Isolation and characterization of a new mutant of Saccharomyces cerevisiae with altered synthesis of 5-aminolevulinic acid.
A new gene, RHM1, required for normal production of 5-aminolevulinic acid by Saccharomyces cerevisiae, was identified by a novel screening method. Ethyl methanesulfonate treatment of a fluorescent porphyric strain bearing the pop3-1 mutation produced nonfluorescent or weakly fluorescent mutants with defects in early stages of tetrapyrrole biosynthesis. Class I mutants defective in synthesis of 5-aminolevulinate regained fluorescence when grown on medium supplemented with 5-aminolevulinate, whereas class II mutants altered in later biosynthetic steps did not. Among six recessive class I mutants, at least three complementation groups were found. One mutant contained an allele of HEM1, the structural gene for 5-aminolevulinate synthase, and two mutants contained alleles of the regulatory gene CYC4. The remaining mutants contained genes complementary to both hem1 and cyc4. Mutant strain DA3-RS3/68 contained mutant gene rhm1, which segregated independently of hem1 and cyc4 during meiosis. 5-Aminolevulinate synthase activity of the rhm1 mutant was 35 to 40% of that of the parental pop3-1 strain, whereas intracellular 5-aminolevulinate concentration was only 3 to 4% of the parental value. Transformation of an rhm1 strain with a multicopy plasmid containing the cloned HEM1 gene restored normal levels of 5-aminolevulinate synthase activity, but intracellular 5-aminolevulinate was increased to only 9 to 10% of normal. We concluded that RHM1 could control either targeting of 5-aminolevulinate synthase to the mitochondrial matrix or the activity of the enzyme in vivo.
Carvajal
E
E
Departamento de Bioquímica, Universidade Federal do Rio de Janeiro, Brazil.
Panek
A D
AD
Mattoon
J R
JR
D000624:5-Aminolevulinate Synthetase / Q000235:genetics*; D000622:Aminolevulinic Acid / Q000378:metabolism; D003580:Cytochromes / Q000032:analysis; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic
D003580:Cytochromes; D000622:Aminolevulinic Acid; D000624:5-Aminolevulinate Synthetase