Characterization of Saccharomycopsis lipolytica mutants that express temperature-sensitive synthesis of isocitrate lyase.
Four mutants specifically deficient in the activity of isocitrate lyase were independently isolated in the alkane yeast Saccharomycopsis lipolytica. Genetic analysis by means of protoplast fusion and mitotic haploidization revealed that the mutations were recessive and non-complementary at a single genetic locus, icl. icl is a structural gene for isocitrate lyase, because some revertants from icl-1 and icl-3 mutants produced thermolabile isocitrate lyase in comparison with the wild-type enzyme, and also because the gene dosage effect was observed on the specific activity of isocitrate lyase in icl+/icl-1 and icl+/icl-3 heterozygotes. The icl-3 mutation also gave rise to temperature-sensitive revertants that could grow on acetate at 23 degrees C but not at 33 degrees C, exhibiting temperature-sensitive synthesis as well as thermostable activity of isocitrate lyase. Studies on purified isocitrate lyase showed that this enzyme is tetrameric and that the enzyme synthesized at 23 degrees C by a temperature-sensitive synthesis mutant was indistinguishable from the wild-type enzyme with respect to the subunit molecular weight (59,000), the isoelectric pH (5.3), the thermostability, and the Km value for threo-Ds-isocitrate (0.2 mM). When induced by acetate at 33 degrees C, the temperature-sensitive synthesis mutant did not express isocitrate lyase activity but did synthesize polypeptides whose electrophoretic mobilities were equal to that of the purified mutant enzyme. Hence, the temperature-sensitive mutation assumed in the structural gene for isocitrate lyase might have prevented the maturation of the polypeptide chains synthesized at the restrictive temperature.
D000085:Acetates / Q000494:pharmacology; D001203:Ascomycota / Q000235:genetics*; D005796:Genes; D005800:Genes, Fungal; D005808:Genes, Recessive; D007522:Isocitrate Lyase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007526:Isoelectric Point; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009154:Mutation; D007652:Oxo-Acid-Lyases / Q000235:genetics*; D012442:Saccharomycopsis / Q000201:enzymology / Q000235:genetics*; D013696:Temperature
D000085:Acetates; D046911:Macromolecular Substances; D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase; D008274:Magnesium