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-u
tilizers 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 r
apid 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 ap
plicable to the selection of peroxisomal assembly mutants in other yea
sts. To take advantage of the pay mutant system, we constructed a geno
mic library in the autonomously replicating vector pINA445 and develop
ed an efficient and rapid electroporation procedure for the functional
complementation of these mutants. We have been successful in function
ally 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.