Dissection of Autophagosome Formation Using Apg5-Deficient Mouse Embryonic Stem Cells

By N. Mizushima, A. Yamamoto, M. Hatano, Yoshinori Kobayashi, Y. Kabeya, Kuninori Suzuki, T. Tokuhisa, Y. Ohsumi, T. Yoshimori. Journal of Cell Biology, 2001

1,455 citations recorded by the source corpus.

In macroautophagy, cytoplasmic components are delivered to lysosomes for degradation via autophagosomes that are formed by closure of cup-shaped isolation membranes. However, how the isolation membranes are formed is poorly understood. We recently found in yeast that a novel ubiquitin-like system, the Apg12-Apg5 conjugation system, is essential for autophagy. Here we show that mouse Apg12-Apg5 conjugate localizes to the isolation membranes in mouse embryonic stem cells. Using green fluorescent protein–tagged Apg5, we revealed that the cup-shaped isolation membrane is developed from a small crescent-shaped compartment. Apg5 localizes on the isolation membrane throughout its elongation process. To examine the role of Apg5, we generated Apg5-deficient embryonic stem cells, which showed defects in autophagosome formation. The covalent modification of Apg5 with Apg12 is not required for its membrane targeting, but is essential for involvement of Apg5 in elongation of the isolation membranes. We also show that Apg12-Apg5 is required for targeting of a mammalian Aut7/Apg8 homologue, LC3, to the isolation membranes. These results suggest that the Apg12-Apg5 conjugate plays essential roles in isolation membrane development.

Subfields: Cell Biology, Epidemiology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/518a23f9fd74548a820215f27c6d9159f54b0ae0

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Cell Biology

1. D. Klionsky
2. N. Mizushima
3. Beth Levine
4. I. Slaninová
5. Y. Ohsumi
6. Guido Kroemer
7. V. Deretic
8. T. Yoshimori

In Epidemiology

1. N. Mizushima
2. D. Klionsky
3. Beth Levine
4. Guido Kroemer
5. I. Slaninová
6. V. Deretic
7. Alec C. Kimmelman
8. E. White

In Molecular Biology

1. N. Mizushima
2. Guido Kroemer
3. Beth Levine
4. D. Klionsky
5. I. Slaninová
6. Alec C. Kimmelman
7. Y. Ohsumi
8. E. White

Citations in the corpus

• ER stress (PERK/eIF2α phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation (2007, background): “ProLC3 is processed to LC3-I by cleavage at the C-terminal region(7) immediately after synthesis, and then LC3-I is targeted to the isolated membrane (LC3-II), dependent on the Atg5–Atg12 complex throughout the course of membrane elongation.(5,8)”
• Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy and mitochondrial fusion dynamics (2015, background): “To further test autophagy’s role in FA delivery to mitochondria, we examined whether Red C12 transfer to mitochondria was affected in starved Atg5 knockout (Atg5KO) MEFs, in which autophagosome biogenesis is blocked (Mizushima et al., 2001).”
• Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. (2006, neutral): “The following antibodies were used: anti-Beclin1 (Santa Cruz, Santa Cruz, CA); anti-ATG5 (Mizushima et al., 2001); anti-BAX and -BAK (Upstate Biotechnology, Lake Placid, NY); anti-BCL-2 (Santa Cruz); anti-p53 (Ab-1); anti-E1A and -actin (Oncogene, Cambridge, MA); anti-HMGB1 (ABCAM, Cambridge, MA);…”
• Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene1 (2002, background): “…has been proven in mammalian cells, because several mammalian APG proteins have been shown to be essential for autophagy (Liang et al., 1999; Mizushima et al., 2001) and to act via a molecular mechanism quite similar to that of yeast (Mizushima et al., 1998b; Kabeya et al., 2000; Tanida et…”
• Autophagy suppresses tumor progression by limiting chromosomal instability. (2007, positive): “technology); anti-ATG5 (Mizushima et al. 2001).”
• A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation (2009, result): “IMs in NIH 3T3 cells formed cup-shaped structures, which morphologically resembled those observed in previous studie”
• Autophagy: molecular machinery for self-eating (2005, background): “In mammalian cells, however, the complex is clearly associated with the forming autophagosome, termed the phagophore or isolation membrane.(64,58) These results lead us to suggest that during the membrane formation stage the Atg12–Atg5-Atg16 com-”
• Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination (2016, background): “Under culture conditions permissive for the propagation of primed ESCs and where there is an abundance of nutrients, autophagy occurs at a basal level and therefore its deficiency has no detrimental effect in both mESCs and hESCs (Mizushima et al., 2001; Cho et al., 2014).”
• Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice (2005, background): “The antibodies for Atg7 (Tanida et al., 1999) and Atg5 (Mizushima et al., 2001) were described previously.”
• Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. (2008, background): “Wortmannin completely inhibited starvation-induced Atg16L1 puncta formation as described previously for the Atg5 puncta (Mizushima et al., 2001).”
• Autophagy: a lysosomal degradation pathway with a central role in health and disease. (2009, neutral): “hagy compartment in mammalian cells [16].”