Abstract
Autophagy is a bulk degradation system highly conserved among eukaryotic cells and plays crucial roles in a wide range of physiological and pathological situations. Remarkably, this process involves two ubiquitin-like (Ubl) conjugation systems. Here, we describe two sodium dodecyl sulfate-polyacrylamide gel electrophoresis techniques to analyze these systems: one that allows separation of the Ubl protein Atg8 conjugated to the lipid phosphatidylethanolamine from its unlipidated form, and the other by which otherwise labile thioester intermediates between Atg8 and either the E1 enzyme Atg7 or the E2 enzyme Atg3 are stably preserved during electrophoresis, and thus easily detected by following protein visualization. Especially, the latter technique is also ubiquitously applicable for studies on conjugation reactions of ubiquitin (Ub) and other Ubl proteins.
*This research was originally published in J. Biol. Chem. Oh-oka K, Nakatogawa H, Ohsumi Y. Physiological pH and acidic phospholipids contribute to substrate specificity in lipidation of Atg8. 2008, 283:21847–21852. ©The American Society for Biochemistry and Molecular Biology (16).
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Acknowledgments
We thank Drs. Kyoko Oh-oka and Hayashi Yamamoto for providing the gel images and helpful comments on the procedures.
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Nakatogawa, H., Ohsumi, Y. (2012). SDS-PAGE Techniques to Study Ubiquitin-Like Conjugation Systems in Yeast Autophagy. In: Dohmen, R., Scheffner, M. (eds) Ubiquitin Family Modifiers and the Proteasome. Methods in Molecular Biology, vol 832. Humana Press. https://doi.org/10.1007/978-1-61779-474-2_37
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DOI: https://doi.org/10.1007/978-1-61779-474-2_37
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