Monitoring autophagy in yeast: the Pho8Delta60 assay.
Autophagy is an ubiquitous degradative process in eukaryotic cells. It is involved in various developmental programs and is also implicated in human pathophysiology. The basic process involves the formation of a cytosolic double membrane vesicle, termed an autophagosome. The autophagosome sequesters bulk cytosol, and after completion its outside 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 micromolecules 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 results from damaged organelles including the mitochondria, or from the accumulation of large protein aggregates, such as occur in certain types of neurodegenerative diseases. 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, Pho8Delta60, are assayed for alkaline phosphatase activity before and after shifting to conditions that induce autophagy.
Klionsky
Daniel J
DJ
Department of Molecular, Cellular, and Developmental Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
D000469:Alkaline Phosphatase / Q000032:analysis*; D001343:Autophagy / Q000502:physiology*; D005656:Fungal Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000502:physiology*; D014617:Vacuoles / Q000201:enzymology
D005656:Fungal Proteins; D000469:Alkaline Phosphatase