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. 2016 Dec 19;215(6):857-874.
doi: 10.1083/jcb.201607039. Epub 2016 Nov 18.

Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation

Thanh Ngoc Nguyen  1 , Benjamin Scott Padman  1 , Joanne Usher  1 , Viola Oorschot  2 , Georg Ramm  1   2 , Michael Lazarou  3
Affiliations

Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation

Thanh Ngoc Nguyen et al. J Cell Biol. .

Abstract

Members of the Atg8 family of proteins are conjugated to autophagosomal membranes, where they have been proposed to drive autophagosome formation and selective sequestration of cargo. In mammals, the Atg8 family consists of six members divided into the LC3 and GABARAP subfamilies. To define Atg8 function, we used genome editing to generate knockouts of the LC3 and GABARAP subfamilies as well as all six Atg8 family members in HeLa cells. We show that Atg8s are dispensable for autophagosome formation and selective engulfment of mitochondria, but essential for autophagosome-lysosome fusion. We find that the GABARAP subfamily promotes PLEKHM1 recruitment and governs autophagosome-lysosome fusion, whereas the LC3 subfamily plays a less prominent role in these processes. Although neither GABARAPs nor LC3s are required for autophagosome biogenesis, loss of all Atg8s yields smaller autophagosomes and a slowed initial rate of autophagosome formation. Our results clarify the essential function of the Atg8 family and identify GABARAP subfamily members as primary contributors to PINK1/Parkin mitophagy and starvation autophagy.

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Figures

Figure 1.
Figure 1.
Characterization of mammalian Atg8 family function during PINK1/Parkin mitophagy and starvation induced autophagy. (A) WT, LC3 TKO, GBRP TKO, and hexa KO (LC3/GBRP KO) lines were confirmed by immunoblotting (IB). Chloroquine (ChQ) treatment prevents lysosomal degradation of LC3s/GBRPs. For LC3C IB, 100 µg of lysates was analyzed. (B and C) The indicated cell lines with and without mCherry (mCh)–Parkin were analyzed by immunoblotting (B) and CoxII levels were quantified (C). (D) Representative images of WT, LC3 TKO, GBRP TKO and hexa KO expressing mCh-Parkin immunostained with mtDNA antibodies (green) and quantified for mitophagy (E). (F and G) WT, LC3 TKO, GBRP TKO, and hexa KO fully fed or starved for 8 h with Earle’s balanced salt solution (EBSS) were analyzed by immunoblotting (F), and p62 levels were quantified (G). (H and I) hexa KO cells expressing mCh-Parkin and individual untagged LC3s and GBRPs after 24-h OA treatment were analyzed by immunoblotting (H), and CoxII levels were quantified (I). Data in C, E, G, and I are mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). ns, not significant. Bar, 10 µm.
Figure 2.
Figure 2.
Analysis of autophagosome initiation and nucleation events in Atg8 knockout lines during PINK1/Parkin mitophagy. (A and D) Representative images of WT, LC3 TKO, GBRP TKO, and hexa KO cells expressing mCh-Parkin and GFP-ULK1 (A) or GFP-DFCP1 (D), immunostained for HSP60 and GFP after 3-h OA treatment (untreated images shown in Fig. S2, G and H). Quantification by image analysis of ULK1 foci structures per cell (B), DFCP1 structures per cell (E), and the proportion of mitochondrially associated (C) ULK1 foci and (F) DFCP1 structures in WT, LC3 TKO, GBRP TKO, and hexa KO cells. Data in B, C, E, and F are mean ± SD from three independent experiments using measurements from >80 cells per sample. *, P < 0.05; **, P < 0.005; ***, P < 0.001 (one-way ANOVA). Bars: 10 µm; (insets) 2 µm.
Figure 3.
Figure 3.
Atg8s are dispensable for autophagosome formation but regulate autophagosome expansion. (A and B) Representative TEM images of autophagosomes containing mitochondria in WT, LC3 TKO, GBRP TKO, and hexa KO cells after incubation for 6 h with OA and BafA1 (A) or for 8 h with EBSS and BafA1 (B). Wider field-of-view images and untreated examples shown in Fig. S3 D. (C and D) TEM quantification of mean autophagosome number per cell (C) and the mean cross-sectional area of autophagosomes in WT and hexa KO cells treated with BafA1 and either OA for 6 h or starved with EBSS for 8 h (D). Scatterplot of measurements provided in Fig. S4 A. (E) 3D rendering of a reconstructed autophagosomal compartment (green) from a hexa KO cell after 6 h OA and BafA1 treatment, shown with sequestered mitochondrion (red) and endoplasmic reticulum (purple). (Source images and further examples shown in Fig. S3, A–C.) Data in C and D are mean ± SD from three independent double-blinded experiments, using measurements from exactly 12 randomly chosen cells (>100 autophagosomes) per sample. **, P < 0.005; ****, P < 0.0001 (one-way ANOVA). Bars, 200 nm.
Figure 4.
Figure 4.
Formation of correctly sealed autophagosomes containing sequestered cargo in Atg8 knockout lines. (A) Schematic of the protease protection assay used to assess packaging of cargo in sealed autophagosomes. Protein cargoes in autophagosomes are protected from the addition of external proteinase K (PK; middle) unless Triton X-100 (TX-100) is present (right). (B–D) Lysates from WT, LC3 TKO, GBRP TKO, and hexa KO cells untreated or treated with OA and BafA1 in the presence or absence of wortmannin (Wort) were subjected to the PK protection assay. (B) Samples were subsequently analyzed by immunoblotting with NDP52. PLEKHM1 served as a cytosolic control. (C) The amount of PK-protected NDP52 for each condition (untreated, OA/BafA1, or OA/BafA1/Wort) was quantified (percentage of NDP52 amount in lanes 2, 5, and 8 relative to NDP52 amount in lanes 1, 4, and 7, respectively). (D) Quantification of the difference in the amount of PK-protected NDP52 in BafA1-treated samples (C, blue bars) versus BafA1/Wort-treated samples (light gray bars in C; represents the amount of NDP52 specifically protected within autophagosomes). (E–G) PK protection assay of WT, LC3 TKO, GBRP TKO, and hexa KO during starvation autophagy. (E) Lysates from the indicated cell lines untreated or treated with EBSS and BafA1 in the presence or absence of wortmannin were incubated with PK with or without 0.2% (vol/vol) Triton X-100 and subsequently immunoblotted with p62 antibodies. (F) Quantification of PK-protected p62 for each condition (untreated, EBSS/BafA1, or EBSS/BafA1/Wort) were performed. (G) The amount of autophagosome-protected p62 was also analyzed. (H) The mean volume of autophagosomes formed during mitophagy and starvation in WT and hexa KO cells was calculated (see Fig. S4 C for calculation) and compared with the proportion of autophagosome-protected NDP52 (mitophagy) and p62 (starvation) obtained from D and G, respectively. Data in C, D, F, and G are mean ± SD from three independent experiments. **, P < 0.005; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). ns, not significant.
Figure 5.
Figure 5.
Atg8s regulate autophagosome capacity, but not the selective sequestration of cargo, during PINK1/Parkin mitophagy. (A) Representative immunogold TEM images of HSP60 labeled WT and hexa KO cells after incubation with OA and BafA1 for 6 h. (B and C) Immunogold TEM quantification of the mean number of mitophagosomes per cell (B) and the mean cross-sectional area of mitophagosomes in WT and hexa KO cells (C) after treatment with OA and BafA1 for 6 h. (Scatterplot of measurements provided in Fig. S4 B.) Data in B and C are mean ± SD from three independent experiments, using measurements from exactly eight randomly chosen cells (>40 mitophagosomes measured per sample). **, P < 0.005 (Student’s t test). Bars, 200 nm.
Figure 6.
Figure 6.
Autophagosome biogenesis is initially slow in the absence of LC3s and GABARAPs during Pink1/Parkin mitophagy. Lysates from WT and hexa KO cells untreated or treated with OA and BafA1 in the presence or absence of wortmannin (Wort) for indicated time points were subjected to the PK protection assay (see Fig. 4 A). (A) Samples were subsequently analyzed by immunoblotting with NDP52. All of the blots were transferred and exposed at the same time. PLEKHM1 served as a cytosolic control. (B) The amount of PK-protected NDP52 for each condition was first quantified (percentage of NDP52 amount PK only treated lanes relative to NDP52 amount in no PK/TX-100 ones). Then the difference in the amount of PK-protected NDP52 in BafA1 treated samples versus BafA1/Wort treated samples (represents the amount of autophagosome-protected NDP52 for each time point) was calculated. The autophagosome formation rates at 2, 4, and 6 h for each cell line were determined by the ratio of the autophagosome protected NDP52% at these time points to the autophagosome protected NDP52% at 8 h; i.e., the autophagosome-protected NDP52% at 8 h was considered to be 100%. Data in B are mean ± SD from three independent experiments. *, P < 0.05; ***, P < 0.001 (one-way ANOVA). ns, not significant.
Figure 7.
Figure 7.
Atg8s are crucial for correct acidification of sequestered cargo. (A and B) WT, LC3 TKO, GBRP TKO, and hexa KO expressing YFP-Parkin and mtKeima were either untreated or treated with OA for indicated times and analyzed for (A) lysosomal-positive mtKeima using fluorescence microscopy (550 nm mtKeima; see Fig. S5 A for 2- and 8-h images), and (B) FACS as the percentage of 561 nm mtKeima positive cells (see Fig. S5 B for FACS plots). (C and D) Hexa KO cells expressing mtKeima and doxycycline (Dox)–inducible HA-LC3C or HA-GBRPL1 were pretreated with OA for 8 h. After being incubated with Dox, Wort, and OA for 6 h, the cells were analyzed by confocal microscopy (C) and FACS (D) for lysosomal-positive mtKeima (see Fig. S5, C and D). Data in B and D are mean ± SD from three independent experiments. ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). ns, not significant. Bars, 10 µm.
Figure 8.
Figure 8.
Atg8s are essential for autophagosome–lysosome fusion, but not lysosomal function. (A and B) Representative images of untreated WT and hexa KO cells stained with LysoTracker green and MitoTracker deep red (A) or loaded with Cascade blue–conjugated dextran (B) for 24 h before immunostaining for LAMP2. (C) Representative images of WT and hexa KO cells expressing untagged Parkin, immunostained for HSP60 and LAMP1 after incubation for 6 h in the presence or absence of OA and protease inhibitors (10 µg/ml Pepstatin A; 10 µg/ml E-64d). (D) Quantification by image analysis of the colocalization between LAMP1 and HSP60 after incubation for 6 h in the presence or absence of OA, Pepstatin A, and E-64d. (E) Representative immunogold TEM images of WT and hexa KO cells, double labeled for LAMP1 (20 nm gold) and HSP60 (10 nm gold) after incubation with OA alone for 6 h (compartment perimeters indicated by offset dashed line). Data in D are mean ± SD from three independent experiments. ***, P < 0.001 (one-way ANOVA). ns, not significant. Bars: (A–C) 10 µm; (A–C, insets) 2 µm; (E) 200 nm.
Figure 9.
Figure 9.
Recruitment of PLEKHM1 by GABARAPs promotes autophagosome–lysosome fusion. (A) Representative images of WT and hexa KO cells expressing mCh-Parkin and GFP-STX17, immunostained for HSP60 and GFP after 3 h OA treatment. (B and C) Lysates from WT, hexa KO, STX17 KO, and Vps39 KO treated for 24 h with OA were analyzed by immunoblotting and (C) CoxII levels were quantified. (D) Deconvolved live-cell time-series images of WT and hexa KO cells stably expressing untagged Parkin and transiently expressing GFP-STX17 and mCherry-LAMP1, showing interactions between STX17 structures and lysosomes (arrowhead) after 6-h OA treatment. (E) Representative images of WT, LC3 TKO, GBRP TKO, and hexa KO cells expressing mCh-Parkin and HA-PLEKHM1, immunostained for HSP60 and HA after 3-h OA treatment. (mCh-Parkin channels and untreated examples are shown in Fig. S2, L and M). (F) Quantification of the mitochondrially associated proportion of PLEKHM1 in WT, LC3 TKO, GBRP TKO, and hexa KO cells. Data in C and F are mean ± SD from three independent experiments. ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). ns, not significant. Bars: (A and E) 10 µm; (A and E, insets) 2 µm; (D) 2 µm.
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