Abstract
The nascent polypeptide-associated complex (NAC) co-translationally screens all nascent proteins and regulates their access to signal recognition particle (SRP) to ensure the fidelity of protein targeting to the endoplasmic reticulum (ER). However, the mechanism by which NAC prevents the mistargeting of nascent mitochondrial proteins remains unclear. Here, we identify a molecular switch in NAC that allows its central barrel domain to adopt a stabilized conformation on ribosomes exposing a mitochondrial targeting sequence (MTS). Mutations of the MTS on the nascent chain or in the NAC switch region increase NAC barrel dynamics and reduce its binding to the ribosome. This impairs the ability of NAC to prevent mistargeting by SRP and causes ER stress in human cells. Our work reveals how NAC detects nascent mitochondrial proteins early in translation and prevents their promiscuous access to SRP, elucidating the structural basis that underlies this role and providing mechanistic insights into protein targeting fidelity with broader implications for cellular proteostasis.
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Data availability
The cryo-EM maps and corresponding atomic models generated in this study have been deposited in the PDB and EMDB under accession code: EMD-48552 and PDB-9MR4 [https://doi.org/10.2210/pdb9mr4/pdb] for the OXA1L NAC-RNCMTS structure; EMD-71310 for HSP60 NAC-RNCMTS; EMD-71286 and EMD-71287 for the H59-undocked and -docked barrel states of the OXA1L∆MTS structure, respectively. All data related to this study are available in the main text or the supplementary material. Source data are provided with this paper.
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Acknowledgements
We thank the Jomaa, Shan, and Qi lab members for the helpful discussions. Cryo-EM data collection was conducted at the molecular electron microscopy core facility (RRID:SCR_019031) at the University of Virginia (UVA) School of Medicine, which was built with NIH grant G20-RR31199. We thank Michael Purdy for assisting with the cryo-EM data collection and additional computational support. We thank Sarah Marks, Kinga Malezyna, and Travis Bishop for assisting with ribosome isolations, NAC purifications, and other biochemical assays. We also thank Shuangcheng Alivia Wu for initial help with preparation of NACβ-KO cells. This work was supported by NIH grant 1R35GM160490, the Owens Family Foundation, by the Searle Scholars Program, Grant #: SSP-2023-106, and aided by Grant # 134088-IRG-19-143-33-IRG from the American Cancer Society and the University of Virginia Comprehensive Cancer Center to A.J., by National Institutes of Health grant R35 GM136321 and National Science Foundation grant 2219287 to S.S., by National Institutes of Health grant R01DK120047, R01DK120330, R35GM130292 to L.Q. We acknowledge the cellular and molecular biology training program at UVA for support provided to E.M. through NIH T32GM139787-3 and the medical scientist training program for support provided to Z.J.L. In addition, the National Ataxia Foundation is acknowledged for support provided to L.L.L and Z.J.L through Post- and Pre-doctoral Fellowships NAF 918037 and 1036307, respectively.
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This study was conceived by A.J., S.S., E.M. and L.Q. Ribosomes were purified by E.M. and R.J.G. for structural and single molecule experiments, respectively. Y.P. provided materials used in various biochemical experiments throughout the study (site directed point mutagenesis, plasmid preps, cultured cells for ribosome isolations, and assisted with the NAC purifications). E.M. collected, processed cryo-EM data, and built the atomic model, and assembled the structural snapshots. R.J.G. conducted the single molecule total internal reflection microscopy studies. L.L.L., L.E.Z. and Z.J.L. generated the NACβ-KO cells and conducted the cell-based experiments. A.J., S.S. and L.Q. supervised the work. E.M., A.J., S.S. and R.J.G. wrote the manuscript. All authors contributed to data analysis and the final version of the manuscript.
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Maldosevic, E., Gora, R.J., Lin, L.L. et al. A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71061-3
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DOI: https://doi.org/10.1038/s41467-026-71061-3
