The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy - PubMed (original) (raw)

The Atg16L complex specifies the site of LC3 lipidation for membrane biogenesis in autophagy

Naonobu Fujita et al. Mol Biol Cell. 2008 May.

Abstract

Two ubiquitin-like molecules, Atg12 and LC3/Atg8, are involved in autophagosome biogenesis. Atg12 is conjugated to Atg5 and forms an approximately 800-kDa protein complex with Atg16L (referred to as Atg16L complex). LC3/Atg8 is conjugated to phosphatidylethanolamine and is associated with autophagosome formation, perhaps by enabling membrane elongation. Although the Atg16L complex is required for efficient LC3 lipidation, its role is unknown. Here, we show that overexpression of Atg12 or Atg16L inhibits autophagosome formation. Mechanistically, the site of LC3 lipidation is determined by the membrane localization of the Atg16L complex as well as the interaction of Atg12 with Atg3, the E2 enzyme for the LC3 lipidation process. Forced localization of Atg16L to the plasma membrane enabled ectopic LC3 lipidation at that site. We propose that the Atg16L complex is a new type of E3-like enzyme that functions as a scaffold for LC3 lipidation by dynamically localizing to the putative source membranes for autophagosome formation.

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Figures

Figure 1.

Figure 1.

The role of the interaction between Atg12 and Atg3 in LC3 lipidation. (A) PC12 cells were either left untreated or infected with adenovirus bearing Atg12 (12), Atg5 (5), or Atg16L (16L). After 40-h incubation, the cells were cultured in HBSS (Starved) for 2 h and collected. Lysates were examined by Western blotting using anti-LC3 antibody. (B) HEK293A cells were transiently cotransfected with Myc-tagged LC3 and Atg12G140A or Atg12G140A,F108 double mutants (F108A, F108L, or F108D). Lysates were subjected to Western blotting with each antibody. Top panel, anti-Atg12; middle panel, anti-myc; bottom panel, anti-α-tubulin. (C) HEK293A cells were transiently transfected with 3xFlag-tagged Atg3 and Atg12G140A or Atg12G140A,F108 double mutants (F108A, F108L, or F108D). Lysates were subjected to anti-Flag immunoprecipitation, and the immune complexes were examined by Western blotting using each antibody. Top panel, anti-Flag; middle panel, anti-Atg12; bottom panel, anti-Atg5. (D) PC12 cells were coinfected using a combination of recombinant adenoviruses as indicated. After 40-h incubation, the cells were collected. Lysate samples were examined by Western blotting using each antibody. From top panel, anti-Flag, anti-RFP, anti-LC3, and anti-α-tubulin.

Figure 2.

Figure 2.

F108 of mouse Atg12 is important for targeting of LC3. (A and B) MDCK cells stably expressing GFP-LC3 were infected with adenovirus bearing mStrawberry (Mock), mStrawberry-Atg12G140A (G140A), or mStrawberry-Atg12F108A,G140A (F108A, G140A) and incubated for 40 h. The cells were then cultured in HBSS (Starved; A and B) or growth medium (Fed; B) for 2 h, fixed, and observed using fluorescence microscopy (A). Bar, 10 μm. More than 100 mStrawberry-positive cells were examined for the number of GFP-LC3 dots per cell (B). Data are represented as means ± SD. (C and D) MDCK cells stably expressing GFP-Atg5 were infected with adenovirus bearing mStrawberry (Mock), mStrawberry-Atg12G140A (G140A), or mStrawberry-Atg12F108A,G140A (F108A, G140A) and incubated for 40 h. The cells were then cultured in HBSS (Starved; C and D) or growth medium (Fed; D) for 2 h and fixed. Three-dimensional image stacks were obtained from sequential optical sections acquired 0.3 μm apart by confocal laser scanning microscopy (FV1000, Olympus) (C). Bar, 10 μm. More than 100 mStrawberry-positive cells were examined for the number of GFP-Atg5 dots per cell (D). Data are represented as means ± SD.

Figure 3.

Figure 3.

The effect of overexpression of the Atg16L coiled-coil domain on autophagy. (A) Schematic diagram of mouse Atg16L and the deletion constructs. The coiled-coil region is indicated by shading, and the WD repeats are shown as black boxes. (B and C) MDCK cells stably expressing GFP-LC3 were infected with adenovirus bearing mStrawberry (Mock) or mStrawberry-fused Atg16L deletion constructs and incubated for 40 h. The cells were then cultured in HBSS (Starved; B and C) or growth medium (Fed; C) for 2 h, fixed, and observed using fluorescence microscopy (B). Bar, 10 μm. More than 100 mStrawberry-positive cells were examined for the number of GFP-LC3 dots per the cell (C). Data are represented as means ± SD. (D) PC12 cells were either left untreated or infected with adenovirus bearing mStrawberry (Mock) or mStrawberry-fused Atg16L deletion constructs. After 40-h incubation, the cells were cultured either in growth medium (F; Fed) or HBSS (S; Starved) for 2 h and collected. Lysates were examined by Western blotting using the indicated antibodies. Anti-RFP antibody reacts with mStrawberry. (E and F) MDCK cells stably expressing GFP-LC3 were infected with adenovirus bearing mStrawberry (E) or mStrawberry-fused Atg16L (F) and incubated for 40 h. The cells were then cultured in HBSS for 2 h, fixed, and subjected to conventional electron microscopic analysis. Autophagic structures are indicated (arrowheads). Bar, 1 μm.

Figure 4.

Figure 4.

Organization of the Atg16L complex in the presence of excess Atg16L. (A) PC12 cells were coinfected using a combination of recombinant adenoviruses as indicated. After 40-h incubation, cells were cultured in growth medium (Fed) or HBSS (Starved) for 2 h, and lysates were analyzed by Western blotting were performed with each antibody. From top panel, anti-Flag, anti-Atg5, anti-Atg12, anti-LC3, and anti-α-tubulin. (B–E) Cytosolic fractions of cell homogenates of control and PC12 cells overexpressing Flag-Atg16L constructs were separated by size exclusion chromatography. Each fraction was subjected to Western blotting using each antibody. Top panel, anti-Atg5; middle panel, anti-Atg16L; bottom panel, anti-Flag (except for B). (B) Mock. (C) Flag-Atg16L. (D) Flag-Atg16L-ΔC. (E) Flag-Atg16L-M. The positions of the molecular mass standards are shown. PC12 cells express only the Atg16L-α and -β isoforms. Vo, void fraction.

Figure 5.

Figure 5.

The effect of Atg16L overexpression on membrane localization of the Atg16L complex and the LC3 lipidation reaction. (A and B) MDCK cells stably expressing GFP-Atg5 were infected with adenovirus bearing mStrawberry (Mock) or mStrawberry-Atg16L-M and incubated for 40 h. The cells were then cultured in HBSS (Starved; A and B) or growth medium (Fed; B) for 2 h and fixed. Three-dimensional image stacks were obtained from sequential optical sections acquired 0.3 μm apart by confocal laser scanning microscopy (FV1000, Olympus) (A). Bar, 10 μm. More than 100 mStrawberry-positive cells were examined for the number of GFP-Atg5 dots per cell (B). Data are represented as means ± SD. (C and D) PC12 cells were coinfected using a combination of recombinant adenoviruses as indicated. After 40-h incubation, cells were collected. Lysates were examined by Western blotting using each antibody. (C) From top panel, anti-myc, anti-RFP, anti-LC3, and anti-GFP. (D) From top panel, anti-Flag, anti-RFP, anti-LC3, and anti-GFP.

Figure 6.

Figure 6.

The effect of ectopic Atg12-Atg5 localized to the plasma membrane on the LC3 lipidation reaction. (A) MCF7 cells stably expressing GFP-LC3 were coinfected using a combination of recombinant adenoviruses. After 40-h incubation, the cells were cultured in HBSS or HBSS with 100 nM wortmannin for 2 h, fixed, and observed by confocal laser-scanning microscopy (FV1000, Olympus). Bar, 10 μm. (B) MCF7 cells stably expressing GFP-LC3 were coinfected with recombinant adenovirus bearing Atg12, Atg5, and Flag-Atg16L-NKras-CAAX. After 40-h incubation, the cells were cultured in HBSS for 2 h, prepermeabilized for 5 min in PEM buffer (80 mM PIPES-KOH, pH 6.8, 5 mM EGTA, 1 mM MgCl2) containing 50 μg/ml digitonin, and fixed. Samples were observed by confocal laser scanning microscopy (FV1000, Olympus). Lateral images reconstituted from Z-sectioning are also shown. Bar, 10 μm. (C) MCF7 cells stably expressing GFP-LC3 were infected using a combination of recombinant adenoviruses as indicated. After 40-h incubation, the cells were grown for 2 h in growth medium (F), HBSS (S), or HBSS with 100 nM wortmannin (W). Lysates were examined by Western blotting using each antibody. From top panel, anti-RFP, anti-Atg5, anti-GFP, anti-LC3, and anti-α-tubulin.

Figure 7.

Figure 7.

The dynamic localization model of LC3 lipidation. On induction of autophagy, the Atg16L complex in the cytosol localizes on a yet undetermined membrane in an Atg16L-dependent manner. The high-energy Atg3-LC3 intermediate activated by Atg7 is recruited to that membrane via the interaction between Atg3 and Atg12. LC3 is thus brought into proximity with PE in the membrane, leading to lipidation. The Atg16L complex functions as a scaffold on which LC3 is transferred from Atg3 to PE.

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