Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens

Abstract

Autophagy is a homeostatic process with multiple functions in mammalian cells. Here, we show that mammalian Atg8 proteins (mAtg8s) and the autophagy regulator IRGM control TFEB, a transcriptional activator of the lysosomal system. IRGM directly interacted with TFEB and promoted the nuclear translocation of TFEB. An mAtg8 partner of IRGM, GABARAP, interacted with TFEB. Deletion of all mAtg8s or GABARAPs affected the global transcriptional response to starvation and downregulated subsets of TFEB targets. IRGM and GABARAPs countered the action of mTOR as a negative regulator of TFEB. This was suppressed by constitutively active RagB, an activator of mTOR. Infection of macrophages with the membrane-permeabilizing microbe Mycobacterium tuberculosis or infection of target cells by HIV elicited TFEB activation in an IRGM-dependent manner. Thus, IRGM and its interactors mAtg8s close a loop between the autophagosomal pathway and the control of lysosomal biogenesis by TFEB, thus ensuring coordinated activation of the two systems that eventually merge during autophagy.

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Fig. 1: IRGM controls TFEB nuclear translocation.
Fig. 2: IRGM and TFEB interact.
Fig. 3: IRGM affects mTOR activity and interacts with PPP3CB.
Fig. 4: mAtg8s affect mTOR and the nuclear translocation of TFEB.
Fig. 5: mAtg8s control the transcriptional activity of TFEB.
Fig. 6: Stx17 affects mTOR and regulates TFEB nuclear translocation.
Fig. 7: IRGM affects TFEB nuclear translocation in cells infected with diverse pathogens associated with tuberculosis, AIDS or CD.

Data availability

RNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE149533. Mass spectrometry proteomics data in this study have been deposited in the MassIVE repository (https://massive.ucsd.edu), with accession number MSV000083251 for Stx17 interactors44 and accession number MSV000085401 for IRGM interactors. All other data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

V.D. dedicates this work to memory of Beth Levine. We thank R. Puertollano, S. Ferguson and A. Ballabio for the TFEB constructs. This work was supported by NIH grants R37AI042999 and R01AI111935 and center grant P20GM121176 to V.D. T.-E.R and A.J. were supported by grants 262652 and 276070 from the Norwegian Research Council.

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Authors

Contributions

Conceptualization: S.K. and V.D. Formal analysis: S.K., A.J., S.W.C., G.P.D.d.S., L.A., M.H.M., R.S.P. and V.D. Investigation and validation: S.K., S.W.C., A.J., M.H.M., M.L., T.-E.R., G.P.D.d.S. and V.D. Resources: V.D., T.-E.R. and M.L. Data curation: L.A. and J.H.A. Writing (original draft): S.K. and V.D. Writing (reviewing and editing): T.-E.R. and M.L. Visualization: S.K., A.J. and V.D. Supervision: V.D. and T.-E.R. Project administration and funding acquisition: V.D.

Corresponding author

Correspondence to Vojo Deretic.

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Extended data

Extended Data Fig. 1 IRGM affects nuclear translocation of TFEB.

a, confocal microscopy analysis of effects of IRGM KD on TFEB nuclear translocation in response to 2 h starvation. Scale bar 5 µm, (n = 3 biologically independent experiments). b,c, HCM images and quantification to test the effect of IRGM KD on nuclear translocation of TFEB. Cells were permeabilized with Triton. Data, means ± SEM (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test; high content microscopy, >500 cells counted per well; minimum number of valid wells 9. Masks; white: algorithm-defined cell boundaries; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Scale bar 10 µm. d,e, HCM images and quantifications to test the effect of IRGM KD on nuclear translocation of TFEB in cells treated with DMSO or pp242. Data, means ± SEM (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test; high content microscopy, >500 cells counted per well; minimum number of valid wells. Masks; white: algorithm-defined cell boundaries; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Scale bar 10 µm. Numerical source data for panels b and d are provided in Source Data Extended Data Fig. 1. Source data

Extended Data Fig. 2 Interactions and localization analyses of IRGM with MiT/TFE family of transcriptional regulators.

a, Confocal microscopy analysis of co-localization between GFP-IRGM and endogenous TFEB. Scale bar 5 µm, (n = 3 biologically independent experiments). b, A screenshot from NCBI showing domain of unknown function (DUF3371) in TFEB. c,d, HCM images and quantifications to analyze the effect of complementation of IRGM KD with GFP-IRGM WT or GFP-IRGM S47N on nuclear translocation of TFEB. Data, means ± SEM (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test; HCM, > 500 cells counted per well; minimum number of valid wells 9, 3 independent experiments. Masks; white: algorithm-defined cell boundaries and computer-identified GFP positive cells; blue outline: computer-identified nuclear stain; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Scale bar 10 µm. The masks in gray scale panels are cloned from the merged images. Inset: western blot showing GFP-IRGM expression IRGM KD cells. e, Co-IP analysis of interactions between GFP-MiTF (H isoform) and FLAG-IRGM in 293T cells, (n = 3 biologically independent experiments). f, Co-IP analysis of interactions between GFP-TFE3 and FLAG-IRGM in 293T cells, (n = 3 biologically independent experiments). g,h, Co-IP analysis of interactions between GFP-IRGM WT or GFP-IRGM S47N with MiTF in 293T cells. Data, means ± SEM of normalized intensities (n = 3 biologically independent experiments) paired t-test. Uncropped blots for panels e, f and g and numerical source data for panels c and h are provided in Source Data Extended Data Fig. 2. Source data

Extended Data Fig. 3 IRGM effects on mTOR and calcineurin and mAtg8s interactions with and effects on TFEB.

a, b, Western blot analysis and quantifications of the effects of IRGM KD on pTFEB (S211) levels in cells treated with pp242. Data, means ± SEM of normalized intensities (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. c-e, western blots analysis of the effects of IRGM on mTOR substrates pS6K and pULK1. Data, means ± SEM of normalized intensities (n = 3 biologically independent experiments) paired t-test. f, HCM image analysis of co-localization between mTOR and LAMP2. Scale bar 10 µm. g,h, HCM analysis of the effects of IRGM KD on LAMP2 puncta. Data, means ± SEM; (n = 3 biologically independent experiments) paired t-test. Scale bar 10 µm. i, HCM analysis of the effect of IRGM expression on cells expressing RagBQ99L and parental 293 T cells on nuclear translocation of TFEB, (n = 3 biologically independent experiments). Scale bar 10 µm. j, confocal microscopy analysis of co-localization between GFP-IRGM and endogenous PPP3CB in HeLa cells (n = 3 biologically independent experiments). Scale bar 5 µm. k,l, HCM analysis of the effect of starvation on colocalization between GFP-IRGM and PPP3CB. Data, means ± SEM (n = 3 biologically independent experiments) paired t-test. Scale bar 10 µm. m, western blot showing PPP3CB KD in HeLa cells (n = 3 biologically independent experiments). n, schematics of LysoIP technique. o, LysoIP to detect indicated proteins on lysosomes (n = 3 biologically independent experiments). p, western blot analysis of the effects of IRGM expression on NFAT mobility shift (n = 3 biologically independent experiments). q, Co-IP analysis of GFP-LC3B and GFP-GABARAP with FLAG-TFEB in 293 T cells. r, GST pull-down analysis of TFEB with WT or LDS mutant of GABARAP. s, HCM images in WT or indicated KO cells in full medium (n = 3 biologically independent experiments). Scale bar 10 µm. Uncropped blots for panels a, c, o, p, q and r and numerical source data for panels b, d, e, g and l are provided in Source Data Extended Data Fig. 3. Source data

Extended Data Fig. 4 GABARAP and GABARAPL1 but not GABARAPL2 control nuclear translocation of TFEB.

a,b, HCM images and quantifications to test the role of mAtg8s on nuclear translocation of GFP-MiTF in response to autophagy induction (EBSS 2 h). Data, means ± SEM (n = 3 biologically independent experiments) paired t-test. Masks; white: algorithm-defined cell boundaries; blue outline: computer-identified nuclear stain; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Scale bar 10 µm. The masks in gray scale panels are cloned from the merged images, (n = 3 biologically independent experiments). c, HCM image analysis of effects of complementation of HexaKO with GFP-GABARAP on nuclear translocation of TFEB. Scale bar 10 µm. d-g, HCM analysis of the effect of complementation of HexaKO cells with GABARAPL1 or GABARAPL2 on nuclear translocation of TFEB. Data, means ± SEM, ANOVA, Tukey’s post hoc test; HCM, > 500 cells counted per well; minimum number of valid wells 9, (n = 3 biologically independent experiments). Scale bar 10 µm. h, HCM analysis of effect of expression of GABARAP in 293 T cells expressing RagBQ99L or parental 293 T cells on nuclear translocation of TFEB. Masks in c, e, g, h; white: algorithm-defined cell boundaries in GFP positive cells; blue outline: computer-identified nuclear stain; yellow outline: computer-identified co-localization between TFEB and Hoechst-33342 nuclear stain), (n = 3 biologically independent experiments). Scale bar 10 µm. Numerical source data for panels a, d, and f are provided in Source Data Extended Data Fig. 4. Source data

Extended Data Fig. 5 mAtg8s affect global gene expression.

a, Volcano plot (RNAseq) showing the effect of pan-mAtg8 knockout on differential gene expression (log2 fold change; ratio HeLa HexaKO/HeLaWT). Red points: down-regulated genes in HexaKO cells. Green points: upregulated in HexaKO cells. A subset of genes not identified as TFEB targets are named. Dotted orange line, significance cuttof (p value < 0.05). P values were calculated using Fisher’s exact test adapted for over-dispersed data; edgeR models read counts with negative binomial (NB) distribution (see Methods). (n = 3 biologically independent experiments). b, Heat map representation of genes upregulated or downregulated in HeLaWT vs. HexaKO cells. c, A volcano plot showing RNAseq analysis of HeLaWT vs. ATG3KO cells. P values were calculated using Fisher’s exact test using R package. Named genes are previously identified TFEB targets those were also down-regulated in HexaKO shown in Fig. 5c. (n = 3 biologically independent experiments). d, A volcano plot (RNAseq) listing upregulated and downregulated autophagy-related genes in HeLaWT vs. HexaKO cells. P values were calculated using Fisher’s exact test adapted for over-dispersed data; edgeR models read counts with negative binomial (NB) distribution (see Methods). (n = 3 biologically independent experiments). e, qRT-PCR analysis of p62, ATG9B and ULK1 in HeLaWT vs. HexaKO cells induced for autophagy in EBSS for 2 h; 18 S was used as an internal control, Data, means ± SEM (n = 3 biologically independent experiments). Numerical source data for panel e are provided in Source Data Extended Data Fig. 5. Source data

Extended Data Fig. 6 mAtg8s affect calcium fluxes and Stx17 affects mTOR and TFEB.

a, A volcano plot showing expression of calcium effectors in HexaKO cells. P values were calculated using Fisher’s exact test adapted for over-dispersed data (see Methods) (n = 3 biologically independent experiments). b,c Flow cytometry using FLUO-3AM to detect intracellular calcium in HeLaWT or HexaKo. Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. d, Confocal microscopy analysis of the effects of Stx17KO on TFEB localization, (n = 3 biologically independent experiments). Scale bar 5 µm. e-g, confocal microscopy (e) and HCM (f,g) analyses of the effects of Stx17KO on colocalization between TFEB and LAMP2. Scale bar 5 µm (e). Scale bar 10 µm (f). Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. h, HCM analysis of the effects of Stx17KO on TFEB puncta. Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. i, Co-IP analysis of interactions between GFP-Stx17 and FLAG-TFEB in 293 T cells (n = 3 biologically independent experiments). j,k, Co-IP analysis of effects of GFP-Stx17 on FLAG-TFEB and IRGM complexes. Data, means ± SEM (n = 3 biologically independent experiments) paired t-test. l,m, MS analysis showing 14-3-3 peptides those interacted with GFP or GFP-Stx17 and GFP-IRGM (n = 3 biologically independent experiments). n-p, Western blot analysis and quantification of the effect of GFP-Stx17 in HeLaWT (full media) or in Stx17KO cells (EBSS 2 h) on mTOR activity. Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. q-s, Western blot analysis and quantification of pULK1 and pS6K to test the effects of GFP-Stx17 expression in WT 293 T cells and cells expressing RagBQ99L. Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. t-w, Co-IP analysis of interactions between RagA and FLAG-p18 (t,u) and Raptor and FLAG-RagA (v-w) in Stx17KO or parental HeLa cells. Data, means ± SEM of normalized intensities (n = 3 biologically independent experiments) paired t-test. Uncropped blots for panels i, j, n, q, t and v and numerical source data for panels b, f, h, k, p, o, r, s, u and w are provided in Source Data Extended Data Fig. 6. Source data

Extended Data Fig. 7 mIR196B affects protective CD variant of IRGM in its role in nuclear translocation of TFEB.

a,b, HCM analysis of the effects of miR196B (shown to downregulate CD protective IRGM variant) and miR20 (control) transfection on TFEB nuclear localization in 293 T cells (c.313 C). HCM (n = 3 biologically independent experiments); >500 primary objects examined per well; minimum number of wells, 9). Masks; white: algorithm-defined cell boundaries; blue: computer-identified nucleus; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Images, a detail from a large database of machine-collected and computer-processed images. Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. Scale bar 10 µm. c, HCM image analysis of the effects of IRGM KD on AIEC LF82 influenced nuclear translocation of TFEB. K12 was used as control, (n = 3 biologically independent experiments). Scale bar 10 µm. d,e, HC microscopy and quantifications to analyze the effect of HIV infection on TFEB localization in HeLa cells transfected with scramble siRNA or IRGM siRNA. HC microscopy (n = 3 biologically independent experiments; >500 primary objects examined per well; minimum number of wells, 12). Masks; white: algorithm-defined cell boundaries; blue: computer-identified nucleus; yellow outline: computer-identified colocalization between TFEB and Hoechst-33342 nuclear stain). Images, a detail from a large database of machine-collected and computer-processed images. Data, means ± SEM; (n = 3 biologically independent experiments) ANOVA, Tukey’s post hoc test. Scale bar 10 µm. f, The model summarizes the effects of IRGM, Stx17 and mAtg8s/GABARAPs on mTOR inhibition and calcineurin (CN) activation promoting nuclear translocation of TFEB. L, lysosome. Numerical source data for panels a and d are provided in Source Data Extended Data Fig. 7. Source data

Supplementary information

Reporting Summary

Supplementary Tables

Supplementary Table 1: RNA-seq analysis (reported as differential gene expression) of HeLa WT and HexaKO cells determining the effects of the deletion of all mAtg8s (HexaKO) on differential gene expression. The cells were incubated in EBSS for 2 h. Previously known TFEB targets are highlighted in yellow. Tab 2 is the same as tab 1, but reporting differential transcript expression (n = 3 biologically independent experiments). Supplementary Table 2: RNA-seq analysis (reported as differential gene expression) of HeLa WT and GABATKO determining the effects of the deletion of all GABARAPs on differential gene expression. The cells were incubated in EBSS for 2 h. Previously known TFEB targets are highlighted in yellow. Tab 2 is the same as tab 1, but reporting differential transcript expression (n = 3 biologically independent experiments). Supplementary Table 3: tab 5 shows RNA-seq analysis (reported as differential gene expression) of HeLa WT and ATG3KO determining the effects of the deletion of ATG3 on differential gene expression. The cells were incubated in EBSS for 2 h. Previously known TFEB targets are highlighted in yellow. Tab 2 is the same as tab 1, but reporting differential transcript expression (n = 3 biologically independent experiments). Supplementary Table 4: mass spectrometry analyses of GFP–IRGM interactors (n = 3 biologically independent experiments).

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Kumar, S., Jain, A., Choi, S.W. et al. Mammalian Atg8 proteins and the autophagy factor IRGM control mTOR and TFEB at a regulatory node critical for responses to pathogens. Nat Cell Biol 22, 973–985 (2020). https://doi.org/10.1038/s41556-020-0549-1

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