3). In the WT strain, YCF1 expression was clearly induced only at the highest Cd2+ concentration tested (400 μM), while PMR1 expression was not induced at 50 μM or 400 μM ( Fig. click here 3A and B). COD1, YVC1 and VCX1 gene expression also did not change significantly in response to Cd2+ presence. Interestingly, PMC1 was the only gene up-regulated at 50 μM Cd2+ in WT strain ( Fig. 3A and B). The cells harboring the YCF1 mutation had increased PMR1 expression after Cd2+ exposure, and a similar pattern was seen for YVC1 and COD1 ( Fig. 3C and D). In addition, in the ycf1Δ mutant, PMC1 up-regulation by Cd2+ was stronger than that observed in WT cells (p < 0.001 at both 50 and 400 μM).
In the pmr1Δ
strain, YCF1 exhibit a clear increase at 400 μM Cd2+ ( Fig. 3E and F). Moreover, PMC1, VCX1, YVC1 and COD1 were also induced by Cd2+ in this mutant, with PMC1 reaching expression levels comparable to that observed for YCF1 at 400 μM ( Fig. 3E and F). In the double mutant pmr1Δycf1Δ, Talazoparib mouse the up-regulation of PMC1, VCX1 and COD1 still persist, but YVC1 is no more induced after Cd2+ stress ( Fig. 3G and H). The early up-regulation of PMC1 at 50 μM Cd2+ in the WT strain as well the strong up-regulation in mutants lacking YCF1, points to the participation of Pmc1p in Cd2+ tolerance. Therefore, we hypothesized that the partial rescue of Cd2+ tolerance in the pmr1Δycf1Δ double mutant ( Fig. 1) could be related to differences in the basal PMC1 expression levels. In fact, its expression in cells lacking Pmr1p is at least 2.5 times higher than in WT cells, even without Cd2+ treatment
( Fig. 4). In ycf1Δ mutants, the basal PMC1 level is increased Carteolol HCl approximately 50%. Also, in pmr1Δ mutants, the basal YCF1 expression is also 70% higher than WT ( Fig. 4). In S. cerevisiae, the detoxification of Cd2+ ions is associated mainly with Ycf1p activity. However, several published studies suggested that additional pathways can help yeast cells to cope with Cd2+ toxicity. For example, Pmr1p participates in Cd2+ tolerance by a mechanism involving the secretory pathway ( Lauer-Júnior et al., 2008). In this work, we showed that in BY4741 the inactivation of PMR1 has a stronger effect upon the over-time profile of Cd2+ uptake ( Fig. 2). In fact, WT cells accumulate Cd2+ for 2 h and then release into the medium some of the ions that previously incorporated; this event seems to allow a new round of Cd2+ uptake. In mutants lacking PMR1, this Cd2+ export capacity is lost; cells accumulate increasing Cd2+ concentrations ( Fig. 2), which confirms that Pmr1p shuttles Cd2+ into the secretory route. Despite this progressive Cd2+ accumulation, the contribution of Pmr1p to Cd2+ tolerance seems to be secondary compared to Ycf1p, since pmr1Δ was relatively insensitive to Cd2+ ( Fig.