Isolation, expression, and regulation of the pgr1+ gene encoding glutathione reductase absolutely required for the growth of Schizosaccharomyces pombe

Abstract

The pgr1+ gene encoding glutathione reductase (GR, EC 1.6.4.2) was isolated from Schizosaccharomyces pombe using a polymerase chain reaction fragment as a probe. The gene consists of two exons and an intron of 55 nucleotides, encoding a polypeptide of 465 amino acids (50,238 Da) with conserved residues characteristic of GR. The transcriptional start site was localized at 239 nucleotides upstream from the ATG initiation codon. The level of transcript as well as the GR enzyme activity increased more than 11- fold when the cloned pgr11 gene was expressed on a multicopy plasmid. This overexpression conferred on S. pombe cells more resistance against menadione, a redox cycling agent, but not against H2O2. The level of pgr1+ transcripts increased by treatment with oxidants such as menadione, cumene hydroperoxide, and diamide. It also increased by treatment with high osmolarity, heat shock, or at the stationary growth phase. The deletion of the pap1+ gene encoding an AP-1 homolog in S. pombe caused reduction in the pgr1+ gene expression. Furthermore, ~pap1 cells lost the inducibility of pgr1+ gene expression by the above stresses, implying that Pap1 is involved in general stress-inducible gene expression. When the pgr1+ gene was disrupted, the haploid spores were not viable. Repression of nmt1 promoter- driven pgr1+ expression by thiamine caused cessation of growth, which was rescued by the episomal pgr1+ gene. These results indicate that GR activity, which efficiently reduces GSSG, is essentially required for the growth of S. pombe, unlike in Saccharomyces cerevisiae or Escherichia coli.