Identification of Pexophagy Genes by Restriction Enzyme-Mediated Integration

Pichia pastoris has proven to be a valuable model for examining the molecular events of the selective degradation of peroxisomes by a process called pexophagy. We have developed a protocol to rapidly identify genes essential for glucose-induced pexophagy. This method utilizes the random integration of a Zeocin resistance cassette vector into the genomic DNA thereby disrupting gene expression. Transformed yeast are selected by growth on Zeocin and pexophagy mutants identified by their inability to degrade the peroxisomal enzyme alcohol oxidase. The Zeocin vector along with flanking genomic DNA is then isolated from the mutants and the disrupted genes identified by sequencing. We have been able to isolate 59 mutants and identify 8 unique genes. The identification of 24 genes in P. pastoris and 7 genes in H. polymorpha have been reported using this approach which has been referred to as restriction-mediated integration.