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Atg12–Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site

Nature Structural & Molecular Biology volume 20, pages 433–439 (2013)Cite this article

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Abstract

Two autophagy-related ubiquitin-like systems have unique features: the E2 enzyme Atg3 conjugates the ubiquitin-like protein Atg8 to the lipid phosphatidylethanolamine, and the other ubiquitin-like protein conjugate Atg12–Atg5 promotes that conjugase activity of Atg3. Here, we elucidate the mode of this action of Atg12–Atg5 as a new E3 enzyme by using Saccharomyces cerevisiae proteins. Biochemical analyses based on structural information suggest that Atg3 requires a threonine residue to catalyze the conjugation reaction instead of the typical asparagine residue used by other E2 enzymes. Moreover, the catalytic cysteine residue of Atg3 is arranged in the catalytic center such that the conjugase activity is suppressed; Atg12–Atg5 induces a reorientation of the cysteine residue toward the threonine residue, which enhances the conjugase activity of Atg3. Thus, this study reveals the mechanism of the key reaction that drives membrane biogenesis during autophagy.

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Figure 1: Thr213 of Atg3 is required for its conjugase activity.
Figure 2: Atg12–Atg5 induces a conformational change in the Atg3 catalytic site.
Figure 3: The conformational change in Atg3 is associated with conjugase activity.
Figure 4: Phe293 is involved in suppression of Atg3 conjugase activity.
Figure 5: His236 is required for both the basal and Atg12–Atg5-stimulated conjugase activities of Atg3.
Figure 6: Model of Atg3 activation by Atg12–Atg5.

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Acknowledgements

We thank the members of our laboratories for materials and helpful discussions. This work was supported in part by the Funding Program for Next Generation World-Leading Researchers HO220017 (to H.N.) and Grants-in-Aid for Scientific Research 23000015 (to Y.O.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Frontier Research Center, Tokyo Institute of Technology, Yokohama, Japan

    Machiko Sakoh-Nakatogawa, Eri Asai, Hiromi Kirisako, Junko Ishii, Hitoshi Nakatogawa & Yoshinori Ohsumi

  2. Institute of Microbial Chemistry, Tokyo, Japan

    Kazuaki Matoba & Nobuo N Noda

  3. Department of Structural Biology, Hokkaido University, Sapporo, Japan

    Fuyuhiko Inagaki

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  1. Machiko Sakoh-Nakatogawa
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Contributions

M.S.-N. performed in vitro and in vivo experiments with the help of E.A., H.K. and J.I.; K.M., N.N.N. and F.I. contributed to structural analysis. M.S.-N., H.N. and Y.O. contributed to experimental design and data interpretation and wrote the manuscript.

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Correspondence to Hitoshi Nakatogawa or Yoshinori Ohsumi.

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Sakoh-Nakatogawa, M., Matoba, K., Asai, E. et al. Atg12–Atg5 conjugate enhances E2 activity of Atg3 by rearranging its catalytic site. Nat Struct Mol Biol 20, 433–439 (2013). https://doi.org/10.1038/nsmb.2527

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