title: Assays to Monitor Pexophagy in Yeast.
abstract: Pexophagy is a selective autophagy process that degrades damaged and/or superfluous peroxisomes in the yeast vacuole or in mammalian lysosomes. The molecular mechanisms of pexophagy are well studied in yeast. Peroxisomes can be rapidly induced by oleate in the budding yeast, Saccharomyces cerevisiae, and by oleate or methanol in the methylotrophic yeast, Pichia pastoris. A number of peroxisomal matrix enzymes, such as 3-ketoacyl CoA thiolase (thiolase) and alcohol oxidase (AOX), are upregulated correspondingly to meet metabolic demands of the cells. Removal of these peroxisome-inducing carbon sources creates conditions wherein peroxisomes are superfluous and results in pexophagy and the degradation of these peroxisomal matrix enzymes. In this chapter, we discuss different assays to monitor pexophagy in yeast. These assays rely on tracking the localization of the BFP-SKL protein (a peroxisomally targeted version of the blue fluorescent protein) by microscopy, biochemical analysis of the degradation of peroxisomal matrix proteins, thiolase and AOX, and/or measuring the reduction of AOX activity during pexophagy.
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, United States.
authors.forename: W
authors.initials: W
authors.lastname: Wang
authors.affiliation: Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, CA, United States. Electronic address: ssubramani@ucsd.edu.
authors.forename: S
authors.initials: S
authors.lastname: Subramani
mesh_terms: D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D001343:Autophagy; D057075:Enzyme Assays / Q000379:methods; D005656:Fungal Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D020675:Peroxisomes / Q000201:enzymology / Q000378:metabolism* / Q000648:ultrastructure; D010843:Pichia / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D059748:Proteolysis; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D000102:Acetyl-CoA C-Acyltransferase
keywords: BFP–SKL; Measuring AOX activity; Pexophagy; Thiolase and AOX degradation; Thiolase–GFP processing