The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice - PubMed (original) (raw)

. 2008 Nov;19(11):4762-75.

doi: 10.1091/mbc.e08-03-0309. Epub 2008 Sep 3.

Satoshi Waguri, Jun-ichi Iwata, Takashi Ueno, Tsutomu Fujimura, Taichi Hara, Naoki Sawada, Akane Yamada, Noboru Mizushima, Yasuo Uchiyama, Eiki Kominami, Keiji Tanaka, Masaaki Komatsu

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The Atg8 conjugation system is indispensable for proper development of autophagic isolation membranes in mice

Yu-shin Sou et al. Mol Biol Cell. 2008 Nov.

Abstract

Autophagy is an evolutionarily conserved bulk-protein degradation pathway in which isolation membranes engulf the cytoplasmic constituents, and the resulting autophagosomes transport them to lysosomes. Two ubiquitin-like conjugation systems, termed Atg12 and Atg8 systems, are essential for autophagosomal formation. In addition to the pathophysiological roles of autophagy in mammals, recent mouse genetic studies have shown that the Atg8 system is predominantly under the control of the Atg12 system. To clarify the roles of the Atg8 system in mammalian autophagosome formation, we generated mice deficient in Atg3 gene encoding specific E2 enzyme for Atg8. Atg3-deficient mice were born but died within 1 d after birth. Conjugate formation of mammalian Atg8 homologues was completely defective in the mutant mice. Intriguingly, Atg12-Atg5 conjugation was markedly decreased in Atg3-deficient mice, and its dissociation from isolation membranes was significantly delayed. Furthermore, loss of Atg3 was associated with defective process of autophagosome formation, including the elongation and complete closure of the isolation membranes, resulting in malformation of the autophagosomes. The results indicate the essential role of the Atg8 system in the proper development of autophagic isolation membranes in mice.

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Figures

Figure 1.

Figure 1.

Generation of _Atg3_-knockout mice. (A) Schematic representation of the targeting vector and the targeted allele of Atg3 gene. The coding exons numbered in accordance with the initiation site as exon 1 are depicted by black boxes. The probe for Southern blot analysis is shown as gray ellipse. The asterisk denotes the essential cysteine residue on exon 10. PstI, PstI sites; neo, neomycin-resistant gene cassette; DT-A, diphtheria toxin gene. (B) Southern blot analysis of genomic DNAs extracted from mice tails. Wild-type and mutant alleles are detected as 5- and 2-kb bands, respectively. (C) Expression of Atg3 transcript. Total RNAs were extracted from each genotyping MEFs. Atg3 transcript was detected by Northern blot with mouse Atg3 cDNA. Actin cDNA was used as an internal control. (D) Immunoblot of Atg3 and Atg8 homologues in MEFs. Primary MEFs of the indicated genotypes were isolated and cultured under nutrient-rich (nondeprived) and nutrient-poor (deprived) conditions. The lysates were immunoblotted with anti-Atg3, anti-LC3, anti-GABARAP, anti-GATE-16, and anti-actin antibodies. Data shown are representative of three separate experiments. (E) Immunofluorescence analysis of MEFs using anti-LC3antibody. Each genotype immortalized MEFs with introduced GFP-tagged Atg5 were cultured in DMEM with fetal calf serum (nondeprived) or Hanks' solution (deprived). The cells were fixed and then immunostained with anti-LC3. Insets show a GFPAtg5-single- (left inset), an LC3-single- (right inset), and a double-positive (middle inset) structure, respectively. Bars; 10 μm.

Figure 2.

Figure 2.

Phenotypes of _Atg3_-deficient mice. (A) Morphology of Atg3+/- and _Atg3_−/− mice. (B) Deficiency of LC3-positive dots in _Atg3_−/− heart. Atg3+/− and _Atg3_−/− mice expressing GFP-LC3 were delivered by Cesarean section and analyzed by florescence microscopy. Representative results obtained from each neonatal heart at 3 h after Cesarean delivery. Bar; 50 μm. (C) The concentrations of total amino acids, essential amino acids, and BCAA in sera of mice of the indicated genotypes. Atg3+/+, Atg3+/− (n = 5) and _Atg3_−/− mice (n = 4) were dissected immediately (top) or at 10 h (bottom) after delivery and the plasma concentrations of amino acids were measured. Data are mean ± SD. ***p < 0.001 by Student's t test.

Figure 3.

Figure 3.

Formation of Atg12–Atg5 conjugates is partially inhibited by Atg3 deficiency. (A) Immunoblot analyses of the Atg12–Atg5 conjugate. Immortalized MEFs of the indicated genotype were lysed, and the lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. As a negative control for detection of Atg12–Atg5 conjugate, we used the lysate of _Atg7_-deficient MEFs. Note the low production of the Atg12–Atg5 conjugate in _Atg3_-deficient cells. (B) Each genotype immortalized MEFs with introduced GFP-tagged Atg5 were lysed, and then the lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. As a negative control for detection of Atg12–GFPAtg5 conjugate, we used the lysate of GFPAtg5-introduced _Atg7_-deficient MEFs. (C) Immortalized wild-type and _Atg3_-deficient MEFs-introduced FLAG-tagged Atg12 or Atg12ΔGly were lysed, and then the lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. As a negative control for detection of FLAGAtg12-Atg5 conjugate, we used the lysate of FLAGAtg12ΔGly-expressing MEFs. Graphs shown in A–C indicate the ratios of Atg12–Atg5, Atg12–GFPAtg5, and FLAGAtg12–Atg5 relative to actin, respectively. Data are mean ± SD of triplicate experiments. **p < 0.01 by Student's t test. (D) Half-lives of GFPAtg5, FLAGAtg12, and GFPAtg5-Atg12 conjugation in _Atg3_-deficient MEFs. Each immortalized wild-type and _Atg3_-deficient MEFs harboring GFPAtg5 or FLAGAtg12 were labeled with [35S]cysteine and methionine for 2 h and chased for the indicated time. For labeling of GFPAtg5-12 conjugate, the cells were labeled with [35S]cysteine and methionine for 24 h. Subsequently, each lysate was immunoprecipitated with anti-GFP and FLAG antibodies and then subjected to SDS-PAGE and visualized by autoradiography. As a negative control for detection of Atg12–GFPAtg5 conjugate, we used the lysate of GFPAtg5-introduced _Atg7_-deficient MEFs. The decay curves of GFPAtg5 (top, right), FLAGAtg12 (middle, right) and Atg12-GFPAtg5 (bottom, right) were generated from quantification of the band shown in the left panels. (E) The MEFs of the indicated genotypes were cultured in the presence or absence of a proteasome inhibitor (epoxomicin) for 16 h (top) or lysosomal inhibitors (E64d and pepstatin A: E/P) for 24 h (bottom). The cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with anti-Atg12 and anti-actin antibodies. (F) Wild-type and _Atg3_-deficient immortalized MEFs were infected with Atg3 and Atg3C264S at 10, or 100 multiplicity of infection (M.O.I.). After 48-h infection, the cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. Bottom graph shows the ratios of Atg12–Atg5 relative to actin, respectively. Data are mean ± SD. **p < 0.01 by Student's t test. Data are representative of three separate experiments.

Figure 4.

Figure 4.

Accumulation of Atg16L-positive structures in _Atg3_-deficient MEFs. (A) Immunofluorescence analysis of Atg16L in GFPAtg5-introduced immortalized MEFs. Each genotype MEFs were cultured in nutrient-rich (nondeprived) or Hanks' solution (deprived). The cells were fixed and then immunostained with anti-Atg16L. Bars; 10 μm. (B) Immunofluorescence analysis of Atg16L in MEFs. MEFs isolated from Atg3+/+ and _Atg3_−/− were cultured in nutrient-rich (nondeprived) or Hanks' solution (deprived). After deprivation of nutrients, the cells were resupplied with nutrients for 10, 30, or 60 min. The cells were fixed and then immunostained with anti-Atg16L. Bars; 10 μm. (C) The number of Atg16L-positive dots in MEF (n = 20) was determined in each genotype. Data are mean ± SD. *p < 0.05 and **p < 0.01 by Student's t test. (D and E) Time-lapse observation of starvation-induced GFPAtg5 dots. Wild-type (D) and _Atg3_-deficient (E) MEFs harboring GFPAtg5 were cultured in Hanks' solution for 1 h and directly observed by time-lapse video microscopy. Bars, 1 μm. (F) Duration of existence of GFPAtg5 dots in wild-type and _Atg3_-deficient MEFs. The time of sustained presence of GFPAtg5 dots in wild-type (n = 9) and mutant (n = 10) was measured. Data are mean ± SD. **p < 0.01 by Student's t test.

Figure 5.

Figure 5.

Induction of autophagosomes under starvation condition in _Atg3_-knockout cells. (A) Electron micrographs of primary MEFs from the indicated genotype mice under nutrient-rich (nondeprived) or -poor (deprived) condition. Bar, 5 μm. Arrowheads, autophagosomes; arrows, autolysosomes. (B) Numbers of autophagosomes (AP) and autolysosomes (AL) in each genotype were counted (n = 20; for details, see Materials and Methods). Data are mean ± SD. *p < 0.01, by Student's t test. (C) Area of autophagosome-like vacuoles (AVs) in MEFs from the indicated genotype under nutrient-rich (nondeprived) and -poor (deprived) conditions. Frequencies (percentages) of the given range of area are expressed in the histogram. The dotted line represents the mean value of the area in each genotype. (D) Immunoelectron micrograph showing labeling of GFP (a and b) and Atg16L (c–h) in wild-type (a–c) and _Atg3_-deficient MEFs (d–h) harboring GFPAtg5 under nutrient deprivation conditions. b is a higher magnification view of the GFPAtg5-positive structure indicated by the arrow in a. Higher magnification views of the Atg16L-positive structures indicated by arrows in d are shown in insets e and f. Arrowheads indicate autophagosome-like structures without GFPAtg5 or Atg16L signal. Asterisks indicate autolysosomes. Bars, 0.5 μm.

Figure 6.

Figure 6.

(A and B) Impaired long-lived protein degradation in _Atg3_-deficient MEFs. Primary MEFs from wild-type, _Atg3_- (A) and _Atg7_-knockout (B) mice were isolated and labeled with [14C]leucine for 24 h, and degradation of long-lived protein in deprived and nondeprived conditions was measured. 3-MA and/or E64d, pepstatin A and ammonium chloride (E64d/Pep + AC), or lactacystin was added as indicated. Data are mean ± SD of triplicate experiments.

Figure 7.

Figure 7.

Impaired elongation and closure in autophagosome formation in _Atg3_-deficient MEFs. Electron microscopic analyses of serial sections of wild-type (A–D) and _Atg3_-deficient MEFs (E–H). Arrows indicate open regions of autophagosome-like structures. Note that the isolation membranes seem to elongate in random directions, thus these membranes sequestrate certain area of the cytoplasm in some parts. Therefore, isolation membranes in mutant cells did not close to form complete autophagosomes. Bars, 1 μm.

Figure 8.

Figure 8.

Suppression of formation of early isolation membranes and appearance of aberrant membranous structures in _Atg3_-deficient hepatocytes. (A) Immunofluorescence analysis of Atg16L in hepatocytes from Atg3+/− (left) and _Atg3_−/− (right) newborn mice. Each newborn mouse was fixed at 6 h after Cesarean delivery. Atg16L-positive structures were observed in Atg3+/−, but only few in _Atg3_−/− neonate hepatocytes. Arrow indicates a pleomorphic structure positive for Atg16L. Bar, 10 μm. (B) Electron microscopic analysis of hepatocytes from Atg3+/− (a and c) and _Atg3_−/− (b, d, and e) newborn mice. Each newborn mouse was fixed for electron microscopy at 6 h after Cesarean delivery. Typical autophagosomes (arrows) were observed in Atg3+/−, but fewer in _Atg3_−/− neonatal hepatocytes. Aberrant membranous structures occurred in _Atg3_−/− neonate hepatocytes (d and e). e is a magnification view of the boxed region in d. Note that the glycogen area (asterisks) is smaller in mutant hepatocytes than heterozygous hepatocytes. N, nucleus. Bars, 10 μm (a and b) and 1 μm (c–e). (C) The number of autophagosomes in a section of hepatocyte in each genotype (mean ± SD; n = 60). *p < 0.001.

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