Abstract
Autophagy is an evolutionarily conserved catabolic process. In a process requiring a cascade of over 35 autophagy-related genes (Atg), a cupped phagophore membrane expands to surround cytoplasmic material, and seals itself to form an autophagosome, which finally fuses with lysosomes. Large numbers of autophagosomes form during stress responses, while simultaneously cells drastically reduce translation to conserve energy. Here, using proximity-labeling and Fluorescence in situ Hybridization we demonstrate that multiple mRNAs encoding proteins required for autophagy preferentially localize in proximity to forming autophagosomes. Polysome fractionation and proteomics of nascent proteins in proximity to forming autophagosomes provides evidence for the local translation of these mRNAs. Translation and the ribosome-binding protein RACK1 were required for the localization of these mRNAs to forming autophagosomes. Inhibition of translation or knockdown of RACK1 caused depletion of several proteins required for autophagy and a reduction in the number of autophagosomes. Local translation may enable a rapid, energy-efficient supply of proteins for autophagy to enable cells to massively induce autophagy while conserving energy during cell stress.
Data availability
All data are available in the main text and the source data file. Source data are provided with this paper. Gene Expression Omnibus (GEO) The RNA-seq data generated in this study have been deposited in the NCBI Gene Expression Omnibus (GEO) under accession code GSE319465. The protein mass spectrometry data generated in this study have been deposited in the PRIDE repository under accession code PXD073558 (“Proteomic analysis of nascent proteins in proximity to DFCP1 using SILAC labeling and APEX2-mediated biotin-streptavidin affinity purification.”) and PXD073592 (“Proteomic analysis of RacK1 immunoprecipitates from Torin1-treated wild-type HeLa cells”). Source data are provided with this paper.
References
Das, S., Vera, M., Gandin, V., Singer, R. H. & Tutucci, E. Intracellular mRNA transport and localized translation. Nat. Rev. Mol. Cell Biol. 22, 483–504 (2021).
Bourke, A. M., Schwarz, A. & Schuman, E. M. De-centralizing the central Dogma: mRNA translation in space and time. Mol. Cell 83, 452–468 (2023).
Schwanhausser, B. et al. Corrigendum: Global quantification of mammalian gene expression control. Nature 495, 126–127 (2013).
Huttelmaier, S. et al. Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438, 512–515 (2005).
Hegde, R. S. & Keenan, R. J. The mechanisms of integral membrane protein biogenesis. Nat. Rev. Mol. Cell Biol. 23, 107–124 (2022).
Reid, D. W. & Nicchitta, C. V. Diversity and selectivity in mRNA translation on the endoplasmic reticulum. Nat. Rev. Mol. Cell Biol. 16, 221–231 (2015).
Kloc, M., Zearfoss, N. R. & Etkin, L. D. Mechanisms of subcellular mRNA localization. Cell 108, 533–544 (2002).
Chouaib, R. et al. A dual protein-mRNA localization screen reveals compartmentalized translation and widespread co-translational RNA targeting. Dev. Cell 54, 773–791 e775 (2020).
Graber, T. E. et al. Reactivation of stalled polyribosomes in synaptic plasticity. Proc. Natl. Acad. Sci. USA. 110, 16205–16210 (2013).
Filipovska, A. & Rackham, O. Specialization from synthesis: how ribosome diversity can customize protein function. FEBS Lett. 587, 1189–1197 (2013).
Shi, Z. et al. Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide. Mol. Cell 67, 71–83 e77 (2017).
Klann, K., Tascher, G. & Munch, C. Functional translatome proteomics reveal converging and dose-dependent regulation by mTORC1 and eIF2alpha. Mol. Cell 77, 913–925 e914 (2020).
Chang, C., Jensen, L. E. & Hurley, J. H. Autophagosome biogenesis comes out of the black box. Nat. Cell Biol. 23, 450–456 (2021).
Melia, T. J., Lystad, A. H. & Simonsen, A. Autophagosome biogenesis: from membrane growth to closure. J Cell Biol. 219, e202002085 (2020).
Alers, S., Loffler, A. S., Wesselborg, S. & Stork, B. Role of AMPK-mTOR-Ulk1/2 in the regulation of autophagy: cross talk, shortcuts, and feedbacks. Mol. Cell Biol. 32, 2–11 (2012).
Kim, J., Kundu, M., Viollet, B. & Guan, K. L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol. 13, 132–141 (2011).
Dikic, I. & Elazar, Z. Mechanism and medical implications of mammalian autophagy. Nat. Rev. Mol. Cell Biol. 19, 349–364 (2018).
Axe, E. L. et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. J. Cell Biol. 182, 685–701 (2008).
Lamb, C. A., Yoshimori, T. & Tooze, S. A. The autophagosome: origins unknown, biogenesis complex. Nat. Rev. Mol. Cell Biol. 14, 759–774 (2013).
Lamark, T. & Johansen, T. Mechanisms of selective autophagy. Annu Rev. Cell Dev. Biol. 37, 143–169 (2021).
Bjorkoy, G. et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 171, 603–614 (2005).
Johansen, T. & Lamark, T. Selective autophagy mediated by autophagic adapter proteins. Autophagy 7, 279–296 (2011).
Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)(1). Autophagy 17, 1–382 (2021).
Biazik, J., Yla-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. Ultrastructural relationship of the phagophore with surrounding organelles. Autophagy 11, 439–451 (2015).
Dunn, W. A. Jr Studies on the mechanisms of autophagy: maturation of the autophagic vacuole. J. Cell Biol. 110, 1935–1945 (1990).
Dunn, W. A. Jr Studies on the mechanisms of autophagy: formation of the autophagic vacuole. J. Cell Biol. 110, 1923–1933 (1990).
Ericsson, J. L. Studies on induced cellular autophagy. II. Characterization of the membranes bordering autophagosomes in parenchymal liver cells. Exp. Cell Res. 56, 393–405 (1969).
Kishi-Itakura, C., Koyama-Honda, I., Itakura, E. & Mizushima, N. Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells. J. Cell Sci. 127, 4089–4102 (2014).
Novikoff, A. B. & Shin, W. Y. Endoplasmic reticulum and autophagy in rat hepatocytes. Proc. Natl. Acad. Sci. USA. 75, 5039–5042 (1978).
Kraft, C., Deplazes, A., Sohrmann, M. & Peter, M. Mature ribosomes are selectively degraded upon starvation by an autophagy pathway requiring the Ubp3p/Bre5p ubiquitin protease. Nat. Cell Biol. 10, 602–610 (2008).
Wyant, G. A. et al. NUFIP1 is a ribosome receptor for starvation-induced ribophagy. Science 360, 751–758 (2018).
Klionsky, D. J. et al. A comprehensive glossary of autophagy-related molecules and processes (2nd edition). Autophagy 7, 1273–1294 (2011).
Voigt, F. et al. Single-molecule quantification of translation-dependent association of mrnas with the endoplasmic reticulum. Cell Rep. 21, 3740–3753 (2017).
Pyhtila, B. et al. Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulum. RNA 14, 445–453 (2008).
Jagannathan, S., Reid, D. W., Cox, A. H. & Nicchitta, C. V. De novo translation initiation on membrane-bound ribosomes as a mechanism for localization of cytosolic protein mRNAs to the endoplasmic reticulum. RNA 20, 1489–1498 (2014).
Fazal, F. M. et al. Atlas of subcellular RNA localization revealed by APEX-Seq. Cell 178, 473–490 e426 (2019).
Le Guerroue, F. et al. Autophagosomal content profiling reveals an LC3C-dependent piecemeal mitophagy pathway. Mol. Cell 68, 786–796 e786 (2017).
Feng, X. et al. Local membrane source gathering by p62 body drives autophagosome formation. Nat. Commun. 14, 7338 (2023).
Kageyama, S. et al. p62/SQSTM1-droplet serves as a platform for autophagosome formation and anti-oxidative stress response. Nat. Commun. 12, 16 (2021).
Wei, Y., Chiang, W. C., Sumpter, R. Jr., Mishra, P. & Levine, B. Prohibitin 2 is an inner mitochondrial membrane mitophagy receptor. Cell 168, 224–238 e210 (2017).
Fielden, J., Popovic, M. & Ramadan, K. TEX264 at the intersection of autophagy and DNA repair. Autophagy 18, 40–49 (2022).
Larsson, E., Moren, B., McMahon, K. A., Parton, R. G. & Lundmark, R. Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization. J Cell Biol. 222 e202205122 (2023).
Zhou, C. et al. Recycling of autophagosomal components from autolysosomes by the recycler complex. Nat. Cell Biol. 24, 497–512 (2022).
Nada, S. et al. The novel lipid raft adaptor p18 controls endosome dynamics by anchoring the MEK-ERK pathway to late endosomes. EMBO J. 28, 477–489 (2009).
Zachari, M. & Ganley, I. G. The mammalian ULK1 complex and autophagy initiation. Essays Biochem. 61, 585–596 (2017).
Hara, T. & Mizushima, N. Role of ULK-FIP200 complex in mammalian autophagy: FIP200, a counterpart of yeast Atg17? Autophagy 5, 85–87 (2009).
Durgan, J. & Florey, O. Many roads lead to CASM: diverse stimuli of noncanonical autophagy share a unifying molecular mechanism. Sci. Adv. 8, eabo1274 (2022).
Guo, H. et al. Atg5 disassociates the V(1)V(0)-ATPase to promote exosome production and tumor metastasis independent of canonical macroautophagy. Dev. Cell 43, 716–730 e717 (2017).
Gardner, J. O., Leidal, A. M., Nguyen, T. A. & Debnath, J. LC3-dependent EV loading and secretion (LDELS) promotes TFRC (transferrin receptor) secretion via extracellular vesicles. Autophagy 19, 1551–1561 (2023).
Ingolia, N. T., Ghaemmaghami, S., Newman, J. R. & Weissman, J. S. Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009).
Ingolia, N. T., Lareau, L. F. & Weissman, J. S. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147, 789–802 (2011).
Pan, P. & van Breukelen, F. Preference of IRES-mediated initiation of translation during hibernation in golden-mantled ground squirrels, Spermophilus lateralis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 301, R370–R377 (2011).
Watanabe-Asano, T., Kuma, A. & Mizushima, N. Cycloheximide inhibits starvation-induced autophagy through mTORC1 activation. Biochem. Biophys. Res. Commun. 445, 334–339 (2014).
Mizushima, N., Yoshimori, T. & Levine, B. Methods in mammalian autophagy research. Cell 140, 313–326 (2010).
Latallo, M. J. et al. Single-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD. Nat. Commun. 14, 5581 (2023).
Fresno, M., Jimenez, A. & Vazquez, D. Inhibition of translation in eukaryotic systems by harringtonine. Eur. J. Biochem. 72, 323–330 (1977).
Sengupta, J. et al. Identification of the versatile scaffold protein RACK1 on the eukaryotic ribosome by cryo-EM. Nat. Struct. Mol. Biol. 11, 957–962 (2004).
Zhao, Y. et al. RACK1 Promotes Autophagy by Enhancing the Atg14L-Beclin 1-Vps34-Vps15 Complex Formation upon Phosphorylation by AMPK. Cell Rep. 13, 1407–1417 (2015).
Kim, H. D., Kong, E., Kim, Y., Chang, J. S. & Kim, J. RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy. Cell Death Dis. 8, e2800 (2017).
Thompson, M. K., Rojas-Duran, M. F., Gangaramani, P. & Gilbert, W. V. The ribosomal protein Asc1/RACK1 is required for efficient translation of short mRNAs. Elife 5, e11154 (2016).
Bai, Y., Zhou, K. & Doudna, J. A. Hepatitis C virus 3’UTR regulates viral translation through direct interactions with the host translation machinery. Nucleic Acids Res. 41, 7861–7874 (2013).
Lee, J. S. et al. RACK1 mediates rewiring of intracellular networks induced by hepatitis C virus infection. PLoS Pathog. 15, e1008021 (2019).
Johnson, A. G. et al. RACK1 on and off the ribosome. RNA 25, 881–895 (2019).
Coyle, S. M., Gilbert, W. V. & Doudna, J. A. Direct link between RACK1 function and localization at the ribosome in vivo. Mol. Cell Biol. 29, 1626–1634 (2009).
Gallo, S. et al. RACK1 specifically regulates translation through its binding to ribosomes. Mol. Cell Biol. 38, e00230-18 (2018).
Yla-Anttila, P., Vihinen, H., Jokitalo, E. & Eskelinen, E. L. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 5, 1180–1185 (2009).
Dunkle, J. A. & Cate, J. H. Ribosome structure and dynamics during translocation and termination. Annu Rev. Biophys. 39, 227–244 (2010).
Eskelinen, E. L., Reggiori, F., Baba, M., Kovacs, A. L. & Seglen, P. O. Seeing is believing: the impact of electron microscopy on autophagy research. Autophagy 7, 935–956 (2011).
Bassham, D. C. & MacIntosh, G. C. Degradation of cytosolic ribosomes by autophagy-related pathways. Plant Sci. 262, 169–174 (2017).
An, H. & Harper, J. W. Ribosome abundance control via the ubiquitin-proteasome system and autophagy. J. Mol. Biol. 432, 170–184 (2020).
An, H., Ordureau, A., Korner, M., Paulo, J. A. & Harper, J. W. Systematic quantitative analysis of ribosome inventory during nutrient stress. Nature 583, 303–309 (2020).
Lopez, A. R. et al. Autophagy-mediated control of ribosome homeostasis in oncogene-induced senescence. Cell Rep. 42, 113381 (2023).
Frankel, L. B., Lubas, M. & Lund, A. H. Emerging connections between RNA and autophagy. Autophagy 13, 3–23 (2017).
Erbil, S. et al. RACK1 is an interaction partner of ATG5 and a novel regulator of autophagy. J. Biol. Chem. 291, 16753–16765 (2016).
Rollins, M. G. et al. Negative charge in the RACK1 loop broadens the translational capacity of the human ribosome. Cell Rep. 36, 109663 (2021).
Kershner, L. & Welshhans, K. RACK1 is necessary for the formation of point contacts and regulates axon growth. Dev. Neurobiol. 77, 1038–1056 (2017).
Romano, N. et al. Ribosomal RACK1 regulates the dendritic arborization by repressing FMRP activity. Int. J. Mol. Sci. 23, 11857 (2022).
Lubas, M.et al. eIF5A is required for autophagy by mediating ATG3 translation. EMBO Rep. 19, e46072 (2018).
Pullmann, R. Jr et al. Analysis of turnover and translation regulatory RNA-binding protein expression through binding to cognate mRNAs. Mol. Cell Biol. 27, 6265–6278 (2007).
Lee, A. S., Kranzusch, P. J. & Cate, J. H. eIF3 targets cell-proliferation messenger RNAs for translational activation or repression. Nature 522, 111–114 (2015).
Shin, S. et al. mTOR inhibition reprograms cellular proteostasis by regulating eIF3D-mediated selective mRNA translation and promotes cell phenotype switching. Cell Rep. 42, 112868 (2023).
Silvestri, F. et al. eIF3d specialized translation requires a RACK1-driven eIF3d binding to 43S PIC in proliferating SH-SY5Y neuroblastoma cells. Cell Signal 125, 111494 (2025).
Arceo, X. G., Koslover, E. F., Zid, B. M. & Brown, A. I. Mitochondrial mRNA localization is governed by translation kinetics and spatial transport. PLoS Comput. Biol. 18, e1010413 (2022).
Lautier, O. et al. Co-translational assembly and localized translation of nucleoporins in nuclear pore complex biogenesis. Mol. Cell 81, 2417–2427 e2415 (2021).
Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).
Ewels, P. A. et al. The nf-core framework for community-curated bioinformatics pipelines. Nat. Biotechnol. 38, 276–278 (2020).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Pelletier, A. R. et al. MealTime-MS: a machine learning-guided real-time mass spectrometry analysis for protein identification and efficient dynamic exclusion. J. Am. Soc. Mass Spectrom. 31, 1459–1472 (2020).
Acknowledgements
The authors acknowledge support from the Canadian Institute for Health Research Project Grant 480523 (D.G.) and National Sciences and Engineering Council Discovery Grant (RGPIN-2019-07234) to D.G. and National Sciences and Engineering Council Discovery Accelerator (RGPAS-2019-00019) to D.G. and a China Council Scholarship (Y.X.). The authors acknowledge technical support from Kristofferson Tandoc. The authors gratefully acknowledge the use and technical support of the following core facilities at the University of Ottawa: Bioinformatics Core Facility (RRID:SCR_022466), Cell Biology and Imaging Core Facility (RRID:SCR_021845), Genome Editing and Molecular Biology Core Facility (RRID:SCR_022954) and the Proteomics Resource Centre.
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Y.X. performed the bulk of experiments, helped design experiments, and drafted the manuscript. K.O. performed and analyzed the Western blot. A.S performed FISH staining. K.N. established the APEX2-DFCP1 vectors and cells and contributed to its validation. K.E.K. generated FIP200 knockout cells. R.C.R provided LC3B-mCheery-GFP cell lines and contributed to interpreting results. C.B. contributed to design of experiments and editing. D.G. conceived the project, designed experiments, and wrote the manuscript. All authors reviewed and approved the manuscript.
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Xue, Y., O’Connor, K., Nalbach, K. et al. Translation in proximity to forming autophagosomes during sustained autophagy. Nat Commun (2026). https://doi.org/10.1038/s41467-026-71551-4
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DOI: https://doi.org/10.1038/s41467-026-71551-4
