Peroxisome Assembly in Yarrowia Lipolytica

Abstract

The primary goal of the research presented in this thesis has been to establish the yeast Yarrowia lipolytica as a model genetic system for the study of peroxisome biogenesis. To facilitate this, three steps were necessary. First, mutant strains of Y. lipolytica that manifested peroxisomal defects were generated; second, a genomic DNA library was created to rescue the mutants and, hence, clone the gene(s) involved; and third, the parameters governing high efficiency DNA transformation of Y. lipolytica were defined to make complementation of mutants with the genomic library feasible. This work culminated in the cloning, sequencing and characterization of a gene, dubbed PAY 4, that is required for peroxisomal assembly in Y. lipolytica. Two of the mutant strains of Y. lipolytica isolated, ole 2 and ole 4, were identified as peroxisome assembly mutants as they; (i) grew in acetate medium but failed to grow on oleic acid medium, and thus were peroxisomal as opposed to mitochondrial mutants; (ii) lacked recognizable peroxisomal structures when observed by immunofluorescence using a polyclonal serum that recognized a number of peroxisomal proteins and; (iii) had several peroxisomal enzyme activities localized to the cytosol, i.e. they manifested mistargeting of otherwise lumenal peroxisomal proteins. These were complemented with a genomic library, which represented at least 19 000 independent recombinants (insert size ~5-7 kilobases) cloned into a vector (piNA445) capable of autonomous replication in Y. lipolytica. Estimations of the size of the Y. lipolytica genome indicate that probability of this library containing a given portion of the genome is .999. To utilize the library effectively, an electro-poration protocol was developed that could transform Y. lipolytica at high efficiency (>5·10^4 transformants·μg-^1 of DNA). Using these resources, the gene complementing the ole 4 mutation was cloned and sequenced. It encodes Pay4p, a protein of ~112 kDa, containing two putative ATPase modules. Preliminary studies indicate that expression of Pay4p is induced slightly by growth on oleic acid.