Isolation and Characterization of Glutathione Biosynthesis-deficient Mutants in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae YNN27 (MATα. trp1 ura3 gal2) was mutagenized with ethyl methanesulfonate to isolate mutants deficient in glutathione biosynthesis. After mutants sensitive to 6.5 mM methylglyoxal were screened for, 12 glutathione biosynthesis-deficient mutants were isolated. Complementation tests found that the 12 mutants were classified into two different classes (YH1, YL1), and that each mutant was recessive and had a single mutation in glutathione biosynthesis. The growth of both strains in minimal medium was slower than that of the parent strain and was restored to the wild type level by supplementation with glutathione. They were also more sensitive to some chemicals such as thiol-reactive agents and metal-chelating agents than the parent strain. The mutant strain YL1 lacked activity of glutathione synthetase, while the deficiencies in γ-glutamylcysteine synthetase in mutant strains YH1 and YL1 were equivocal because the activity of the parent strain was very weak. However, supplementation with glutathione component amino acids and γ-glutamylcysteine to the medium showed that strain YH1, but not YL1, was deficient in γ-glutamylcysteine synthetase.