Summary
Autophagy is an ubiquitous degradative process in eukaryotic cells (1,2) . It is involved in various developmental programs and is also implicated in human pathophysiology (3,4). The basic process involves the formation of a cytosolic double-membrane vesicle, termed an autophagosome (5,6) . The autophagosome sequesters bulk cytosol, and after completion its outer membrane fuses with the limiting membrane of the lysosome/vacuole. The fusion event releases the inner single-membrane vesicle into the lysosome/vacuole lumen, where the vesicle is now termed an autophagic body. The autophagic body and its cargo are typically degraded, and the resulting macromolecules are recycled for subsequent use in the cytosol. Autophagy is the only pathway with the capacity to degrade entire organelles. Accordingly, it may play a critical role in preventing pathologies that result from damaged organelles including the mitochondria (7), or from the accumulation of large protein aggregates, such as occur in certain types of neurodegenerative diseases (8) . This article describes an assay to monitor bulk autophagy in yeast. The marker protein is a cytosolic derivative of the vacuolar enzyme alkaline phosphatase, Pho8. Following uptake into the vacuole, the precursor enzyme is cleaved at the C terminus to generate the active form. Cells expressing the cytosolic form of Pho8, Pho8 Δ60, are assayed for alkaline phosphatase activity before and after shifting to conditions that induce autophagy.
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Acknowledgments
The author would like to thank Su Chen, Heesun Cheong, Zhiping Xie and Drs. Julie Legakis and Usha Nair for helpful comments. This work was supported by Public Health Service grant GM53396 from the National Institutes of Health.
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Klionsky, D.J. (2007). Monitoring Autophagy in Yeast. In: van der Giezen, M. (eds) Protein Targeting Protocols. Methods in Molecular Biology™, vol 390. Humana Press. https://doi.org/10.1007/978-1-59745-466-7_24
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DOI: https://doi.org/10.1007/978-1-59745-466-7_24
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