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Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy

Nature Cell Biology volume 20, pages 135–143 (2018)Cite this article

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Abstract

Ribosomes are abundant cellular machines1,2 that are regulated by assembly, supernumerary subunit turnover and nascent chain quality control mechanisms1,2,3,4,5. Moreover, nitrogen starvation in yeast has been reported to promote selective ribosome delivery to the vacuole in an autophagy conjugation system dependent manner, a process called ‘ribophagy’6,7. However, whether ribophagy in mammals is selective or regulated is unclear. Using Ribo–Keima flux reporters, we find that starvation or mTOR inhibition promotes VPS34-dependent ribophagic flux, which, unlike yeast, is largely independent of ATG8 conjugation and occurs concomitantly with other cytosolic protein autophagic flux reporters8,9. Ribophagic flux was not induced upon inhibition of translational elongation or nascent chain uncoupling, but was induced in a comparatively selective manner under proteotoxic stress induced by arsenite10 or chromosome mis-segregation11, dependent upon VPS34 and ATG8 conjugation. Unexpectedly, agents typically used to induce selective autophagy also promoted increased ribosome and cytosolic protein reporter flux, suggesting significant bulk or ‘bystander’ autophagy during what is often considered selective autophagy12,13. These results emphasize the importance of monitoring non-specific cargo flux when assessing selective autophagy pathways.

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Fig. 1: Construction of a system for monitoring ribophagy flux in human cells.
Fig. 2: mTOR inhibition promotes ribophagy flux in a VPS34-dependent manner.
Fig. 3: Ribophagy in response to mTOR inhibition in HEK293 cells is ATG5-independent but BECN1-dependent.
Fig. 4: A screen of ribosome stress agents identifies sodium arsenite and reversine as ribophagy inducers.
Fig. 5: Quantitative western blot analyses of various Keima reporter cell lines reveal selective capture of ribosomes during AS and reversine treatment and the relative quantity of bystander autophagy during selective autophagy.

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Acknowledgements

This work was supported by the National Institutes of Health (grants R37NS083524 and RO1GM095567 to J.W.H.). The authors acknowledge the Nikon Imaging Center and the Imaging and Data Analysis Core (Harvard Medical School) for imaging assistance.

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  1. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Heeseon An & J. Wade Harper

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  1. Heeseon An
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Contributions

J.W.H. and H.A. conceived the study. H.A. performed all experiments. H.A. and J.W.H. analysed the data and wrote the paper.

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Correspondence to J. Wade Harper.

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The authors declare no competing financial interests.

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Supplementary information

Supplementary Information

Supplementary Figures 1–6, Legends

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Supplementary Table 1

Sequences of guide RNAs used for CRISPR tagging and knock-out, and primers for genotyping and next-generation sequencing.

Supplementary Table 2

Statistics Source Data. The source data for statistical analyses of Figs. 2b, 2f-h, 3b, 4b, 4d-e, 4g, 4l, 5e-f, and Supplemental Figs. 2j, 3a, 5c, and 5h.

Supplementary Table 3

Information of antibodies used in the study.

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An, H., Harper, J.W. Systematic analysis of ribophagy in human cells reveals bystander flux during selective autophagy. Nat Cell Biol 20, 135–143 (2018). https://doi.org/10.1038/s41556-017-0007-x

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