Identification of Pexophagy Genes by Restriction Enzyme-Mediated Integration

Abstract

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 pro-tocol 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 disrupt-ing 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 vec-tor 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.