Lap4, a vacuolar aminopeptidase I, is involved in cadmium-glutathione metabolism.

In Saccharomyces cerevisiae, accumulation of cadmium-glutathione complex in cytoplasm inhibits cadmium absorption, glutathione transferase 2 is required for the formation of the complex and the vacuolar gamma-glutamyl transferase participates of the first step of glutathione degradation. Here, we proposed that Lap4, a vacuolar amino peptidase, is involved in glutathione catabolism under cadmium stress. Saccharomyces cerevisiae cells deficient in Lap4 absorbed almost 3-fold as much cadmium as the wild-type strain (wt), probably due to the lower rate of cadmium-glutathione complex synthesis in the cytoplasm. In wt, but not in lap4 strain, the oxidized/reduced GSH ratio and the Gtt activity increased in response to cadmium, confirming that the mutant is deficient in the synthesis of the complex probably because the degradation of vacuolar glutathione is impaired. Thus, under cadmium stress, Lap4 and gamma-glutamyl transferase seem to work together to assure an efficient glutathione turnover stored in the vacuole.

Vacuolar compartmentation of the cadmium-glutathione complex protects Saccharomyces cerevisiae from mutagenesis.

In the yeast Saccharomyces cerevisiae, gamma-glutamyl transferase (gamma-GT; EC 2.3.2.2) is a vacuolar-membrane bound enzyme. In this work we verified that S. cerevisiae cells deficient in gamma-GT absorbed almost 2.5-fold as much cadmium as the wild-type (wt) cells, suggesting that this enzyme might be responsible for the recycle of cadmium-glutathione complex stored in the vacuole. The mutant strain showed difficulty in keeping constant levels of glutathione (GSH) during the stress, although the GSH-reductase activity was practically the same in both wt and mutant strains, before and after metal stress. This difficulty to maintain the GSH levels in the gamma-GT mutant strain led to high levels of lipid peroxidation and carbonyl proteins in response to cadmium, higher than in the wt, but lower than in a mutant deficient in GSH synthesis. Although the increased levels of oxidative stress, gamma-GT mutant strain showed to be tolerant to cadmium and showed similar mutation rates to the wt, indicating that the compartmentation of the GSH-cadmium complex in vacuole protects cells against the mutagenic action of the metal. Confirming this hypothesis, a mutant strain deficient in Ycf1, which present high concentrations of GSH-cadmium in cytoplasm due to its deficiency in transport the complex to vacuole, showed increased mutation rates.

The role of glutathione transferases in cadmium stress.

Using Saccharomyces cerevisiae as experimental model, we observed that cells mutated in the GTT1 or GTT2 genes showed twice as much cadmium absorption than the control strain. We proposed that the formation of the cadmium-glutathione complex is dependent on that transferase, since it was previously demonstrated that the cytoplasmic levels of this complex affect cadmium uptake. The addition of glutathione monoethyl ester (GME), a drug that mimics glutathione (GSH), to gtt1Delta cells restored the levels of metal absorption to those of the control strain. However, with respect to gtt2Delta cells, addition of GME did not alter the capacity of removing cadmium from the medium. Taken together, these results suggest that Gtt1 and Gtt2 play different roles in the mechanism of cadmium detoxification. By analyzing the toxic effect of this metal, we verified that gtt2Delta and gsh1Delta cells showed, respectively, higher and lower tolerance to cadmium stress than control cells, suggesting that although GSH plays a relevant role in cell protection, formation of the GSH-Cd conjugate is deleterious to the mechanism of defense.

The effect of superoxide dismutase deficiency on cadmium stress.

Saccharomyces cerevisiae mutant strains deficient in superoxide dismutase (Sod), an antioxidant enzyme, were used to analyze cadmium absorption and the oxidation produced by it. Cells lacking the cytosolic Sod1 removed twice as much cadmium as the control strain, while those deficient in the mitochondrial Sod2 exhibited poor metal absorption. Interestingly, the sod1 mutant did not become more oxidized after exposure to cadmium, as opposed to the control strain. We observed that the deficiency of Sod1 increases the expression of both Cup1 (a metallothionein) and Ycf1 (a vacuolar glutathione S-conjugate pump), proteins involved with protection against cadmium. Furthermore, when sod1 cells were exposed to cadmium, the ratio glutathione oxidized/glutathione reduced did not increase as expected. We propose that a high level of metallothionein expression would relieve glutathione under cadmium stress, while an increased level of Ycf1 expression would favor compartmentalization of this metal into the vacuole. Both conditions would reduce the level of glutathione-cadmium complex in cytosol, contributing to the high capacity of absorbing cadmium by the sod1 strain. Previous results showed that the glutathione-cadmium complex regulates cadmium uptake. These results indicate that, even indirectly, metallothionein also regulates cadmium transport.

Factors involved with cadmium absorption by a wild-type strain of Saccharomyces cerevisiae

At the concentration used in this work (10 ppm), cadmium was efficiently removed from the environment by stationary yeast cells. While exponential phase cells showed low capacity of cadmium absorption, stationary cells removed 97 percent of the original metal in 24 hours. Total cadmium absorption shown by dry cells was lower than that of fresh ones, although both cells removed 50 percent of metal during the first hour of treatment. We also verified that only viable cells were capable of absorbing cadmium. Independently of the growth phase, cells showed high tolerance to 10 ppm CdSO4 and about 80 percent of cells remained viable after 24 hours exposure to cadmium. However, when stationary phase cells were previously dehydrated and then exposed to cadmium, they exhibited poor survival. By using an oxidation-dependent fluorescent probe, we observed that, once absorbed by cells, cadmium increases the intracellular level of oxidation, which may be responsible for its toxic effect. Crude extracts from stationary phase cells exposed to cadmium showed a 10-fold increase in fluorescence, while extracts from cells of exponential phase did not increase in fluorescence. Dry cells treated with the metal showed a high increase in fluorescence, mainly caused by dehydration.