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. 2020 Oct 30;295(44):15045-15053.
doi: 10.1074/jbc.RA120.013999. Epub 2020 Aug 26.

ATG16L1 autophagy pathway regulates BAX protein levels and programmed cell death

Fenfen Chen  1 , Dulguun Amgalan  2 , Richard N Kitsis  3 , Jeffrey E Pessin  4 , Daorong Feng  5
Affiliations

ATG16L1 autophagy pathway regulates BAX protein levels and programmed cell death

Fenfen Chen et al. J Biol Chem. .

Abstract

Previously we reported that adipocyte SNAP23 (synaptosome-associated protein of 23 kDa) deficiency blocks the activation of macroautophagy, leading to an increased abundance of BAX, a pro-death Bcl-2 family member, and activation and adipocyte cell death both in vitro and in vivo Here, we found that knockdown of SNAP23 inhibited the association of the autophagosome regulators ATG16L1 and ATG9 compartments by nutrient depletion and reduced the formation of ATG16L1 membrane puncta. ATG16L1 knockdown inhibited autophagy flux and increased BAX protein levels by suppressing BAX degradation. The elevation in BAX protein had no effect on BAX activation or cell death in the nutrient-replete state. However, following nutrient depletion, BAX was activated with a concomitant induction of cell death. Co-immunoprecipitation analyses demonstrated that SNAP23 and ATG16L1 proteins form a stable complex independent of nutrient condition, whereas in the nutrient-depleted state, BAX binds to SNAP23 to form a ternary BAX-SNAP23-ATG16L1 protein complex. Taken together, these data support a model in which SNAP23 plays a crucial function as a scaffold for ATG16L1 necessary for the suppression of BAX activation and induction of the intrinsic cell death program.

Keywords: ATG16L1; ATG9; BAX; SNAP23; SNARE proteins; adipocyte; apoptosis; autophagy; cell death; programmed.

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Conflict of interest statement

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.
SNAP23 deficiency decreases co-localization of ATG9 vesicles with ATG16L1 vesicles under nutrient depletion. A, NIH3T3 NM shRNA and NIH3T3 SNAP23 shRNA cells were co-transfected with cDNAs encoding CFP-ATG16L1 and RFP-ATG9. The cells were then maintained in NR medium as described under “Experimental procedures.” 24 h later, the cells were placed in ND medium for 1 h. The cells were then visualized by fluorescence microscopy. Scale bars, 25 μm. B, co-localization of CFP-ATG16L1 and RFP-ATG9 under nutrient-depleted conditions was determined by Pearson's coefficient from three independent replicate experiments. C, the average number of RFP-ATG16L1 vesicles per cell with the standard error of the mean was quantified from three independent replicate experiments. D, the average size of RFP-ATG16L1 vesicles/cell (AU) were quantified from three independent replicate experiments. All data represent the means ± standard deviation. *, P ≤ 0.05; ***, P ≤ 0.005 by unpaired two-tailed Student's t test.
Figure 2.
Figure 2.
SNAP23-deficient adipocytes decrease the number and size of ATG16L1 vesicles under nutrient-depleted conditions. A, NM shRNA and SNAP23 shRNA expressing differentiated 3T3L1 adipocytes were placed under NR or ND conditions for 1 h, immune-labeled with the ATG16L1 antibody, and then visualized by confocal fluorescence microscopy. White arrows point to ATG16L1 representative vesicles. Scale bars, 20 μm. B, the quantitation of the number of ATG16L1 vesicles per adipocyte under ND conditions. The data are the averages with standard error of the mean from two independent replicated experiments. C, the average size of RFP-ATG16L1 vesicles per adipocyte (AU) with standard error of the mean was quantified from three independent replicated experiments. All data represent the means ± standard deviation. *, P ≤ 0.05; ***, P ≤ 0.005 by unpaired two-tailed Student's t test.
Figure 3.
Figure 3.
ATG16L deficiency inhibits BAX protein degradation. A, cell extracts from NM shRNA and ATG16L1 shRNA NIH3T3 cells maintained under NR conditions and cell extracts were immunoblotted for BAX, ACTIN, and ATG16L1 in two sets of cells. B and C, quantitation of expression of BAX (B) and ATG16L1 (C) protein normalized to ACTIN from four independent experiments. D, NM shRNA and ATG16L1 shRNA cells were treated with 20 µg/ml cycloheximide (CHX), and at various times cell extracts were immunoblotted for BAX and ACTIN. E and F, quantitation of expression of BAX protein at 0, 2, 4, 6, and 8 h after CHX treatment in NM shRNA (E) and ATG16L1 shRNA (F) cells from five independent experiments. G, NM shRNA and ATG16L1 shRNA cells were subjected to ND conditions for 2 h in the absence and presence of the lysosomotropic agents NH4Cl and leupeptin and were immunoblotted for LC3. H, net LC3-II flux was calculated as the difference between LC3II protein levels in the presence and absence of the lysosomotropic agents as described under “Experimental procedures” from four independent experiments. I, NM shRNA and ATG16L1 shRNA cells were subjected to ND conditions for 2 h in the absence and presence of the lysosomotropic agents NH4Cl and leupeptin and were immunoblotted for p62. J, net p62 flux was calculated as the difference between p62 protein levels in the presence and absence of the lysosomotropic agents from four independent experiments. These are representative immunoblots that have been replicated in four independent experiments. All data represent the means ± standard deviation. *, P ≤ 0.05 by unpaired two-tailed Student's t test.
Figure 4.
Figure 4.
ATG16L1 deficiency induces BAX activation. A, NM shRNA and ATG16L1 shRNA NIH3T3 cells were maintained under NR or ND conditions for 1 h. The cells were fixed and subjected to immunofluorescence microscopy using the BAX activation–specific mAb 6A7 (green, panels 1–4), the mitochondria-specific antibody ATP5α (red, panels 5–8), and merged images with DAPI (blue, panels 9–12). These are representative images from four independent determinations. Scale bars, 25 μm. B, the percentage of 6A7 staining positive cells in NM shRNA and ATG16L1 shRNA NIH3T3 cells cultured under NR or NR conditions for 1 h were quantified from ∼300 cells in four independent experiments. All data represent the means ± standard deviation. ****, P ≤ 0.001 by unpaired two-tailed Student's t test.
Figure 5.
Figure 5.
ATG16L1 deficiency–induced cell death. A, NM shRNA and ATG16L1 shRNA NIH3T3 cells maintained under NR or ND conditions for 6 h were subjected to PI and DAPI labeling as described under “Experimental procedures.” Images are representative of two independent experiments. Scale bars, 100 μm. B, quantification of the percentage of PI-positive cells from more than 400 cells in four independent experiments. All data represent the means ± standard deviation. **, P ≤ 0.01 by unpaired two-tailed Student's t test.
Figure 6.
Figure 6.
BAX–SNAP23–ATG16L1 form a ternary complex in the nutrient-depleted state. NIH3T3 cells were cultured in NR or ND medium for 1 h and cell lysates generated using a buffer that does not activate BAX as described under “Experimental procedures.” A, the lysates were directly immunoblotted or immunoprecipitated (IP) with SNAP23 antibody and then blotted with the ATG16L1 or the SNAP23 antibody. B, same culture condition as in A, were immunoprecipitated with the BAX antibody and immunoblotted with the SNAP23 or BAX antibody. C, the same cell lysates were immunoprecipitated with the BAX antibody and immunoblotted for ATG16L1 and BAX. D, the NM shRNA, SNAP23 shRNA, and ATG16L1 shRNA NIH3T3 cells were cultured in nutrient-depleted medium for 1 h, and cell lysate was generated, immunoprecipitated with the BAX antibody, and immunoblotted for ATG16L1, SNAP23, and BAX. These are representative immunoblots independently performed three times. E and F, the ratio of ATG16L1 (E) and SNAP23 (F) protein co-precipitated with BAX from four independent experiments. All data represent the means ± standard deviation. *, P ≤ 0.05; ****, P ≤ 0.001 by unpaired two-tailed Student's t test.
Figure 7.
Figure 7.
Proposed model predicting the interaction of ATG16L1, SNAP23, and BAX required for BAX degradation. SNAP23 consistently interacts with ATG16L1. Following nutrient depletion, BAX interacts with SNAP23 to form a ternary ATG16L1–SNAP23–BAX complex. This allows BAX to associate with ATG16L1 and ATG9 membrane compartments that form an autophagosome. The BAX-associated autophagosome can then fuse with the lysosome to mediate BAX degradation.
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