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ATG12–ATG3 interacts with Alix to promote basal autophagic flux and late endosome function

Nature Cell Biology volume 17, pages 300–310 (2015)Cite this article

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Abstract

The ubiquitin-like molecule ATG12 is required for the early steps of autophagy. Recently, we identified ATG3, the E2-like enzyme required for LC3 lipidation during autophagy, as an ATG12 conjugation target. Here, we demonstrate that cells lacking ATG12–ATG3 have impaired basal autophagic flux, accumulation of perinuclear late endosomes, and impaired endolysosomal trafficking. Furthermore, we identify an interaction between ATG12–ATG3 and the ESCRT-associated protein Alix (also known as PDCD6IP) and demonstrate that ATG12–ATG3 controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis and viral budding. Similar to ATG12–ATG3, Alix is functionally required for efficient basal, but not starvation-induced, autophagy. Overall, these results identify a link between the core autophagy and ESCRT machineries and uncover a role for ATG12–ATG3 in late endosome function that is distinct from the canonical role of either ATG in autophagosome formation.

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Figure 1: ATG12–ATG3 promotes basal autophagic flux.
Figure 2: Cells lacking ATG12–ATG3 accumulate enlarged perinuclear late endosomes.
Figure 3: ATG12–ATG3 promotes late endosome to lysosome trafficking.
Figure 4: ATG12–ATG3 interacts with Alix.
Figure 5: Loss of ATG12–ATG3 conjugation phenocopies loss of Alix.
Figure 6: ATG12–ATG3 conjugation promotes multiple Alix functions.
Figure 7: Loss of Alix specifically impairs basal autophagic flux.

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Acknowledgements

Confocal microscopy was performed in the Biological Imaging Development Center at UCSF. Grant support to J.D. includes the NIH (CA126792, CA188404) and a Howard Hughes Medical Institute Physician-Scientist Early Career Award. This material is based on work supported by the National Science Foundation Graduate Research Fellowship to L.M. under grant DGE-1144247.

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Authors and Affiliations

  1. Department of Pathology and Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, San Francisco, California 94143, USA

    Lyndsay Murrow, Ritu Malhotra & Jayanta Debnath

  2. Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, California 94143, USA

    Lyndsay Murrow & Jayanta Debnath

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  1. Lyndsay Murrow
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Contributions

L.M. and J.D. conceived the study and designed the experiments. L.M. performed the experiments and analysed the data. R.M. performed the mass spectrometry analysis. L.M. and J.D. wrote the manuscript.

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Correspondence to Jayanta Debnath.

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Integrated supplementary information

Supplementary Figure 1 Generation of pBABE, WTATG3, and KR cells and analysis of mCherry-GFP-LC3 in atg3+/+ and atg3−/− MEFs.

(a) Overview of the ubiquitin-like conjugation systems required for autophagy and formation of the ATG12-ATG3 conjugate. ATG12 is conjugated to lysine 243 (K243) of ATG3. (b) Stable pools of atg3−/− MEFs expressing an empty vector control (pBabe), wild-type mouse ATG3 (WTATG3) or the ATG3 K243R (KR) mutant were transduced with FLAG-HA-ATG12 (FHA-ATG12). Lysates were immunoblotted for anti-HA to detect ATG12 conjugates. (c) Lysates from stable pools of atg3−/− MEFs expressing an empty vector control (pBabe), V5-tagged wild-type mouse ATG3 (WTATG3) or V5-tagged mutant K243R ATG3 (KR) were immunoprecipitated with anti-V5. Immune complexes (IP V5) were resolved by SDS-PAGE and immunoblotted with anti-ATG3 and anti-ATG12 to detect the ATG12-ATG3 conjugate. (d) atg3−/− MEFs expressing mCherry-GFP-LC3 were grown in full media (Full), starved in Hank’s buffered saline solution (HBSS) for 2 h, or treated with bafilomycin A (Baf A, 50 nM). Scale bar, 20 μm. (e) atg3+/+ MEFs expressing mCherry-GFP-LC3 were grown in full media (Full), HBSS starved for 2 h, or treated with Baf A (50 nM) for 2 h. Scale bar, 20 μm. (f) Quantification of the percentage of mature autolysosomes, delineated as mCherry-positive, GFP-negative (mCherry-only) puncta as described in e and Fig. 1a–c (mean ± s.e.m.; n = 200 cells pooled from three independent experiments). Data for WTATG3 cells is the same as that presented in Fig. 1c. Statistical significance calculated using ANOVA, followed by Tukey’s HSD test. (g) Indicated cell types expressing mCherry-GFP-LC3 were treated for 2 h with bafilomycin A (Baf A, 50 nM) to block lysosome function. Scale bar, 20 μm. Uncropped images of blots are shown in Supplementary Fig. 6.

Supplementary Figure 2 Cells lacking ATG12-ATG3 accumulate perinuclear late endosomes.

(a) Quantification of the number of LBPA puncta as described in Fig. 2d (mean ± s.e.m.; n = 60 cells pooled from three independent experiments). Statistical significance calculated using ANOVA, followed by Tukey’s HSD test (∗∗∗P < 0.001). (b) Cells were immunostained with anti-CD63 and DAPI. The perinuclear CD63+ fraction was defined as the fraction of CD63 area located within 5 μm of the nucleus. Cells with completely clustered CD63 localization have a perinuclear CD63+ fraction close to 1. Scale bar, 10 μm. (c) Quantification of the perinuclear CD63+ fraction as described in b (mean ± s.e.m.; n = 60 cells pooled from three independent experiments). Statistical significance calculated using ANOVA, followed by Tukey’s HSD test (∗∗∗P < 0.001).

Supplementary Figure 3 Endolysosomal trafficking of Bodipy-LDL.

Stable pools of atg3−/− MEFs expressing an empty vector control (pBabe), wild-type mouse ATG3 (WTATG3) or the ATG3 KR mutant were used for experiments as indicated. (a) atg3−/− MEFs stably expressing wild-type mouse ATG3 (WTATG3) were incubated with Bodipy-LDL for 15 min at 37 °C in serum-free medium followed by a chase in full media for the indicated times. Cells were immunostained with anti-EEA1 to mark early endosomes, anti-lysobisphosphatidic acid (LBPA) to mark late endosomes, and anti-LAMP1 to mark lysosomes. Colocalized pixels are highlighted in white using the Colocalization plugin in ImageJ. Scale bar, 5 μm. (b) Indicated cell types were incubated with Bodipy-LDL for 15 min at 37 °C in serum-free medium followed by a 2 h chase in full media. When indicated, Baf A (50 nM) was used during the chase to block lysosome function. Quantification of the percentage of LDL puncta colocalized with LAMP1 is shown. Data for untreated cells (CTL) is the same as Fig. 3d. Data are presented as median (horizontal line), interquartile range (box), and 10–90th percentile (whiskers); n = 100 cells pooled from three independent experiments. Statistical significance calculated using ANOVA, followed by Tukey’s HSD test (∗∗P < 0.01). (c) Stable pools of reconstituted atg3−/− MEFs were incubated with Bodipy-LDL for 15 min at 37 °C followed by a 2 h chase in full media. Cells were immunostained with anti-LBPA to mark late endosomes. Colocalized pixels were highlighted in white using the Colocalization plugin in ImageJ. Scale bar, 5 μm. (d) Indicated cell types were incubated with Bodipy-LDL for 15 min at 37 °C in serum-free media followed by a 2 h chase in full media. Cells were immunostained with anti-EEA1 to mark early endosomes. Quantification of the percentage of LDL puncta colocalized with EEA1 is shown. Data are presented as interquartile range (box) and 90th percentile (whiskers); n = 100 cells pooled from three independent experiments. Statistical significance calculated using ANOVA, followed by Tukey’s HSD test.

Supplementary Figure 4 ATG12-ATG3 conjugation does not affect early endosome or lysosome function.

(a) Indicated cell types were incubated with fluorescently labeled transferrin (Tfn, red) for 15 min at 37 °C in serum-free media followed by a chase in full media for the indicated times. Cells were immunostained for EEA1 (green) to mark early endosomes. Scale bar, 10 μm. (b) Indicated cell types were incubated with Tfn as described in a and chased in full media for the indicated times. Tfn uptake as measured by fluorescence at time 0 and Tfn recycling as measured by a decrease in fluorescence over time were quantified by flow cytometry. Data are presented as mean ± s.e.m. relative fluorescence intensity; n = 3 independent experiments. Statistical significance calculated using ANOVA, followed by Tukey’s HSD test. (c) Indicated cell types were incubated with LysoSensor DND-189 or DND-153 to measure acidic (pH < 5.2) and total lysosomal content, respectively. Data is presented as mean ± s.e.m. relative fluorescence intensity; n = 3 independent experiments. Statistical significance calculated using ANOVA, followed by Tukey’s HSD test. (d) Indicated cell types were incubated with fluorogenic Magic Red cathepsin B or cathepsin L substrates to measure cathepsin activity. Data is presented as mean ± s.e.m. relative fluorescence intensity; n = 3 independent experiments. Statistical significance calculated using ANOVA, followed by Tukey’s HSD test.

Supplementary Figure 5 ATG12-ATG3 interacts with Alix (also known as PDCD6IP).

ATG12 was immunoprecipitated from atg5−/− MEFs stably expressing tandem tagged FLAG-HA-ATG12. Eluted proteins were resolved by SDS-PAGE, and Coomassie-stained protein bands were subject to LC-MS/MS analysis. Alix was identified (n = 2 independent experiments); bold underlined sequences correspond to independent peptides identified by mass spectrometry.

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Murrow, L., Malhotra, R. & Debnath, J. ATG12–ATG3 interacts with Alix to promote basal autophagic flux and late endosome function. Nat Cell Biol 17, 300–310 (2015). https://doi.org/10.1038/ncb3112

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