Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP‐1 transcriptional regulation

Abstract

Glutathione (GSH) is an abundant cellular thiol which has been implicated in many cellular processes including protection against xenobiotics, carcinogens and free radicals. Utilization of GSH in both enzymic and non-enzymic defence mechanisms results in its conversion to the oxidized form (GSSG), and it must be recycled to GSH to maintain the high intracellular ratio of GSH to GSSG. Glutathione reductase (GLR) is a flavoenzyme, which catalyses reduction of GSSG to GSH using the reducing power of NADPH. We show that yeast mutants deleted for GLR1, encoding glutathione reductase, lack GLR activity and accumulate increased levels of GSSG. In addition, the glr1 mutant strain was unaffected in the inducible adaptive response to hydrogen peroxide, but showed increased sensitivity to oxidants including both peroxides and superoxide, indicating a requirement for GLR in protection against oxidative stress. Furthermore, GLR1 expression was elevated two to threefold in the presence of oxidants, and regulation was dependent upon the yAP-1 transcriptional activator protein. Thus, GLR1 is one of a growing number of genes involved in the protection of yeast cells against oxidative stress and regulated by yAP-1.

Discrimination between Paralogs using Microarray Analysis: Application to the Yap1p and Yap2p Transcriptional Networks

“…A detailed description of all the clusters produced from this analysis can be found at http://genetics.med.harvard.edu/ ϳcohen/yaps/Yaps.html. A Yap binding site weight matrix (Stormo et al, 1982) was constructed using sites from four promoters known to be regulated by Yap1p (Kuge and Jones, 1994;Wu and Moye-Rowley, 1994;Wemmie et al, 1994;Grant et al, 1996). This weight matrix was used as an input to the computer program ScanACE (Hughes et al, 2000a) to determine the distribution of Yap sites among all of the expression clusters.…”

The YeastSaccharomyces cerevisiaeContains Two Glutaredoxin Genes That Are Required for Protection against Reactive Oxygen Species

“…Glutathione reductase (Glr) is the primary enzyme that controls the redox state of GSH and is required for protection against oxidative stress (Grant et al, 1996a). Glr activity is regulated in response to ROS (Grant et al, 1996a) but was unaffected in the glutaredoxin mutants, indicating that there was no increased demand for the enzyme in these strains (Table 3).…”

“…Glr activity is regulated in response to ROS (Grant et al, 1996a) but was unaffected in the glutaredoxin mutants, indicating that there was no increased demand for the enzyme in these strains (Table 3). Cellular redox state, as measured by the reaction of DTNB with free thiol groups, was also unaffected in the glutaredoxin mutants (Table 3).…”

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine.

“…Exogenous GSH is required for growth of strains deleted for GSH1 and GSH2. Strains CY4 (wt), CY4p (petite), CY8 (gshl), and CY97 (gsh2) were streaked for single colonies on minimal medium (SD) and SD supplemented with 1 mM GSH or 1 mM y-glutamylcysteine (y-GC (Kosower and Kosower, 1995) and has been shown to induce the expression of TRX2 and GLR1 in yeast (Kuge and Jones, 1994;Grant et al, 1996a). Strains lacking GSH (gshl and gsh2) were found to be resistant to diamide (Figure 4) and could grow on concentrations of diamide as high as 5 mM, whereas the highest permissible concentration for the wild-type strain was 1.5 mM.…”