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An avoidance segment resolves a lethal nuclear–mitochondrial targeting conflict during ribosome assembly

Nature Cell Biology volume 27, pages 336–346 (2025) Cite this article

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Abstract

The correct sorting of nascent ribosomal proteins from the cytoplasm to the nucleus or to mitochondria for ribosome production poses a logistical challenge for cellular targeting pathways. Here we report the discovery of a conserved mitochondrial avoidance segment (MAS) within the cytosolic ribosomal protein uS5 that resolves an evolutionary lethal conflict between the nuclear and mitochondrial targeting machinery. MAS removal mistargets uS5 to the mitochondrial matrix and disrupts the assembly of the cytosolic ribosome. The resulting lethality can be rescued by impairing mitochondrial import. We show that MAS triages nuclear targeting by disabling a cryptic mitochondrial targeting activity within uS5 and thereby prevents fatal capture by mitochondria. Our findings identify MAS as an essential acquisition by the primordial eukaryote that reinforced organelle targeting fidelity while developing an endosymbiotic relationship with its mitochondrial progenitor.

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Fig. 1: Acquisition of MAS by the eukaryotic uS5.
The alternative text for this image may have been generated using AI.
Fig. 2: MAS removal mistargets uS5 to the mitochondrial matrix.
The alternative text for this image may have been generated using AI.
Fig. 3: MAS triages nuclear targeting of uS5 for ribosome assembly.
The alternative text for this image may have been generated using AI.
Fig. 4: MAS resolves a targeting conflict during ribosome assembly.
The alternative text for this image may have been generated using AI.
Fig. 5: MAS requirement is bypassed by impairing mitochondrial uptake.
The alternative text for this image may have been generated using AI.

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Data availability

All data are presented in the main text and figures or supplementary information. Detailed protocols can be requested from the corresponding author. Proteomic data generated in this study have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository (https://www.proteomexchange.org) with the dataset identifier PXD035295 and are summarized in Supplementary Table 3. The databases used in this study are the SGD (https://www.yeastgenome.org/) and the BLAST database (https://blast.ncbi.nlm.nih.gov/Blast.cgi). All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

References

  1. Margulis, L. Symbiotic theory of the origin of eukaryotic organelles; criteria for proof. Symp. Soc. Exp. Biol. 29, 21–38 (1975).

    Google Scholar 

  2. Youle, R. J. Mitochondria—striking a balance between host and endosymbiont. Science 365, eaaw9855 (2019).

    Article  CAS  PubMed  Google Scholar 

  3. Muñoz-Gómez, S. A. et al. Site-and-branch-heterogeneous analyses of an expanded dataset favour mitochondria as sister to known Alphaproteobacteria. Nat. Ecol. Evol. 6, 253–262 (2022).

    Article  PubMed  Google Scholar 

  4. Wiedemann, N. & Pfanner, N. Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 86, 685–714 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Bykov, Y. S. et al. Widespread use of unconventional targeting signals in mitochondrial ribosome proteins. EMBO J. 41, e109519 (2022).

    Article  CAS  PubMed  Google Scholar 

  6. Song, J., Herrmann, J. M. & Becker, T. Quality control of the mitochondrial proteome. Nat. Rev. Mol. Cell Biol. 22, 54–70 (2021).

    Article  CAS  PubMed  Google Scholar 

  7. von Heijne, G. Mitochondrial targeting sequences may form amphiphilic helices. EMBO J. 5, 1335–1342 (1986).

    Article  Google Scholar 

  8. Backes, S. et al. Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences. J. Cell Biol. 217, 1369–1382 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Stan, T. et al. Mitochondrial protein import: recognition of internal import signals of BCS1 by the TOM complex. Mol. Cell. Biol. 23, 2239–2250 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Rimmer, K. A. et al. Recognition of mitochondrial targeting sequences by the import receptors Tom20 and Tom22. J. Mol. Biol. 405, 804–818 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Sirrenberg, C., Bauer, M. F., Guiard, B., Neupert, W. & Brunner, M. Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature 384, 582–585 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Steger, H. F. et al. Import of ADP/ATP carrier into mitochondria: two receptors act in parallel. J. Cell Biol. 111, 2353–2363 (1990).

    Article  CAS  PubMed  Google Scholar 

  13. Hines, V. et al. Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70. EMBO J. 9, 3191–3200 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Woellhaf, M. W., Hansen, K. G., Garth, C. & Herrmann, J. M. Import of ribosomal proteins into yeast mitochondria1. Biochem. Cell Biol. 92, 489–498 (2014).

    Article  CAS  PubMed  Google Scholar 

  15. Rout, M. P., Blobel, G. & Aitchison, J. D. A distinct nuclear import pathway used by ribosomal proteins. Cell 89, 715–725 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Peña, C., Hurt, E. & Panse, V. G. Eukaryotic ribosome assembly, transport and quality control. Nat. Struct. Mol. Biol. 24, 689–699 (2017).

    Article  PubMed  Google Scholar 

  17. Klinge, S. & Woolford, J. L. Ribosome assembly coming into focus. Nat. Rev. Mol. Cell Biol. 20, 116–131 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Pillet, B., Mitterer, V., Kressler, D. & Pertschy, B. Hold on to your friends: dedicated chaperones of ribosomal proteins: dedicated chaperones mediate the safe transfer of ribosomal proteins to their site of pre-ribosome incorporation. Bioessays 39, 1–12 (2017).

    Article  CAS  PubMed  Google Scholar 

  19. Pausch, P. et al. Co-translational capturing of nascent ribosomal proteins by their dedicated chaperones. Nat. Commun. 6, 7494 (2015).

    Article  CAS  PubMed  Google Scholar 

  20. Black, J. J., Musalgaonkar, S. & Johnson, A. W. Tsr4 is a cytoplasmic chaperone for the ribosomal protein Rps2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 39, e00094-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Rössler, I. et al. Tsr4 and Nap1, two novel members of the ribosomal protein chaperOME. Nucleic Acids Res. 47, 6984–7002 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Sardana, R. et al. The DEAH-box helicase Dhr1 dissociates U3 from the Pre-rRNA to promote formation of the central pseudoknot. PLoS Biol. 13, 1002083 (2015).

    Article  Google Scholar 

  23. Ben-Shem, A. et al. The structure of the eukaryotic ribosome at 3.0 Å resolution. Science 334, 1524–1529 (2011).

    Article  CAS  PubMed  Google Scholar 

  24. Coureux, P. D., Lazennec-Schurdevin, C., Bourcier, S., Mechulam, Y. & Schmitt, E. Cryo-EM study of an archaeal 30S initiation complex gives insights into evolution of translation initiation. Commun. Biol. 3, 58 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nürenberg‐Goloub, E. et al. Molecular analysis of the ribosome recycling factor ABCE 1 bound to the 30S post‐splitting complex. EMBO J. 39, e103788 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. Preprint at bioRxiv https://doi.org/10.1101/2021.10.04.463034 (2022).

  28. Türker, C. et al. B-fabric: the Swiss army knife for life sciences. In Advances in Database Technology EDBT 2010 13th International Conference on Extending Database Technology Proceedings 717–720 (Association for Computing Machinery, 2010).

  29. Huang, D. W., Sherman, B. T. & Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Milkereit, P. et al. A Noc complex specifically involved in the formation and nuclear export of ribosomal 40S subunits. J. Biol. Chem. 278, 4072–4081 (2003).

    Article  CAS  PubMed  Google Scholar 

  31. Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 23, 102–107 (2005).

    Article  CAS  PubMed  Google Scholar 

  32. Ruan, L. et al. Cytosolic proteostasis through importing of misfolded proteins into mitochondria. Nature 543, 443–446 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kalderon, D., Roberts, B. L., Richardson, W. D. & Smith, A. E. A short amino acid sequence able to specify nuclear location. Cell 39, 499–509 (1984).

    Article  CAS  PubMed  Google Scholar 

  34. van Leeuwen, J. et al. Systematic analysis of bypass suppression of essential genes. Mol. Syst. Biol. 16, e9828 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. López-Llano, J., Campos, L. A. & Sancho, J. Alpha-helix stabilization by alanine relative to glycine: roles of polar and apolar solvent exposures and of backbone entropy. Proteins 64, 769–778 (2006).

    Article  PubMed  Google Scholar 

  36. Oborská-Oplová, M., Fischer, U., Altvater, M. & Panse, V. G. Eukaryotic ribosome assembly and nucleocytoplasmic transport. Methods Mol. Biol. 2533, 99–126 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Moy, T. I. & Silver, P. A. Requirements for the nuclear export of the small ribosomal subunit. J. Cell Sci. 115, 2985–2995 (2002).

    Article  CAS  PubMed  Google Scholar 

  38. Fischer, U. et al. A non-canonical mechanism for Crm1-export cargo complex assembly. eLife 2015, 1–20 (2015).

    CAS  Google Scholar 

  39. Altvater, M., Schütz, S., Chang, Y. & Panse, V. G. in Methods in Cell Biology Vol. 122, 437–461 (Academic, 2014).

  40. Eisenberg-Bord, M. et al. Cnm1 mediates nucleus–mitochondria contact site formation in response to phospholipid levels. J. Cell Biol. 220, e202104100 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  PubMed Central  Google Scholar 

  42. Okreglak, V. & Walter, P. The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins. Proc. Natl Acad. Sci. USA 111, 8019–8024 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Metzger, M. B., Scales, J. L., Dunklebarger, M. F., Loncarek, J. & Weissman, A. M. A protein quality control pathway at the mitochondrial outer membrane. eLife 9, e51065 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  45. Wang, L. & Walter, P. Msp1/ATAD1 in protein quality control and regulation of synaptic activities. Annu. Rev. Cell Dev. Biol. 36, 141–164 (2020).

    Article  PubMed  Google Scholar 

  46. Chen, Y. et al. Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail-anchored proteins. EMBO J. 33, 1548–1564 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Plitzko, B. & Loesgen, S. Measurement of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in culture cells for assessment of the energy metabolism. Bio-Protoc. 8, e2850 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Song, J. & Becker, T. Fidelity of organellar protein targeting. Curr. Opin. Cell Biol. 75, 102071 (2022).

    Article  CAS  PubMed  Google Scholar 

  49. Juszkiewicz, S. & Hegde, R. S. Quality control of orphaned proteins. Mol. Cell 71, 443–457 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hurt, E. C. & Schatz, G. A cytosolic protein contains a cryptic mitochondrial targeting signal. Nature 325, 499–503 (1987).

    Article  CAS  PubMed  Google Scholar 

  51. Margulis, L. & Bermudes, D. Symbiosis as a mechanism of evolution: status of cell symbiosis theory. Symbiosis 1, 101–124 (1985).

    CAS  PubMed  Google Scholar 

  52. Lang, B. F., Gray, M. W. & Burger, G. Mitochondrial genome evolution and the origin of eukaryotes. Annu. Rev. Genet. 33, 351–397 (1999).

    Article  CAS  PubMed  Google Scholar 

  53. Sagan, L. On the origin of mitosing cells. J. Theor. Biol. 14, 255–274 (1967).

    Article  CAS  PubMed  Google Scholar 

  54. Mills, D. B. et al. Eukaryogenesis and oxygen in Earth history. Nat. Ecol. Evol. 6, 520–532 (2022).

    Google Scholar 

  55. Bader, G. et al. Assigning mitochondrial localization of dual localized proteins using a yeast bi-genomic mitochondrial-split-gfp. eLife 9, 1–24 (2020).

    Article  Google Scholar 

  56. Emanuelsson, O. & von Heijne, G. Prediction of organellar targeting signals. Biochim. Biophys. Acta 1541, 114–119 (2001).

    Article  CAS  PubMed  Google Scholar 

  57. Zheng, N. & Gierasch, L. M. Signal sequences: the same yet different. Cell 86, 849–852 (1996).

    Article  CAS  PubMed  Google Scholar 

  58. Zhang, X. & Shan, S. O. Fidelity of cotranslational protein targeting by the signal recognition particle. Annu. Rev. Biophys. 43, 381–408 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Hegde, R. S. & Zavodszky, E. Recognition and degradation of mislocalized proteins in health and disease Cold Spring Harb. Perspect. Biol. 11, a033902 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  PubMed  Google Scholar 

  61. Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).

    Article  CAS  PubMed  Google Scholar 

  62. Wolski, W., Grossmann, J. & Panse, C. protViz/SRMService. GitHub https://github.com/protViz/SRMService (2018).

  63. Meurer, M. et al. Genome-wide C-SWAT library for high-throughput yeast genome tagging. Nat. Methods 15, 598–600 (2018).

    Article  CAS  PubMed  Google Scholar 

  64. Cohen, Y. & Schuldiner, M. Advanced methods for high-throughput microscopy screening of genetically modified yeast libraries. Methods Mol. Biol. 781, 127–159 (2011).

    Article  CAS  PubMed  Google Scholar 

  65. Tong, A. H. Y. & Boone, C. Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol. Biol. 313, 171–192 (2006).

    CAS  PubMed  Google Scholar 

  66. Hanscho, M. et al. Nutritional requirements of the BY series of Saccharomyces cerevisiae strains for optimum growth. FEMS Yeast Res. 12, 796–808 (2012).

    Article  CAS  PubMed  Google Scholar 

  67. Schindelin, J. et al. Fiji—an open source platform for biological image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  PubMed  Google Scholar 

  68. Bareth, B. et al. Oms1 associates with cytochrome c oxidase assembly intermediates to stabilize newly synthesized Cox1. Mol. Biol. Cell 27, 1570–1580 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Wittig, I., Braun, H. P. & Schägger, H. Blue native PAGE. Nat. Protoc. 1, 418–428 (2006).

    Article  CAS  PubMed  Google Scholar 

  70. Wenger, C., Oeljeklaus, S., Warscheid, B., Schneider, A. & Harsman, A. A trypanosomal orthologue of an intermembrane space chaperone has a non-canonical function in biogenesis of the single mitochondrial inner membrane protein translocase. PLoS Pathog. 13, e1006550 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  71. Michel, E., Cucuzza, S., Mittl, P. R. E., Zerbe, O. & Plückthun, A. Improved repeat protein stability by combined consensus and computational protein design. Biochemistry 62, 318–329 (2023).

    Article  CAS  PubMed  Google Scholar 

  72. Delano, W. L. PyMOL: an open-source molecular graphics tool.

  73. Petterson, Eric. et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci. 30, 70–82 (2021).

    Article  Google Scholar 

  74. Cherry, J. M. et al. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. 40, D700 (2012).

    Article  CAS  PubMed  Google Scholar 

  75. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  76. Weill, U. et al. Genome-wide SWAp-Tag yeast libraries for proteome exploration. Nat. Methods 15, 617–622 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank M. Peter, D. Rappaport and R. Li for generously sharing yeast strains and plasmids. We thank R. Pillai, H. Hilbi, M. Seeger, H. Meyer, M. Pilhofer and all members of the Panse laboratory for enthusiastic discussions, the Center for Microscopy and Image analysis, University of Zurich (ZMB, UZH) for maintaining the imaging equipment and the Functional Genomic Center Zurich (FGCZ) for proteomic analysis. We thank C. Pena for performing a comparative analysis of ribosomal proteins, F. Willenborg for technical support during the maternity leave of M.O.-O. M.O.-O. was supported by a Boehringer Ingelheim Fonds PhD fellowship, and a Pregnancy and Maternity Leave Compensation Grant from National Center of Competence in Research (NCCR) RNA and Disease. V.G.P. is supported by grants from the Swiss National Science Foundation (SNF 188527), NCCR RNA and Disease (182880), Eidgenössische Technische Hochschule Zürich (ETHZ), Novartis Foundation, Olga Mayenfisch Stiftung and a Starting Grant Award from the European Research Council (ERC) (EURIBIO260676). Work in the laboratory of A.S. was supported in part by NCCR RNA and Disease (205601) and by project grant SNF 205200 both funded by the Swiss National Science Foundation. Research of P.R. was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany’s Excellence Strategy EXC 2067/1-390729940, SFB1565 (P14), the ERC Advanced Grant MiXpress (ERCAdG no. 101095062) and the Max Planck Society. Work in the Schuldiner laboratory is supported by the ERC CoG OnTarget (864068). M.S. is an Incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics. Funding: the Swiss National Science Foundation (VGP); NCCR in RNA and Disease (205601) (V.G.P. and A.S.); Novartis Science Foundation (V.G.P.); Olga Mayenfisch Stiftung (V.G.P.); ERC Starting Grant Award EURIBIO260676 (V.G.P.); Boehringer Ingelheim Fonds PhD fellowship (M.O.-O.); Swiss National Science Foundation (SNF 205200) (A.S.); DFG, German Research Foundation, EXC 2067/1-390729940, SFB1565 (P.R.); ERC Advanced Grant MiXpress (ERCAdG No. 101095062) (P.R.); ERC Consolidator Grant OnTarget (864068) (M.S.); and Max Planck Society (P.R.).

Author information

Authors and Affiliations

  1. Institute of Biochemistry, ETH Zurich, Zurich, Switzerland

    Michaela Oborská-Oplová

  2. Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland

    Michaela Oborská-Oplová, Alexander Gregor Geiger, Purnima Klingauf-Nerurkar & Vikram Govind Panse

  3. Department of Biochemistry, University of Zurich, Zurich, Switzerland

    Erich Michel

  4. Department of Cellular Biochemistry, University Medical Center Goettingen, Goettingen, Germany

    Sven Dennerlein & Peter Rehling

  5. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Yury S. Bykov & Maya Schuldiner

  6. Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland

    Simona Amodeo & André Schneider

  7. Max-Planck Institute for Multidisciplinary Sciences, Goettingen, Germany

    Peter Rehling

  8. Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’, University of Goettingen, Goettingen, Germany

    Peter Rehling

  9. Faculty of Science, University of Zurich, Zurich, Switzerland

    Vikram Govind Panse

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  1. Michaela Oborská-Oplová
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  2. Alexander Gregor Geiger
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Contributions

Experimental design: M.O.-O., V.G.P., S.D., P.R., S.A., A.S. and M.S. Experiment execution: M.O.-O., P.K.-N., A.G.G., E.M., S.D., S.A. and Y.S.B. Data analysis: M.O.-O., A.G.G., E.M., S.D. and S.A. Supervision: V.G.P., A.S., P.R. and M.S. Writing—original draft: M.O.-O. and V.G.P. Writing—review and editing: M.O.-O., V.G.P., M.S., Y.S.B., P.R. and A.S.

Corresponding author

Correspondence to Vikram Govind Panse.

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Extended data

Extended Data Fig. 1 Eukaryote-specific segments (ESSs) of r-proteins.

Structures of 46 r-proteins from S. cerevisiae (PDB ID: 4V7R) with marked ESSs based on the sequence alignment between S. cerevisiae and archaeal species. N-terminal ESSs in blue, insertions in green, and C-terminal ESSs in red. The shared sequence between S. cerevisiae and archaea in yellow.

Extended Data Fig. 2 MAS recruits Tsr4 for helix-chaperoning.

a, An AlphaFold generated multimer model of Tsr4-uS5 complex. The inter-residue distance errors in the model were low (pTM = 0.68), and the model’s confidence in the positions of individual residues was high (pLDDT = 67.82). MAS (blue) and Tsr4 (pink) and the G128 residue (green) in the α-helix of the uS5 RNA-binding domain (grey) surrounded by Tsr4. b, Western analysis of tsr4∆ cells expression uS5-GFP or uS5GA-GFP fusion proteins using indicated antibodies. Gsp1 was used as a loading control. Representative blot of n = 3 independent experiments is shown. c, Localisation of uS5-GFP or uS5GA-GFP fusions in tsr4∆ cells Scale bar = 5 µm. Representative images of n = 3 biological replicates are shown. Source unprocessed blots are available in source data.

Source data

Extended Data Fig. 3 MAS triages nuclear targeting of uS5 for ribosome assembly across eukaryotes.

a, Fluorescent images of split-GFP assay performed with MTS-mCherry-GFP1-10 strain expressing huS5 or huS5ΔMAS fused to GFP11 fragment grown in selective 3% glycerol-containing media. Scale bar = 5 µm. b, Fluorescence imaging of HeLa Flp-In T-Rex cells transiently expressing uS5-Venus or uS5∆MAS-Venus. Mitochondria were stained by Mitotracker Red. Scale bar = 20 μm. c, upper panel: Immunofluorescence assays of T. brucei cells expressing TbuS5-HA or TbuS5∆MAS-HA fusion proteins. ATOM40 was used as a mitochondrial marker. Scale bar = 5 µm. lower panel: Digitonin extractions of crude mitochondrial fractions from T. brucei cells expressing TbuS5-HA or TbuS5∆MAS-HA fusion proteins were analysed by immunoblots using the indicated antibodies. WC, whole cell extract; S, cytosol-containing supernatant; P, mitochondria-enriched pellet. d, Upper panel: Cartoon depicting uS5 domain organization from archaea Thermoprotei archaeon. Lower panel: Fluorescence imaging of wild-type yeast cells expressing uS5-GFP and AE-GFP from archaea Thermoprotei archaeon (Ta). MTS-mCherry was used as a mitochondrial marker. The nucleus is indicated by a white arrow. Scale bar = 5 µm. Source unprocessed blots are available in source data.

Source data

Extended Data Fig. 4 Truncational analysis of MAS.

Top: Cartoon depicting uS5GA domain organization. Sequence of N-terminal MAS in blue, region shared between eukaryotes and archaea (AE) in yellow, universally conserved RNA-binding domain in grey, C-terminal eukaryotic-specific extension (CE) in red. G128A point mutation in green. Bottom: yrb2∆ cells expressing C-terminal GFP fusions of uS5GA, uS5GA∆MAS, uS5GA∆N22, uS5GA∆N25 and uS5GA∆N28. Representative images of n = 3 biological replicates are shown. Mitochondria were stained by Mitotracker Red. Scale bar = 5 µm.

Extended Data Fig. 5 Functional analysis of uS5ΔMAS cells.

a, rps2∆ubx2∆, rps2∆msp1∆, or rps2∆cmn1∆ shuffle strains transformed with empty vector or plasmids encoding uS5 or uS5∆MAS were spotted in 10-fold serial dilutions on selective SD and 5-FOA containing plates and grown at 30 °C for 3-8 days. b, Non-native extracts derived from mitochondria isolated from uS5 WT, uS5GA, and uS5GA∆MAS were separated by SDS-PAGE and subjected to Western blotting analysis using indicated antibodies. c, mtDNA amplification of COX1 gene from rps2∆ or rps2∆ tom70∆ cells expressing indicated uS5 variants. Source unprocessed blots are available in source data.

Source data

Extended Data Fig. 6 Bi-genomic split GFP assay55.

visualizes the mitochondrial fraction of any protein by using a yeast strain encoding GFP1-10 within the mitochondrial (mt) DNA and 3xGFP11 fused to a protein of interest. A genomically tagged collection of all available cytosolic ribosomal proteins (RPs) was created using the SWAp-Tag (SWAT) approach76. The collection was grown in YPD media, and representative images of n = 3 individual experiments for r-proteins with mitochondrial fractions are shown along with a no GFP11 control. mCherry fused to Su9MTS was used as a mitochondrial marker. Scale bar 5 µm.

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Oborská-Oplová, M., Geiger, A.G., Michel, E. et al. An avoidance segment resolves a lethal nuclear–mitochondrial targeting conflict during ribosome assembly. Nat Cell Biol 27, 336–346 (2025). https://doi.org/10.1038/s41556-024-01588-4

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