Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. Supplementary Fig. 1 contains verification of yeast strains described in the Methods section (‘Yeast strain construction’). The full version of all gels and blots is provided in Supplementary Fig. 2. Source data for the overexpression screen have been provided in Supplementary Table 1. Mass spectrometry (associated with Fig. 3d and Extended Data Fig. 6f) have been deposited to the ProteomeXchange Consortium via the PRIDE86 with the dataset identifier PXD066688 (publicly available at https://www.ebi.ac.uk/pride/archive/projects/PXD066688). Source data for the mass spectrometry have also been provided in Supplementary Table 2. RNA-seq data (associated with Fig. 4b and Extended Data Fig. 6c) can be accessed via the Gene Expression Omnibus with the accession number GSE303345. Source data for these RNA-seq analysis have also been provided in Supplementary Tables 3–5. Source data for all graphs have been provided. There are no restrictions on data availability. Source data are provided with this paper.

References

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). 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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

Article Google Scholar 1.

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 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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

Article Google Scholar 1.

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

Article Google Scholar 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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

Article Google Scholar 1.

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

Article CAS Google Scholar 1.

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

Article CAS Google Scholar 1.

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

Article CAS Google Scholar 1.

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 1.

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

Article CAS Google Scholar 1.

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

Article Google Scholar 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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 1.

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

Article Google Scholar 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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

Article CAS Google Scholar 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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 1.

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

CAS Google Scholar 1.

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 1.

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 1.

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 1.

Demichev, V., Messner, C. B., Vern