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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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Fasting response of Atg7-deficient liver. (A and B) Livers from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice fed ad libitum (Fed) or fasted for 1 d (Fast) at 20 d after pIpC injection were dissected, and the amount of total protein (A) and SDH activity (B) per liver were measured. Data are mean ± SD values of five mice in each group; *, P < 0.01. (C) Cytochrome c levels in the cytosolic and mitochondria/lysosomal fractions of the liver at 20 d after injection. Equal amount of PNS fractions were centrifuged at 8,000 g for 10 min and the pellets were used as the mitochondrial/lysosomal fraction (ML). The supernatants were further centrifuged at 100,000 g for 1 h and the supernatant was used as the cytosolic fraction (C). Actin was blotted as control. Data shown are representative of two separate experiments. (D) Turnover of long-lived protein. Hepatocytes from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice were isolated and labeled with [14C]leucine for 24 h, and degradation of long-lived protein in deprived or nondeprived condition was measured. Monomethylamine (MA) and/or E64d and pepstatin (E/P) or epoxomicin (epoxo) was added as indicated. Data are the mean ± SD of triplicate experiments.
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fig4: Fasting response of Atg7-deficient liver. (A and B) Livers from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice fed ad libitum (Fed) or fasted for 1 d (Fast) at 20 d after pIpC injection were dissected, and the amount of total protein (A) and SDH activity (B) per liver were measured. Data are mean ± SD values of five mice in each group; *, P < 0.01. (C) Cytochrome c levels in the cytosolic and mitochondria/lysosomal fractions of the liver at 20 d after injection. Equal amount of PNS fractions were centrifuged at 8,000 g for 10 min and the pellets were used as the mitochondrial/lysosomal fraction (ML). The supernatants were further centrifuged at 100,000 g for 1 h and the supernatant was used as the cytosolic fraction (C). Actin was blotted as control. Data shown are representative of two separate experiments. (D) Turnover of long-lived protein. Hepatocytes from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice were isolated and labeled with [14C]leucine for 24 h, and degradation of long-lived protein in deprived or nondeprived condition was measured. Monomethylamine (MA) and/or E64d and pepstatin (E/P) or epoxomicin (epoxo) was added as indicated. Data are the mean ± SD of triplicate experiments.

Mentions: Given that autophagosome formation was impaired in Atg7-deficient liver, we next examined its effects on the bulk degradation of proteins and organelles under fasting condition. After 1-d fasting in control Atg7F/+:Mx1 and mutant Atg7F/F:Mx1 mice, the liver was dissected and the amount of total protein per whole liver was measured. The amount of total liver proteins decreased to ∼66% by 1-d fasting in the control liver (Fig. 4 A). In contrast, fasting did not significantly decrease the amount of total liver proteins in the mutant liver. Moreover, the amount of total proteins in the mutant liver was 1.5-fold that of control. These results indicate that the decrease of total proteins is dependent on Atg7 and autophagosome formation.


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)

Fasting response of Atg7-deficient liver. (A and B) Livers from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice fed ad libitum (Fed) or fasted for 1 d (Fast) at 20 d after pIpC injection were dissected, and the amount of total protein (A) and SDH activity (B) per liver were measured. Data are mean ± SD values of five mice in each group; *, P < 0.01. (C) Cytochrome c levels in the cytosolic and mitochondria/lysosomal fractions of the liver at 20 d after injection. Equal amount of PNS fractions were centrifuged at 8,000 g for 10 min and the pellets were used as the mitochondrial/lysosomal fraction (ML). The supernatants were further centrifuged at 100,000 g for 1 h and the supernatant was used as the cytosolic fraction (C). Actin was blotted as control. Data shown are representative of two separate experiments. (D) Turnover of long-lived protein. Hepatocytes from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice were isolated and labeled with [14C]leucine for 24 h, and degradation of long-lived protein in deprived or nondeprived condition was measured. Monomethylamine (MA) and/or E64d and pepstatin (E/P) or epoxomicin (epoxo) was added as indicated. Data are the mean ± SD of triplicate experiments.
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Related In: Results  -  Collection

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fig4: Fasting response of Atg7-deficient liver. (A and B) Livers from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice fed ad libitum (Fed) or fasted for 1 d (Fast) at 20 d after pIpC injection were dissected, and the amount of total protein (A) and SDH activity (B) per liver were measured. Data are mean ± SD values of five mice in each group; *, P < 0.01. (C) Cytochrome c levels in the cytosolic and mitochondria/lysosomal fractions of the liver at 20 d after injection. Equal amount of PNS fractions were centrifuged at 8,000 g for 10 min and the pellets were used as the mitochondrial/lysosomal fraction (ML). The supernatants were further centrifuged at 100,000 g for 1 h and the supernatant was used as the cytosolic fraction (C). Actin was blotted as control. Data shown are representative of two separate experiments. (D) Turnover of long-lived protein. Hepatocytes from Atg7F/+:Mx1 and Atg7F/F:Mx1 mice were isolated and labeled with [14C]leucine for 24 h, and degradation of long-lived protein in deprived or nondeprived condition was measured. Monomethylamine (MA) and/or E64d and pepstatin (E/P) or epoxomicin (epoxo) was added as indicated. Data are the mean ± SD of triplicate experiments.
Mentions: Given that autophagosome formation was impaired in Atg7-deficient liver, we next examined its effects on the bulk degradation of proteins and organelles under fasting condition. After 1-d fasting in control Atg7F/+:Mx1 and mutant Atg7F/F:Mx1 mice, the liver was dissected and the amount of total protein per whole liver was measured. The amount of total liver proteins decreased to ∼66% by 1-d fasting in the control liver (Fig. 4 A). In contrast, fasting did not significantly decrease the amount of total liver proteins in the mutant liver. Moreover, the amount of total proteins in the mutant liver was 1.5-fold that of control. These results indicate that the decrease of total proteins is dependent on Atg7 and autophagosome formation.

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