The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway

By T. Kirisako, Yoshinobu Ichimura, H. Okada, Y. Kabeya, N. Mizushima, T. Yoshimori, M. Ohsumi, Toshifumi Takao, Takeshi Noda, Y. Ohsumi. Journal of Cell Biology, 2000

1,009 citations recorded by the source corpus.

Autophagy and the Cvt pathway are examples of nonclassical vesicular transport from the cytoplasm to the vacuole via double-membrane vesicles. Apg8/Aut7, which plays an important role in the formation of such vesicles, tends to bind to membranes in spite of its hydrophilic nature. We show here that the nature of the association of Apg8 with membranes changes depending on a series of modifications of the protein itself. First, the carboxy-terminal Arg residue of newly synthesized Apg8 is removed by Apg4/Aut2, a novel cysteine protease, and a Gly residue becomes the carboxy-terminal residue of the protein that is now designated Apg8FG. Subsequently, Apg8FG forms a conjugate with an unidentified molecule “X” and thereby binds tightly to membranes. This modification requires the carboxy-terminal Gly residue of Apg8FG and Apg7, a ubiquitin E1-like enzyme. Finally, the adduct Apg8FG-X is reversed to soluble or loosely membrane-bound Apg8FG by cleavage by Apg4. The mode of action of Apg4, which cleaves both newly synthesized Apg8 and modified Apg8FG, resembles that of deubiquitinating enzymes. A reaction similar to ubiquitination is probably involved in the second modification. The reversible modification of Apg8 appears to be coupled to the membrane dynamics of autophagy and the Cvt pathway.

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1. N. Mizushima
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5. Xiaohua Li
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8. Naonobu Fujita

Citations in the corpus

• An Overview of the Molecular Mechanism of Autophagy (2009, background): “The liberated Atg8 is recycled and used in another conjugation reaction to allow efficient progression of autophagy and the Cvt pathway (Kirisako et al. 2000).”
• Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4 (2007, background): “The active site, Cys77, is conserved from yeast to man, whereas Cys81 is conserved only in tetrapod homologues of Atg4A and Atg4B.”
• Autophagosome formation: core machinery and adaptations (2007, background): “Before attachment, the C terminus of Atg8 is processed by a cysteine protease, Atg4, to expose the glycine residu”
• Mechanisms of Selective Autophagy (2016, unknown): “First, Atg8 undergoes a proteolytic cleavage mediated by Atg4, which exposes the C-terminal glycine employed for the subsequent conjugation reaction [74].”
• Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. (2007, methodology): “Urea-SDS-PAGE and Immunoblotting Analysis All SDS-PAGE was performed with 13.5% polyacrylamide gels containing 6 M urea to separate Atg8-PE from unconjugated Atg8 (Kirisako et al., 2000).”
• FYCO1 is a Rab7 effector that binds to LC3 and PI3P to mediate microtubule plus end–directed vesicle transport (2010, background): “All members of the family are membrane associated via a phosphatidylethanolamine lipid anchor attached to a C-terminal glycine (Kirisako et al., 2000; Kabeya et al., 2004).”
• Protein turnover via autophagy: implications for metabolism. (2007, background): “Atg8 (LC3 in mammals) and Atg12 are ubiquitin-like proteins.”
• Leaf Senescence and Starvation-Induced Chlorosis Are Accelerated by the Disruption of an Arabidopsis Autophagy Gene1 (2002, methodology): “Apg8p is then activated by Apg7p and is subsequently attached to Apg3p, another E2-like enzyme, after which the C-terminal Gly of Apg8p is conjugated to phosphatidylethanolamine by an amide bond (Ichimura et al., 2000; Kirisako et al., 2000).”
• A genomic and functional inventory of deubiquitinating enzymes. (2005, unknown): “For instance, newly synthesized Ubl Atg8 is processed by a distinct protease (Apg4) of which five family members are found in the genome (Kirisako et al., 2000).”
• Mouse Apg16L, a novel WD-repeat protein, targets to the autophagic isolation membrane with the Apg12-Apg5 conjugate (2003, background): “The Aut7/Apg8 system, in which Aut7 is conjugated to phosphatidylethanolamine, is catalyzed by Apg7 and Aut1/Apg3 (Kirisako et al., 2000; Ichimura et al., 2000).”
• LC3 and GATE‐16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis (2010, background): “Atg4 is also responsible for the priming of Atg8 by cleaving its C-terminus to expose a glycine residue (Kirisako et al, 2000; Scherz-Shouval et al, 2007).”
• Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response (2006, background): “Atg8 is a cytosolic protein that becomes lipidated [54,55] and accumulates in pre-autophagosomal structures (PASs) that are in close proximity to the vacuole and can be visualized as dots by fluorescent microscopy in cells expressing green fluorescent protein (GFP)Atg8 fusion proteins [56–58].”