Elucidation of the mode of action of Atg12-Atg5 conjugate in autophagy

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Autophagy is a bulk degradation system, which involves the formation of a double-membrane vesicle called autophagosome. Autophagosome formation requires two ubiquitin-like proteins, Atg8 and Atg12. They are conjugated to the phosphatidylethanolamine (PE) and Atg5, respectively, via a series of enzymatic reactions with E1 and E2 enzyme. In this study, we elucidated the mode of action of Atg12-Atg5 as an E3 enzyme in the Atg8-PE conjugation reaction. We established a biochemical assay based on the structural information to determine the configuration of the catalytic center of Atg3, which is an E2 enzyme in Atg8-PE conjugation reaction. This approach revealed that Atg12-Atg5 conjugate induces a conformational change in the catalytic center of Atg3 to enhance its E2 activity. Moreover, mutational analyses indicated how the activity of Atg3 is suppressed in the absence of Atg12-Atg5 conjugate. We are attempting to analyze the crystal structure of Atg12-Atg5 conjugate/Atg3 complex.