Chapter 3 The Quantitative Pho8Δ60 Assay of Nonspecific Autophagy

The measurement of autophagic flux is critical in understanding the regulation of autophagy. The Pho8Delta60 assay employs a very sensitive enzymatic assay that provides a high signal-to-noise ratio and allows for precise quantification of autophagic flow in yeast. Pho8, alkaline phosphatase, is a resident vacuolar enzyme that is delivered to the vacuole membrane through a portion of the secretory pathway. The assay utilizes a genetically engineered version of Pho8 that lacks the N-terminal transmembrane domain that allows for translocation into the endoplasmic reticulum. Accordingly, Pho8Delta60 remains in the cytosol and is delivered to the vacuole only through autophagy. Once in the vacuole lumen, the C-terminal propeptide is proteolytically removed, which results in activation. Thus, the alkaline phosphatase activity reflects the amount of the cytosol delivered to the vacuole through nonspecific autophagy.