MitoPho8Δ60 assay as a tool to quantitatively measure mitophagy activity

Abstract

Mitophagy, a selective type of macroautophagy (hereafter referred to as autophagy), specifically mediates the vacuole/lysosome-dependent degradation of damaged or surplus mitochondria. Because this process regulates the number and quality of mitochondria, it is vital for proper cellular homeostasis. Mitophagy also plays critical roles in the clearance of paternal mitochondria in C. elegans embryos, in erythroid cell maturation, and in the prevention of neurodegenerative disease and cancer. In order to study the molecular mechanism and regulation of mitophagy, sensitive assays are necessary to quantitatively measure mitophagy activity. In the budding yeast, Saccharomyces cerevisiae, a “mitoPho8Δ60” assay was developed to study mitophagy. In this assay, Pho8, a vacuolar phosphatase protein, is genetically engineered to be targeted to mitochondria. When mitophagy is induced, the phosphatase protein, along with mitochondria, is conveyed to the vacuole, where its C-terminal propeptide is removed and the phosphatase activity becomes activated; under growing conditions only a background level of delivery occurs. For this reason, the enzymatic activity of mitoPho8Δ60 is correlated with the amount of mitochondria delivered to the vacuole. Thus, this assay serves as a very convenient tool to quantitatively monitor mitophagy activity in yeast.