title: Quantitative Assay of Macroautophagy Using Pho8△60 Assay and GFP-Cleavage Assay in Yeast.
abstract: It is intrinsically difficult to directly measure a specific protein reduction that is mediated by nonselective autophagy, because the degradation proceeds at a relatively slow pace, and the residual nondegraded part becomes the background. Several methods for measuring nonselective autophagy have been reported in the past. One classical simple method is called bulk degradation assay, which measures the release of degraded amino acids after radioisotope labeling of the total cellular proteins. In 1995, we developed a quantitative Pho8△60 assay in the yeast, Saccharomyces cerevisiae, for studying autophagy, which is widely used for its advantages that are described in the following sections. Another method used in recent times is the GFP-based processing assay in yeast. We will describe these two methods in this chapter.
authors.affiliation: Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Araki
authors.affiliation: Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kira
authors.affiliation: Center for Frontier Oral Science, Graduate School of Dentistry, Osaka University, Osaka, Japan; Graduate School of Frontier BioSciences, Osaka University, Osaka, Japan. Electronic address: takenoda@dent.osaka-u.ac.jp.
authors.forename: T
authors.initials: T
authors.lastname: Noda
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy; D000071190:Autophagy-Related Protein 8 Family / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000235:genetics / Q000378:metabolism
chemical_list: C113757:ATG8 protein, S cerevisiae; D000071190:Autophagy-Related Protein 8 Family; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase
keywords: Alkaline phosphatase; GFP-Atg8; Nonselective autophagy; Pho8△60; Vacuole; Yeast