3A and B). The inactivation of PMR1 also delays the initial Cd2+ capture compared to WT cells. In this sense, pmr1Δ mutants have depletion of Ca2+ in secretory compartments, which stimulates the initial rate of Ca2+ influx through Cch1p/Mid1p, a cell membrane high affinity Ca2+-channel ( Locke et al., 2000 and Kellermayer et al., 2003). This phenomenon does not occur in WT cells; moreover, it is not related to increased expression of Cch1p/Mid1p neither with its relocation from internal compartments

to the cell surface ( Locke et al., 2000). Knowing that Cd2+ and Ca2+ can compete for this channel ( Gardarin et al., 2010), we hypothesized that the high-affinity of Cch1p/Mid1p by Ca2+ ions, as well some kind of intracellular Doxorubicin cell line signaling that improves this affinity, could favor the early uptake of Ca2+ instead of Cd2+. In this sense, it was demonstrated that Cch1p/Mid1p activity is influenced by proteins of intracellular signaling pathways as calcineurin and the MAP kinases Mpk1p and Bck1p ( Bonilla and Cunningham, 2003). With time, competition between

Ca2+ and Cd2+ should be reduced due to alteration in the proportional concentration of these cations and, in turn, Cd2+ uptake becomes more effective. A set of kinetic experiments are necessary to confirm this hypothesis. The amount of Cd2+ incorporated by the ycf1Δ strain does not vary greatly GW-572016 mw over time ( Fig. 2), possibly because the metal accumulates in the cytosol, forming Cd-[GS]2 complexes that has a feedback negative effect upon Cd2+ uptake ( Gomes et al., 2002), and because these complexes are not substrates for Pmr1p, which transports only why divalent metals ( Sorin et al., 1997 and Missiaen et al., 2007). In the expression analysis, we observed that YCF1 and PMC1 were the genes whose expression was more affected by Cd2+ ( Fig. 3A and H). Interestingly, PMC1 was activated earlier than YCF1, since it was the only gene up-regulated at 50 μM Cd2+ in the WT strain, and was even higher in ycf1Δ cells ( Fig. 3A–D). PMC1 encodes

a vacuolar Ca2+ transporter not essential for viability under normal growth conditions; however, it plays an essential role in yeast tolerance to high Ca2+ stress ( Cunningham and Fink, 1994 and Miseta et al., 1999). The ionic similarities between Ca2+ and Cd2+, and the prominent induction of PMC1 in response to Cd2+ in the ycf1Δ strain ( Fig. 3C and D), allow us to infer that Pmc1p can help yeast cells cope with Cd2+ toxicity, although we have not detected great sensitivity to Cd2+ in pmc1Δ cells (data not shown). In addition, strains lacking functional Ycf1p can also activate the PMR1 gene as an accessory pathway to remove Cd2+ from the cytosol ( Fig. 3C and D). A remarkable observation from this work was that deletion of the PMR1 gene can overcome the Cd2+ sensitivity produced by the absence of Ycf1p, as demonstrated by the pmr1Δycf1Δ cells ( Fig. 1).