Expression, characterization and regulation of a Saccharomyces cerevisiae monothiol glutaredoxin (Grx6) gene in Schizosaccharomyces pombe

Abstract

Glutaredoxins (Grxs), also known as thioltransferases (TTases), are thiol oxidoreductases that regulate cellular redox state in a variety of organisms. In the budding yeast Saccharomyces cerevisiae, Grx1 and 2 are cytosolic dithiol Grxs, while Grx3, 4 and 5 are monothiol Grxs. A gene encoding a new monothiol Grx, Grx6, was cloned from the genomic DNA of S. cerevisiae by PCR. Its DNA sequence contains 1,080 bp, and encodes a putative protein of 203 amino acid residues containing Cys-Phe-TyrSer at the active site. Grx6 is similar to other monothiol Grxs in the same organism and to Grx3 in the fission yeast Schizosaccharomyces pombe. and its predicted three-dimensional structure resembles that of S. pombe Grx3. S. pombe cells harboring plasmid pFGRX6 containing the Grx6 gene had about 1.3-fold elevated Grx activity in the exponential phase, and grew better than the control cells under some stressful conditions. Synthesis of beta-galactosidase from a Grx6-lacZ fusion gene in S. pombe was enhanced by potassium chloride, aluminum chloride and heat (37 degrees C) treatment. S. pombe cells harboring plasmid pFGRX6 had elevated ROS levels whereas S. pombe cells harboring extra copies of Grx3 had reduced ROS levels.