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.

Several groups have reported the cloning and sequencing of genes involved in the biogenesis of yeast peroxisomes. Yeast strains bearing mutations in these genes are unable to grow on carbon sources whose metabolism requires peroxisomes, and these strains lack morphologically normal peroxisomes. We report the cloning of Pichia pastoris PAS1, the homologue (based on a high level of protein sequence similarity) of the Saccharomyces cerevisiae PAS1. We also describe the creation and characterization of P. pastoris pas1 strains. Electron microscopy on the P. pastoris pas1 cells revealed that they lack morphologically normal peroxisomes, and instead contain membrane-bound structures that appear to be small, mutant peroxisomes, or "peroxisome ghosts." These "ghosts" proliferated in response to induction on peroxisome-requiring carbon sources (oleic acid and methanol), and they were distributed to daughter cells. Biochemical analysis of cell lysates revealed that peroxisomal proteins are induced normally in pas1 cells. Peroxisome ghosts from pas1 cells were purified on sucrose gradients, and biochemical analysis showed that these ghosts, while lacking several peroxisomal proteins, did import varying amounts of several other peroxisomal proteins. The existence of detectable peroxisome ghosts in P. pastoris pas1 cells, and their ability to import some proteins, stands in contrast with the results reported by Erdmann et al. (1991) for the S. cerevisiae pas1 mutant, in which they were unable to detect peroxisome-like structures. We discuss the role of PAS1 in peroxisome biogenesis in light of the new information regarding peroxisome ghosts in pas1 cells.