Endo, T. & Wiedemann, N. Molecular machineries and pathways of mitochondrial protein transport. Nat. Rev. Mol. Cell Biol. https://doi.org/10.1038/s41580-025-00865-w (2025).

Pfanner, N., den Brave, F. & Becker, T. Mitochondrial protein import stress. Nat. Cell Biol. 27, 188–201 (2025).

Article 
CAS 

Google Scholar
 

Balzarini, M., Kim, J. & Weidberg, H. Quality control of un-imported mitochondrial proteins at a glance. J. Cell Sci. 138, jcs263757 (2025).

Article 
CAS 

Google Scholar
 

Lee-Glover, L. P. & Shutt, T. E. Mitochondrial quality control pathways sense mitochondrial protein import. Trends Endocrinol. Metab. 35, 308–320 (2024).

Article 
CAS 

Google Scholar
 

Suomalainen, A. & Nunnari, J. Mitochondria at the crossroads of health and disease. Cell 187, 2601–2627 (2024).

Article 
CAS 

Google Scholar
 

Nashed, S. et al. Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import. PLoS Genet. 19, e1010848 (2023).

Article 
CAS 

Google Scholar
 

Vögtle, F.-N. et al. Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability. Cell 139, 428–439 (2009).

Article 

Google Scholar
 

Craig, E. A. Hsp70 at the membrane: driving protein translocation. BMC Biol. 16, 11 (2018).

Article 

Google Scholar
 

Boos, F., Labbadia, J. & Herrmann, J. M. How the mitoprotein-induced stress response safeguards the cytosol: a unified view. Trends Cell Biol. 30, 241–254 (2020).

Article 
CAS 

Google Scholar
 

Nowicka, U. et al. Cytosolic aggregation of mitochondrial proteins disrupts cellular homeostasis by stimulating the aggregation of other proteins. eLife 10, e65484 (2021).

Article 
CAS 

Google Scholar
 

Boos, F. et al. Mitochondrial protein-induced stress triggers a global adaptive transcriptional programme. Nat. Cell Biol. 21, 442–451 (2019).

Article 
CAS 

Google Scholar
 

Fessler, E. et al. A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature 579, 433–437 (2020).

Article 
CAS 

Google Scholar
 

Fessler, E., Krumwiede, L. & Jae, L. T. DELE1 tracks perturbed protein import and processing in human mitochondria. Nat. Commun. 13, 1853 (2022).

Article 
CAS 

Google Scholar
 

Guo, X. et al. Mitochondrial stress is relayed to the cytosol by an OMA1-DELE1-HRI pathway. Nature 579, 427–432 (2020).

Article 
CAS 

Google Scholar
 

Haakonsen, D. L. et al. Stress response silencing by an E3 ligase mutated in neurodegeneration. Nature 626, 874–880 (2024).

Article 
CAS 

Google Scholar
 

Krämer, L. et al. MitoStores: chaperone-controlled protein granules store mitochondrial precursors in the cytosol. EMBO J. 42, e112309 (2023).

Article 

Google Scholar
 

Münch, C. & Harper, J. W. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation. Nature 534, 710–713 (2016).

Article 

Google Scholar
 

Shakya, V. P. et al. A nuclear-based quality control pathway for non-imported mitochondrial proteins. eLife 10, e61230 (2021).

Article 
CAS 

Google Scholar
 

Sutandy, F. X. R., Gößner, I., Tascher, G. & Münch, C. A cytosolic surveillance mechanism activates the mitochondrial UPR. Nature 618, 849–854 (2023).

Article 
CAS 

Google Scholar
 

Wang, X. & Chen, X. J. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524, 481–484 (2015).

Article 
CAS 

Google Scholar
 

Weidberg, H. & Amon, A. MitoCPR — a surveillance pathway that protects mitochondria in response to protein import stress. Science 360, eaan4146 (2018).

Article 

Google Scholar
 

Wrobel, L. et al. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488 (2015).

Article 
CAS 

Google Scholar
 

Coyne, L. P. et al. Mitochondrial protein import clogging as a mechanism of disease. eLife 12, e84330 (2023).

Article 
CAS 

Google Scholar
 

Hsu, M.-C. et al. Mitochondrial YME1L1 governs unoccupied protein translocase channels. Nat. Cell Biol. 27, 309–321 (2025).

Article 
CAS 

Google Scholar
 

Kim, J. et al. ATAD1 prevents clogging of TOM and damage caused by un-imported mitochondrial proteins. Cell Rep. 43, 114473 (2024).

Article 
CAS 

Google Scholar
 

Krakowczyk, M. et al. OMA1 protease eliminates arrested protein import intermediates upon mitochondrial depolarization. J. Cell Biol. 223, e202306051 (2024).

Article 
CAS 

Google Scholar
 

Mårtensson, C. U. et al. Mitochondrial protein translocation-associated degradation. Nature 569, 679–683 (2019).

Article 

Google Scholar
 

Buechel, E. R. & Pinkett, H. W. Transcription factors and ABC transporters: from pleiotropic drug resistance to cellular signaling in yeast. FEBS Lett. 594, 3943–3964 (2020).

Article 
CAS 

Google Scholar
 

Delahodde, A., Pandjaitan, R., Corral-Debrinski, M. & Jacq, C. Pse1/Kap121-dependent nuclear localization of the major yeast multidrug resistance (MDR) transcription factor Pdr1. Mol. Microbiol. 39, 304–313 (2001).

Article 
CAS 

Google Scholar
 

Bursać, D. & Lithgow, T. Jid1 is a J-protein functioning in the mitochondrial matrix, unable to directly participate in endoplasmic reticulum associated protein degradation. FEBS Lett. 583, 2954–2958 (2009).

Article 

Google Scholar
 

Lytovchenko, O. et al. The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo-ATP synthase. EMBO J. 33, 1624–1638 (2014).

Article 
CAS 

Google Scholar
 

Westermann, B., Prip-Buus, C., Neupert, W. & Schwarz, E. The role of the GrpE homologue, Mge1p, in mediating protein import and protein folding in mitochondria. EMBO J. 14, 3452–3460 (1995).

Article 
CAS 

Google Scholar
 

Nguyen Ba, A. N., Pogoutse, A., Provart, N. & Moses, A. M. NLStradamus: a simple hidden Markov model for nuclear localization signal prediction. BMC Bioinformatics 10, 202 (2009).

Article 

Google Scholar
 

Kimura, M. & Imamoto, N. Biological significance of the importin-β family-dependent nucleocytoplasmic transport pathways. Traffic 15, 727–748 (2014).

Article 
CAS 

Google Scholar
 

Veatch, J. R., McMurray, M. A., Nelson, Z. W. & Gottschling, D. E. Mitochondrial dysfunction leads to nuclear genome instability via an iron-sulfur cluster defect. Cell 137, 1247–1258 (2009).

Article 

Google Scholar
 

Devaux, F., Carvajal, E., Moye-Rowley, S. & Jacq, C. Genome-wide studies on the nuclear PDR3-controlled response to mitochondrial dysfunction in yeast. FEBS Lett. 515, 25–28 (2002).

Article 
CAS 

Google Scholar
 

Rödl, S. et al. A protein-specific priority code in presequences determines the efficiency of mitochondrial protein import. PLoS Biol. 23, e3003298 (2025).

Article 

Google Scholar
 

Gallas, M. R., Dienhart, M. K., Stuart, R. A. & Long, R. M. Characterization of Mmp37p, a Saccharomyces cerevisiae mitochondrial matrix protein with a role in mitochondrial protein import. Mol. Biol. Cell 17, 4051–4062 (2006).

Article 
CAS 

Google Scholar
 

Tamura, Y. et al. Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria. J. Cell Biol. 174, 631–637 (2006).

Article 
CAS 

Google Scholar
 

Tamura, Y. et al. Tam41 is a CDP-diacylglycerol synthase required for cardiolipin biosynthesis in mitochondria. Cell Metab. 17, 709–718 (2013).

Article 
CAS 

Google Scholar
 

Kutik, S. et al. The translocator maintenance protein Tam41 is required for mitochondrial cardiolipin biosynthesis. J. Cell Biol. 183, 1213–1221 (2008).

Article 
CAS 

Google Scholar
 

Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).

Article 
CAS 

Google Scholar
 

Banerjee, A., Rahman, H., Prasad, R. & Golin, J. How fungal multidrug transporters mediate hyper resistance through DNA amplification and mutation. Mol. Microbiol. 118, 3–15 (2022).

Article 
CAS 

Google Scholar
 

Ducett, J. K. et al. Unfolding of the C-terminal domain of the J-protein Zuo1 releases autoinhibition and activates Pdr1-dependent transcription. J. Mol. Biol. 425, 19–31 (2013).

Article 
CAS 

Google Scholar
 

DeRisi, J. et al. Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants. FEBS Lett. 470, 156–160 (2000).

Article 
CAS 

Google Scholar
 

Thakur, J. K. et al. A nuclear receptor-like pathway regulating multidrug resistance in fungi. Nature 452, 604–609 (2008).

Article 
CAS 

Google Scholar
 

Wu, G.-Z. et al. Control of retrograde signalling by protein import and cytosolic folding stress. Nat. Plants 5, 525–538 (2019).

Article 
CAS 

Google Scholar
 

Narendra, D. P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, e1000298 (2010).

Article 

Google Scholar
 

Nargund, A. M., Pellegrino, M. W., Fiorese, C. J., Baker, B. M. & Haynes, C. M. Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337, 587–590 (2012).

Article 
CAS 

Google Scholar
 

Rolland, S. G. et al. Compromised mitochondrial protein import acts as a signal for UPRmt. Cell Rep. 28, 1659–1669.e5 (2019).

Article 
CAS 

Google Scholar
 

Shpilka, T. et al. UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans. Nat. Commun. 12, 479 (2021).

Article 
CAS 

Google Scholar
 

Konovalova, S. et al. Redox regulation of GRPEL2 nucleotide exchange factor for mitochondrial HSP70 chaperone. Redox Biol. 19, 37–45 (2018).

Article 
CAS 

Google Scholar
 

Marada, A. et al. Mge1, a nucleotide exchange factor of Hsp70, acts as an oxidative sensor to regulate mitochondrial Hsp70 function. Mol. Biol. Cell 24, 692–703 (2013).

Article 
CAS 

Google Scholar
 

DiGiovanni, L. F. et al. ROS transfer at peroxisome-mitochondria contact regulates mitochondrial redox. Science 389, 157–162 (2025).

Article 
CAS 

Google Scholar
 

McMinimy, R. et al. Reactive oxygen species control protein degradation at the mitochondrial import gate. Mol. Cell 84, 4612–4628.e13 (2024).

Article 
CAS 

Google Scholar
 

Savojardo, C., Bruciaferri, N., Tartari, G., Martelli, P. L. & Casadio, R. DeepMito: accurate prediction of protein sub-mitochondrial localization using convolutional neural networks. Bioinformatics 36, 56–64 (2020).

Article 
CAS 

Google Scholar
 

Srivastava, S. et al. Regulation of mitochondrial protein import by the nucleotide exchange factors GrpEL1 and GrpEL2 in human cells. J. Biol. Chem. 292, 18075–18090 (2017).

Article 
CAS 

Google Scholar
 

Calvo, S. E. et al. Comparative analysis of mitochondrial N-termini from mouse, human, and yeast. Mol. Cell. Proteomics 16, 512–523 (2017).

Article 
CAS 

Google Scholar
 

Bayne, A. N., Dong, J., Amiri, S., Farhan, S. M. K. & Trempe, J.-F. MTSviewer: a database to visualize mitochondrial targeting sequences, cleavage sites, and mutations on protein structures. PLoS ONE 18, e0284541 (2023).

Article 
CAS 

Google Scholar
 

Myers, A. M., Pape, L. K. & Tzagoloff, A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 4, 2087–2092 (1985).

Article 
CAS 

Google Scholar
 

Schrott, S. & Osman, C. Two mitochondrial HMG-box proteins, Cim1 and Abf2, antagonistically regulate mtDNA copy number in Saccharomyces cerevisiae. Nucleic Acids Res. 51, 11813–11835 (2023).

Article 
CAS 

Google Scholar
 

Smoyer, C. J. et al. Analysis of membrane proteins localizing to the inner nuclear envelope in living cells. J. Cell Biol. 215, 575–590 (2016).

Article 
CAS 

Google Scholar
 

Fukasawa, Y. et al. MitoFates: improved prediction of mitochondrial targeting sequences and their cleavage sites. Mol. Cell. Proteomics 14, 1113–1126 (2015).

Article 
CAS 

Google Scholar
 

Pfanner, N., Müller, H. K., Harmey, M. A. & Neupert, W. Mitochondrial protein import: involvement of the mature part of a cleavable precursor protein in the binding to receptor sites. EMBO J. 6, 3449–3454 (1987).

Article 
CAS 

Google Scholar
 

Wiley, J. C., Wailes, L. A., Idzerda, R. L. & McKnight, G. S. Role of regulatory subunits and protein kinase inhibitor (PKI) in determining nuclear localization and activity of the catalytic subunit of protein kinase A. J. Biol. Chem. 274, 6381–6387 (1999).

Article 
CAS 

Google Scholar
 

Bragoszewski, P., Turek, M. & Chacinska, A. Control of mitochondrial biogenesis and function by the ubiquitin-proteasome system. Open Biol. 7, 170007 (2017).

Article 

Google Scholar
 

Valenti, R. et al. A proteome-wide yeast degron collection for the dynamic study of protein function. J. Cell Biol. 224, e202409050 (2025).

Article 
CAS 

Google Scholar
 

Morawska, M. & Ulrich, H. D. An expanded tool kit for the auxin-inducible degron system in budding yeast. Yeast 30, 341–351 (2013).

Article 
CAS 

Google Scholar
 

Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).

Article 
CAS 

Google Scholar
 

Gallego, O. et al. Detection and characterization of protein interactions in vivo by a simple live-cell imaging method. PLoS ONE 8, e62195 (2013).

Article 
CAS 

Google Scholar
 

Haruki, H., Nishikawa, J. & Laemmli, U. K. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol. Cell 31, 925–932 (2008).

Article 
CAS 

Google Scholar
 

Gelperin, D. M. et al. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 19, 2816–2826 (2005).

Article 
CAS 

Google Scholar
 

Young, B. P. & Loewen, C. J. Balony: a software package for analysis of data generated by synthetic genetic array experiments. BMC Bioinformatics 14, 354 (2013).

Article 

Google Scholar
 

Storey, J. D. & Tibshirani, R. Statistical methods for identifying differentially expressed genes in DNA microarrays. Methods Mol. Biol. 224, 149–157 (2003).

CAS 

Google Scholar
 

Meisinger, C., Pfanner, N. & Truscott, K. N. Isolation of yeast mitochondria. Methods Mol. Biol. 313, 33–39 (2006).

CAS 

Google Scholar
 

Glowczewski, L., Waterborg, J. H. & Berman, J. G. Yeast chromatin assembly complex 1 protein excludes nonacetylatable forms of histone H4 from chromatin and the nucleus. Mol. Cell. Biol. 24, 10180 (2004).

Article 
CAS 

Google Scholar
 

Hughes, C. S. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 14, 68–85 (2019).

Article 
CAS 

Google Scholar
 

Rappsilber, J., Mann, M. & Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2, 1896–1906 (2007).

Article 
CAS 

Google Scholar
 

Demichev, V., Messner, C. B., Vernardis, S. I., Lilley, K. S. & Ralser, M. DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput. Nat. Methods 17, 41–44 (2020).

Article 
CAS 

Google Scholar
 

Teo, G. et al. SAINTq: scoring protein-protein interactions in affinity purification-mass spectrometry experiments with fragment or peptide intensity data. Proteomics 16, 2238–2245 (2016).

Article 
CAS 

Google Scholar
 

Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25, 402–408 (2001).

Article 
CAS 

Google Scholar
 

Sprouffske, K. & Wagner, A. Growthcurver: an R package for obtaining interpretable metrics from microbial growth curves. BMC Bioinformatics 17, 172 (2016).

Article 

Google Scholar
 

Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

Article 
CAS 

Google Scholar
 

Waterhouse, A. M., Procter, J. B., Martin, D. M. A., Clamp, M. & Barton, G. J. Jalview Version 2 — a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009).

Article 
CAS 

Google Scholar
 

Creighton, T. E. Proteins: Structures and Molecular Properties (W. H. Freeman, 1993).

Perez-Riverol, Y. et al. The PRIDE database at 20 years: 2025 update. Nucleic Acids Res. 53, D543–D553 (2025).

Article 

Google Scholar
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