Stress-dependent regulation of a monothiol glutaredoxin gene from Schizosaccharomyces pombe

Glutaredoxin (Grx) is a small, heat-stable protein acting as a multi-functional glutathione-dependent disulfide oxidoreductase. In this work, a gene encoding the monothiol glutaredoxin Grx4 was cloned from the genomic DNA of the fission yeast Schizosaccharomyces pombe. The determined DNA sequence carries 1706 bp, which is able to encode the putative 244 amino acid sequence of Grx with 27 099 Da. It does not contain an intron, and the sequence CGFS is found in the active site. Grx activity was increased 1.46-fold in S. pombe cells harboring the cloned Grx4 gene, indicating that the Grx4 gene is in vivo functioning. Although aluminum, cadmium, and hydrogen peroxide marginally enhanced the synthesis of beta-galactosidase from the Grx4-lacZ fusion gene, NO-generating sodium nitroprusside (0.5 mmol/L and 1.0 mmol/L) and potassium chloride (0.2 mol/L and 0.5 mol/L) significantly enhanced it. The Grx4 mRNA level was also enhanced after the treatment with sodium nitroprusside and potassium chloride. The synthesis of beta-galactosidase from the Grx4-lacZ gene was increased by fermentable carbon sources, such as glucose (lower than 2%) and sucrose, but not by nonfermentable carbon sources such as acetate and ethanol. The basal expression of the S. pombe Grx4 gene did not depend on the presence of Pap1. These results imply that the S. pombe monothiol Grx4 gene is genuinely functional and regulated by a variety of stresses.     

Pap1-mediated regulation of thioredoxin gene from Schizosaccharomyces pombe

The genomic DNA encoding thioredoxin (TRX) was previously isolated from the fission yeast Schizosaccharomyces pombe. In this investigation, regulation of the S. pombe TRX gene was studied in lacZ translational fusions. The synthesis of beta-galactosidase from the fusion plasmid pYKT24 was significantly enhanced by treatments with cadmium chloride, zinc chloride, and high temperatures. Synthesis of beta-galactosidase from the fusion plasmid was significantly decreased by higher concentrations (5 microM, 10 microM) of mercuric chloride, whereas it was enhanced by its lower concentration (1 microM). Diamide affected the synthesis of beta-galactosidase in the same manner with mercuric chloride. However, high osmolarity had no effect on the beta-galactosidase synthesis from the fusion plasmid pYKT24. Various fusion plasmids were constructed to carry serially deleted upstream regions of the TRX gene. Pap1 mediates the regulation of the S. pombe TRX gene. The upstream region, between 987 and 1,270 bp from the translational initiation point, is responsible for the regulation.     

Pap1-dependent regulation of the GSTII gene from the fission yeast

The genomic DNA encoding a second glutathione S-transferase (GSTII) was previously isolated from the fission yeast Schizosaccharomyces pombe. Its expression was shown to be induced by menadione, mercuric chloride, o-dinitrobenzene, and NO-generating S-nitroso-N-acetylpenicillamine using the GSTII-lacZ fusion harboring the 910 bp upstream region from the translational initiation point. In this study, the additional fusion plasmids pGST50-590 and pGST50-6R-590 were constructed to carry the 590 bp upstream region in the vectors YEp357 and YEp367R, respectively. The synthesis of beta-galactosidase from the fusion plasmid pGST50-590 was about 3-fold higher than that from the fusion plasmid pGST50-F, indicating the presence of negatively activating sequence in the -910 to approximately -590 region. It was also enhanced by the same agents, which induced the synthesis of beta-galactosidase from the fusion plasmid pGST50-F. The synthesis of beta-galactosidase from both fusion plasmids pGST50-F and pGST50-590 was enhanced by the overexpressed Pap1 protein. The synthesis of beta-galactosidase from the two YEp367R derivatives pGST50-6R-F and pGST50-6R-590 was greatly decreased in the Pap1-negative strain TP108-3C. These results propose the Pap1-dependent regulation of the GSTII gene from the fission yeast.     

Cellular functions and transcriptional regulation of a third thioredoxin from Schizosaccharomyces pombe

The structural gene encoding a third thioredoxin (Trx) homologue, TRX3, of the fission yeast Schizosaccharomyces pombe was characterized and its regulation was studied. The determined DNA sequence encoded a putative 290 amino acid sequence of Trx with a molecular mass of 31,889 Da. The TRX3 mRNA level was increased in S. pombe cells harboring plasmid pTRX3, suggesting that the cloned TRX3 gene was functional. Yeast cultures harbouring plasmid pTRX3 exhibited shorter generation times and higher survival on solid minimal media plates incorporating mercury chloride (0.01 mmol/L) or hydrogen peroxide (1 mmol/L) compared with control cultures. Yeast cells containing extra copies of TRX3, but not TRX1 and TRX2, gave rise to lower reactive oxygen species levels than control cells. Oxidative stress owing to hydrogen peroxide and menadione enhanced the synthesis of beta-galactosidase from the TRX3-lacZ fusion gene in Pap1-positive cells but not in Pap1-negative cells. The TRX3 mRNA level was increased by oxidative stress only in Pap1-positive cells. Basal expression of the TRX3 gene also depended on Pap1. We concluded that S. pombe TRX3 is linked with yeast growth and oxidative stress response, with its expression being regulated by oxidative stress in a Pap1-dependent manner.     

Cloning, characterization and regulation of a protein disulfide isomerase from the fission yeast Schizosaccharomyces pombe

To elucidate the physiological roles and regulation of a protein disulfide isomerase (PDI) from the fission yeast Schizosaccharomyces pombe, the full-length PDI gene was ligated into the shuttle vector pRS316, resulting in pPDI10. The determined DNA sequence carries 1,636 bp and encodes the putative 359 amino acid sequence of PDI with a molecular mass of 39,490 Da. In the amino acid sequence, the S. pombe PDI appears to be very homologous to A. thaliana PDI. The S. pombe cells harboring pPDI10 showed increased PDI activity and accelerated growth, suggesting that the cloned PDI gene is functioning and involved in the yeast growth. The 460 bp upstream region of the PDI gene was fused into promoterless β-galactosidase gene of the shuttle vector YEp367R to generate pYUPDI10. The synthesis of β-galactosidase from the PDI–lacZ fusion gene was enhanced by oxidative stress, such as superoxide anion and hydrogen peroxide. It was also induced by some non-fermentable and fermentable carbon sources. Nitrogen starvation was able to enhance the synthesis of β-galactosidase from the PDI–lacZ fusion gene. The enhancement by oxidative stress and fermentable carbon sources did not depend on the presence of Pap1. The PDI mRNA levels were increased in both Pap1-positive and Pap1-negative cells treated with glycerol. Taken together, the S. pombe PDI gene is involved in cellular growth and response to nutritional and oxidative stress.     

Molecular characterization of yeast regulatory gene CAT3 necessary for glucose derepression and nuclear localization of its product

The yeast regulatory gene CAT3 has an essential function for the depression of several glucose-repressible enzymes. Therefore, cat3 mutants are unable to grow on maltose or on non-fermentable carbon sources. Unlike the point mutants isolated previously, cat3 null allele strains also failed to utilize raffinose or galactose as sole carbon sources. Sequencing of an 1.6-kb HindIII-BglII fragment complementing cat3 mutations revealed an open reading frame of 322 codons, size of which is in good agreement with the 1.3-kb size of mRNA. No significant similarities with previously sequenced genes could be detected. CAT3-lacZ fusions confirmed the proposed reading frame. A CAT3-lacZ fusion encoding 307 amino acids of CAT3 was able to complement the growth defects of cat3 point mutants and null allele strains. Assay of beta-galactosidase activity under different growth conditions indicated a constitutive expression of the CAT3 gene product. Cellular fractionation studies showed the nuclear localization of the CAT3 protein.     

Characterization and regulation of a second gene encoding thioredoxin from the fission yeast

A genomic DNA encoding a second thioredoxin (TRX2) was isolated from the chromosomal DNA of the fission yeast Schizosaccharomyces pombe. The cloned sequence contains 1823 bp and encodes a protein of 121 amino acids. It has extra N-terminal 17 amino acid residues compared to previously identified thioredoxin (TRX1), which are positively charged and hydrophobic amino acids. The additional N-terminal region contains a plausible prepeptidase cleavage site, indicating that the TRX2 protein exists in mitochondria. The cloned TRX2 gene produced functional TRX estimated with insulin reduction assay. The upstream region of the TRX2 gene was fused into the promoterless beta-galactosidase gene of the shuttle vector YEp357R. The 782 bp sequence in the region further upstream of the TRX2 gene was found to be inhibitory in its expression. Synthesis of beta-galactosidase from the fusion plasmid pYFX135-HRL was enhanced by the addition of aluminum chloride and ferrous chloride, indicating that the TRX2 protein is involved in stress response.