=3 independent experiments. Statistical analysis was assessed by one-way ANOVA with Dunnett’s multiple comparisons test (* p<0.05; ** p<0.01; ns, not significant). (c) HeLa6-TetOn-IDF cells were treated with Dox/MTX or chloramphenicol (Cam) for 24 hours. The cell lysates were analysed by IB. All blots are representative of three biological replicates. (d) Quantification of YME1L1, TIMM17A, TIMM23 and ATF4 in (c) determined by densitometry, normalized first to HSP90 and subsequently to untreatment (lane 1). The bars are displayed as mean ± SD from n=3 independent experiments. Statistical analysis was assessed by one-way ANOVA with Dunnett’s multiple comparisons test (** p<0.01; *** p<0.001; ns, not significant). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 7 Mitochondrial localization of FLAG-tagged TIMM17A and TIMM17B.
pBABE-EGFP-P2A-TIMM17 stable HeLa cells were analysed by immunofluorescent staining with anti-FLAG and TOMM20 antibodies. Bars, 10 μm. All images are representative of two independent experiments.
Extended Data Fig. 8 Overexpression of mitochondrial bipartite signals fused to DHFR induce YME1L1-dependent TIMM17A degradation.
(a) Schematic illustration of Dox-inducible proteins consisting of hDHFR and the indicated N-terminal mitochondrial precursors that contain a bipartite signal of mitochondrial targeting sequence (MTS) and a single transmembrane domain (TMD). MIC60 is identical to IDF (Fig. 1a). PISD is a mitochondria-localized enzyme that converts phosphatidylserine to phosphatidylethanolamine (UniProt: Q9UG56). DHODH has an uncleavable MTS at the N terminus followed by TMD anchored to IMM (UniProt: Q02127), and SCO2 is known as a cytochrome c oxidase assembly factor (UniProt: O43819). (b) WT and YME1L1 KO (KO) HeLa cells stably expressing the indicated construct were treated with or without Dox/MTX for 24 hours. The cell lysates were analysed by IB. All blots are representative of three independent experiments. p, precursor; m, mature. Asterisks denote DHFR moieties partially degraded or translated from a second methionine or later. (c) Quantification of TIMM17A (n=3 independent experiments) in (b) determined by densitometry, normalized first to Actin and subsequently to untreated for each substrate. The bars are displayed as mean ± SD. Statistical analysis was assessed by one-way ANOVA with Dunnett’s multiple comparisons test (* p<0.05; ** p<0.01; *** p<0.001). Source numerical data and unprocessed blots are available in source data.
Supplementary information
Supplementary Table 1
MS data file related to Fig. 5a.
Supplementary Table 2
Materials including antibodies, cells, plasmids etc.
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Hsu, MC., Kinefuchi, H., Lei, L. et al. Mitochondrial YME1L1 governs unoccupied protein translocase channels. Nat Cell Biol 27, 309–321 (2025). https://doi.org/10.1038/s41556-024-01571-z
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DOI: https://doi.org/10.1038/s41556-024-01571-z
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