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Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T - J. Cell Biol. (2005)

Bottom Line: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation.Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies.Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.

ABSTRACT
Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

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Generation of Atg7F/F mice. (A) Schematic representation of the targeting vector and the targeted allele of Atg7 gene. The coding exons numbered in accordance with the initiation site as exon 1 are depicted by black boxes. Green and red boxes indicate Atg7 cDNA fragment (aa 1786–2097) and Atg7 cDNA fragment (aa 1669–1698) with stop codon, respectively. The open triangles denote loxP sequence. A probe for Southern blot analysis is shown as a gray ellipse. Arrows indicate the positions of PCR primers. The asterisk denotes the essential cysteine residue on exon 14. EcoRV, EcoRV 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 Flox alleles are detected as 17.5- and 7.5-kb bands, respectively. (C) Immunoblot of Atg7 in MEFs. The lysates of MEFs of indicated genotypes were immunoblotted with Atg7 and actin.
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fig1: Generation of Atg7F/F mice. (A) Schematic representation of the targeting vector and the targeted allele of Atg7 gene. The coding exons numbered in accordance with the initiation site as exon 1 are depicted by black boxes. Green and red boxes indicate Atg7 cDNA fragment (aa 1786–2097) and Atg7 cDNA fragment (aa 1669–1698) with stop codon, respectively. The open triangles denote loxP sequence. A probe for Southern blot analysis is shown as a gray ellipse. Arrows indicate the positions of PCR primers. The asterisk denotes the essential cysteine residue on exon 14. EcoRV, EcoRV 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 Flox alleles are detected as 17.5- and 7.5-kb bands, respectively. (C) Immunoblot of Atg7 in MEFs. The lysates of MEFs of indicated genotypes were immunoblotted with Atg7 and actin.

Mentions: To investigate the physiological roles of autophagy in mammals, we generated Atg7 conditional knockout mice. Mouse Atg7 gene is encoded by 17 exons that span 216-kb long genomic DNA. The active site cysteine residue essential for activation of the substrates is encoded by exon 14 and the targeting vector is designed to conditionally disrupt this exon by Cre-loxP technology. The targeted exon 14 was modified so that it could express Atg7 even in the presence of neo-resistant gene cassette in intron 14 (Fig. 1 A). Mice homozygous for the Atg7Flox allele (referred to as Atg7F/F mice), which were expected to express intact Atg7, were born healthy and fertile without any noticeable pathological phenotypes. Fig. 1 B shows Southern blots of mice with the indicated genotypes. Immunoblot analysis revealed the presence of Atg7 protein in Atg7F/F mouse embryonic fibroblasts (MEFs; Fig. 1 C), suggesting that Atg7 is efficiently expressed from the Atg7Flox allele.


Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice.

Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, Ezaki J, Mizushima N, Ohsumi Y, Uchiyama Y, Kominami E, Tanaka K, Chiba T - J. Cell Biol. (2005)

Generation of Atg7F/F mice. (A) Schematic representation of the targeting vector and the targeted allele of Atg7 gene. The coding exons numbered in accordance with the initiation site as exon 1 are depicted by black boxes. Green and red boxes indicate Atg7 cDNA fragment (aa 1786–2097) and Atg7 cDNA fragment (aa 1669–1698) with stop codon, respectively. The open triangles denote loxP sequence. A probe for Southern blot analysis is shown as a gray ellipse. Arrows indicate the positions of PCR primers. The asterisk denotes the essential cysteine residue on exon 14. EcoRV, EcoRV 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 Flox alleles are detected as 17.5- and 7.5-kb bands, respectively. (C) Immunoblot of Atg7 in MEFs. The lysates of MEFs of indicated genotypes were immunoblotted with Atg7 and actin.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171928&req=5

fig1: Generation of Atg7F/F mice. (A) Schematic representation of the targeting vector and the targeted allele of Atg7 gene. The coding exons numbered in accordance with the initiation site as exon 1 are depicted by black boxes. Green and red boxes indicate Atg7 cDNA fragment (aa 1786–2097) and Atg7 cDNA fragment (aa 1669–1698) with stop codon, respectively. The open triangles denote loxP sequence. A probe for Southern blot analysis is shown as a gray ellipse. Arrows indicate the positions of PCR primers. The asterisk denotes the essential cysteine residue on exon 14. EcoRV, EcoRV 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 Flox alleles are detected as 17.5- and 7.5-kb bands, respectively. (C) Immunoblot of Atg7 in MEFs. The lysates of MEFs of indicated genotypes were immunoblotted with Atg7 and actin.
Mentions: To investigate the physiological roles of autophagy in mammals, we generated Atg7 conditional knockout mice. Mouse Atg7 gene is encoded by 17 exons that span 216-kb long genomic DNA. The active site cysteine residue essential for activation of the substrates is encoded by exon 14 and the targeting vector is designed to conditionally disrupt this exon by Cre-loxP technology. The targeted exon 14 was modified so that it could express Atg7 even in the presence of neo-resistant gene cassette in intron 14 (Fig. 1 A). Mice homozygous for the Atg7Flox allele (referred to as Atg7F/F mice), which were expected to express intact Atg7, were born healthy and fertile without any noticeable pathological phenotypes. Fig. 1 B shows Southern blots of mice with the indicated genotypes. Immunoblot analysis revealed the presence of Atg7 protein in Atg7F/F mouse embryonic fibroblasts (MEFs; Fig. 1 C), suggesting that Atg7 is efficiently expressed from the Atg7Flox allele.

Bottom Line: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation.Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies.Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan.

ABSTRACT
Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

Show MeSH
Related in: MedlinePlus