BIOCHEMICAL METHODS TO MONITOR AUTOPHAGY-RELATED PROCESSES IN YEAST

An increasing number of reports have elucidated the importance of macroautophagy in cell physiology and pathology. Macroautophagy occurs at a basal level and participates in the turnover of cytoplasmic constituents including long-lived proteins to maintain cellular homeostasis, but it also serves as an adaptive response to protect cells from various intra- or extracellular stresses. In addition, macroautophagy plays a rote in development and aging and acts to protect against cancer, microbial invasion, and neurodegeneration. The machinery involved in carrying out this process, the autophagy-related (Atg) proteins were identified and characterized in various fungal systems, in particular because of the powerful tools available for genetic manipulation and the relative abundance of good biochemical assays in these model organisms. The analysis of these Atg proteins has allowed us to begin to understand the molecular mechanism of this process. Furthermore, many of the autophagy genes are functionally conserved in higher eukaryotes, including mammals, allowing the findings in fungi to be applied to other systems. Here, we discuss three biochemical methods to measure autophagy-related activities and to examine individual steps of the corresponding process. These methods rely on the detection of different modification states of certain marker proteins. Processing of the precursor form of the resident vacuolar hydrolase aminopeptidase I (Ape1) is applicable to fungi, whereas cleavage of the GFP-Atg8 and Pex14-GFP chimeras can be used in a wide array of systems.