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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. Access through your institution

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SYNCHRONIZES THE MITOCHONDRIAL IMPORT PATHWAYS FOR METABOLIC REWIRING Article Open access 20 June 2024 MOLECULAR PATHWAY OF MITOCHONDRIAL PREPROTEIN IMPORT THROUGH THE TOM–TIM23 SUPERCOMPLEX

Article 11 September 2023 STRUCTURAL BASIS OF MITOCHONDRIAL PROTEIN IMPORT BY THE TIM23 COMPLEX Article 21 June 2023 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. REFERENCES * Yamano, K., Kinefuchi, H. & Kojima, W.

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220, e202006180 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references 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.). AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA Meng-Chieh Hsu, 

Linlin Lei & Richard J. Youle * Department of Animal Science and Technology, National Taiwan University, Taipei City, Taiwan Meng-Chieh Hsu * Department of Biomolecular Pathogenesis,

Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan Hiroki Kinefuchi, Reika Kikuchi & Koji Yamano * Department of Biomolecular Pathogenesis, Medical Research

Laboratory, Institute of Integrated Research, Institute of Science Tokyo, Tokyo, Japan Hiroki Kinefuchi, Reika Kikuchi & Koji Yamano Authors * Meng-Chieh Hsu View author publications You

can also search for this author inPubMed Google Scholar * Hiroki Kinefuchi View author publications You can also search for this author inPubMed Google Scholar * Linlin Lei View author

publications You can also search for this author inPubMed Google Scholar * Reika Kikuchi View author publications You can also search for this author inPubMed Google Scholar * Koji Yamano

View author publications You can also search for this author inPubMed Google Scholar * Richard J. Youle View author publications You can also search for this author inPubMed Google Scholar

CONTRIBUTIONS 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. CORRESPONDING AUTHORS Correspondence to Koji Yamano or

Richard J. Youle. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature Cell Biology_ thanks Cole Haynes, Thomas

Becker, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer

Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 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.

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. 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. 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. 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. 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. Source data SUPPLEMENTARY

INFORMATION REPORTING SUMMARY PEER REVIEW FILE SUPPLEMENTARY TABLE 1 MS data file related to Fig. 5a. SUPPLEMENTARY TABLE 2 Materials including antibodies, cells, plasmids etc. SOURCE DATA

SOURCE DATA FIG. 1 Unprocessed western blots. SOURCE DATA FIG. 1 Statistical source data. SOURCE DATA FIG. 2 Unprocessed western blots. SOURCE DATA FIG. 2 Statistical source data. SOURCE

DATA FIG. 3 Unprocessed western blots. SOURCE DATA FIG. 3 Statistical source data. SOURCE DATA FIG. 4 Unprocessed western blots. SOURCE DATA FIG. 4 Statistical source data. SOURCE DATA FIG.

5 Unprocessed western blots. SOURCE DATA FIG. 5 Statistical source data. SOURCE DATA FIG. 6 Unprocessed western blots. SOURCE DATA FIG. 6 Statistical source data. SOURCE DATA FIG. 7

Unprocessed western blots. SOURCE DATA FIG. 7 Statistical source data. SOURCE DATA FIG. 8 Unprocessed western blots. SOURCE DATA FIG. 8 Statistical source data. SOURCE DATA EXTENDED DATA

FIG. 2 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 3 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 4 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 5

Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 6 Unprocessed western blots. SOURCE DATA EXTENDED DATA FIG. 6 Statistical source data. SOURCE DATA EXTENDED DATA FIG. 8 Unprocessed

western blots. SOURCE DATA EXTENDED DATA FIG. 8 Statistical source data. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received:

26 January 2024 * Accepted: 04 November 2024 * Published: 07 January 2025 * Issue Date: February 2025 * DOI: https://doi.org/10.1038/s41556-024-01571-z SHARE THIS ARTICLE Anyone you share

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