Role of Ycf1 protein in Cadmium Sensibility During Respiratory Metabolism in Saccharomyces cerevisiae

Objective: The main pathway of cadmium (Cd) detoxification in S. cerevisiae consist of  Cd sequestering by glutathione (GSH) followed by Ycf1p-mediated transport of Cd-GSH complexes to vacuole. The generation of oxidative stress as a consequence of reduction in the GSH intracellular pool is one of the mechanisms related to the Cd toxicity (Free Radic Biol Med. 2:  321; 1995). In S. cerevisiae the transition between fermentative and respiratory metabolism (diauxic shift) is followed by an increase of the oxidative stress resistance. The objective of this work was to investigate the effects of diauxic shift in the resistance to oxidative stress induced by Cd in yeast and a possible contribution of Ycf1p for this process. Methods and Results:  Both strains BY4741 (wild-type) and ycf1D  were analyzed for its sensitivity to Cd⁺² (drops tests and curves of survival performed after 24h treatments) in synthetic medium containing a range of concentrations between 12 and 384mM. Additionally to Cd⁺², the synthetic medium contained glucose or glycerol/ethanol to simulate, respectively, the pre and post-diauxic shift metabolism. The results of survival curves showed that in medium containing glucose the  ycf1D  strain was more sensitive than BY4741 strain to Cd (survival of 13% against 48% after treatment with 192mM of Cd⁺²). In glycerol, the  ycf1D  survival was around 91% and BY4741 around 73%. Moreover, in the drops tests carried in plates containing glucose,  ycf1D  was more sensible than BY4741 in Cd concentrations above 48µM. In glycerol or ethanol plates, both strains were able to growth only in Cd concentrations bellow 48mM but ycf1D  were more resistant to Cd than BY4741. This resistance can be reverted by the addition of DNTB (a GSH sequestering compound) in the medium with glicerol/ethanol. Conclusions:  The data indicate that Ycf1p is important for Cd resistance during fermentative metabolism. However, the importation of conjugated Cd-GSH by Ycf1p can result in a reduction of intracellular GSH pool, that has more severe effects in cells submitted to cadmium stress during respiratory metabolism. These results allow to infer that a Ycf1p-independent pathways should be acting in Cd detoxification in post-diauxic phase. More experiments are being conduced in order to quantify the GSH intracellular pool in both ycf1D  and BY4741 strains.
Financial support: CNPq