Effects of oxidative stress on the phosphoglycerate kinase gene promoter of saccharomyces cerevisiae

Abstract

Yeast cells exposed to physical or chemical alterations in their environment respond by employing a number of defence mechanisms in order to successfully adapt to such changes and sustain a high proliferation rate. In such stressful circumstances, the cell is often subjected to increased energy demands. The cellular ATP pool is therefore determinant in enabling the cell to mount an effective stress response. In previous work, a possible stimulation by the redox active compound paraquat (PQ) of the glycolytic 3-phosphoglycerate kinase gene (PGKl) promoter controlling the Eschericia coli iron superoxide dismutase gene had been observed in yeast cells (Agius et al., 1998). In this study the effects of oxidative stress, generated by PQ, on the PGKl promoter were investigated. This was done by subcloning the yeast PGKl promoter in-frame with the lacZ gene of E.coli, thus constructing a multi copy β-galactosidase (β-gal) reporter plasmid. Wild-type Saccharomyces cerevisiae cells were then transformed with the reporter gene vector. After exposure oftransformants to PQ, a combination of β-gal activity assays and β-gal ELISA were carried out. This enabled direct information as to effects at promoter level to be obtained. The results showed that moderate, as opposed to excessive, doses of PQ induced increased stimulation of the PGKl promoter, at mid-exponential phase of growth. Plasmid copy numbers from cells exposed to PQ and from control cells were determined at mid-log phase, and found to be similar. This ruled out the possibility that the results were due to a selective effect of PQ on yeast cells synthesising a higher amount of β-gal, because of harbouring a greater number of β-gal reporter plasmids. The induction effect by 1 mM PQ was not apparent at stationary phase. Addition of the thiol antioxidant N-acetylcysteine (NAC) at mid-log phase attenuated the stimulatory effect of PQ on the PGKl promoter. Furthermore, one hour exposure of yeast transformants to PQ was insufficient to enhance stimulation of the promoter.

In order to corroborate some aspects of the results obtained using PQ, an alternative source of oxidative stress was used, namely, plumbagin. As determined by β-gal activity assays, plumbagin induced increased stimulation of the PGKl promoter at mid-log phase but not at stationary phase. One hour exposure to plumbagin was also insufficient to further stimulate the promoter. These results thus mirrored those obtained using PQ.

Results from this work may therefore suggest one aspect of a more general role for glycolysis in maintaining the energy pools of yeast cells under stress.