RAPID IDENTIFICATION AND CHARACTERIZATION OF PEROXISOMAL ASSEMBLY MUTANTS IN YARROWIA-LIPOLYTICA

We describe the isolation and characterization of peroxisomal assembly mutants in the genetically manipulable yeast Yarrowia lipolytica (pay mutants). These mutants were initially identified as oleic acid-non-utilizers by their inability to grow on oleic acid, the utilization of which requires peroxisomal beta-oxidation enzymes. Identification of a subset of oleic acid-non-utilizers as pay mutants was obtained by a rapid immunofluorescence procedure using antibodies to the peroxisomal targeting signal Ser-Lys-Leu-CO2H. Punctate structures characteristic of peroxisomes were not detected in pay mutants using this technique. This rapid identification by immunofluorescence should be generally applicable to the selection of peroxisomal assembly mutants in other yeasts. To take advantage of the pay mutant system, we constructed a genomic library in the autonomously replicating vector pINA445 and developed an efficient and rapid electroporation procedure for the functional complementation of these mutants. We have been successful in functionally complementing two independent pay mutants. Molecular analysis of these and other complementing genes will allow for characterization of some of the cellular elements involved in peroxisomal assembly.