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

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SIMILAR CONTENT BEING VIEWED BY OTHERS COUPLING OF RIBOSOME BIOGENESIS AND TRANSLATION INITIATION IN HUMAN MITOCHONDRIA Article Open access 17 April 2025 DISTINCT MECHANISMS OF THE HUMAN

MITORIBOSOME RECYCLING AND ANTIBIOTIC RESISTANCE Article Open access 14 June 2021 A ROADMAP FOR RIBOSOME ASSEMBLY IN HUMAN MITOCHONDRIA Article Open access 11 July 2024 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

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proteome exploration. _Nat. Methods_ 15, 617–622 (2018). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references 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 * Institute

of Biochemistry, ETH Zurich, Zurich, Switzerland Michaela Oborská-Oplová * Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland Michaela Oborská-Oplová, Alexander

Gregor Geiger, Purnima Klingauf-Nerurkar & Vikram Govind Panse * Department of Biochemistry, University of Zurich, Zurich, Switzerland Erich Michel * Department of Cellular Biochemistry,

University Medical Center Goettingen, Goettingen, Germany Sven Dennerlein & Peter Rehling * Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel Yury S.

Bykov & Maya Schuldiner * Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Bern, Switzerland Simona Amodeo & André Schneider * Max-Planck

Institute for Multidisciplinary Sciences, Goettingen, Germany Peter Rehling * Cluster of Excellence ‘Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells’,

University of Goettingen, Goettingen, Germany Peter Rehling * Faculty of Science, University of Zurich, Zurich, Switzerland Vikram Govind Panse Authors * Michaela Oborská-Oplová View author

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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. ETHICS DECLARATIONS COMPETING INTERESTS All authors declare that they have no competing interests. PEER REVIEW PEER REVIEW

INFORMATION _Nature Cell Biology_ thanks the anonymous reviewers 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 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 _h_uS5 or _h_uS5ΔMAS 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 _Tb_uS5-HA or _Tb_uS5∆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 _Tb_uS5-HA or _Tb_uS5∆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. SUPPLEMENTARY INFORMATION REPORTING SUMMARY PEER REVIEW FILE SUPPLEMENTARY TABLES 1–4 MS

data and list of oligonucleotides. SOURCE DATA SOURCE DATA ALL Statistical source data. SOURCE DATA ALL Uncropped gels. RIGHTS AND PERMISSIONS Springer Nature or its licensor (e.g. a

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version of this article is solely governed by the terms of such publishing agreement and applicable law. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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).

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