In Saccharomyces cerevisiae, bis(glutathionato)cadmium (Cd-[GS]2) is removed

from the cytosol to the vacuole by specific proteins such as the glutathione-conjugated transporter Ycf1p ( Li et al., 1997), an ATP-binding cassette protein analogous to the human multidrug resistance associated protein 1 (MRP1) ( Szczypka et al., 1994). Upon Cd2+ stress, selleck inhibitor YCF1 is regulated by Yap1p and by GSH availability ( Wemmie et al., 1994 and Mielniczki-Pereira et al., 2008). At the post-translational level, Ycf1p activity is controlled by phosphorylation ( Eraso et al., 2004 and Paumi et al., 2008). In addition to Ycf1p, the Ca2+- pump Pmr1p located at Golgi membrane, promotes Cd2+ detoxification by a mechanism associated with the secretory pathway ( Missiaen et al., 2007 and Lauer-Júnior et al., 2008). Ca2+ is an essential element that has a central role as intracellular cell messenger in eukaryotes, regulating a broad variety of processes like morphogenesis and proliferation (Chattopadhyay and Brown, 2000 and Schaub and Heizmann, 2008). In aqueous solution, Cd2+ and Ca2+ ions have similar ionic radii; consequently, several proteins containing Ca2+ binding motifs can also bind Cd2+ (Chao et al., 1990, Akiyama et al., 1990 and Liu and Templeton, learn more 2007). Considering that Pmr1p is the major Ca2+ ATPase of S. cerevisiae ( Marchi et al., 1999), its activity in Cd2+ detoxification may alter the Ca2+ intracellular levels and,

therefore, the function of other Ca2+-carriers found in these cells. Multiple Ca2+ transporters have been identified in S. cerevisiae, including Pmr1p and the vacuolar transporters Ca2+-ATPase Pmc1p, Ca2+/H+ exchanger Vcx1p/Hum1p, and Yvc1p ionic channel ( Bonilla and Cunningham, 2002), which respond to the calmodulin/calcineurin-signaling pathway and are controlled by the transcription factor complex Tcn1p/Crz1p ( Stathopoulos and Cyert, 1997 and Matheos et al., 1997). In addition, the endoplasmic reticulum (ER) ATPase Cod1p/Spf1p also contributes to maintenance of Ca2+ levels in yeast ( Cronin et al.,

2002). In this work, we investigate the relative contribution of Ycf1p and Pmr1p to Cd2+ tolerance in S. cerevisiae. We performed cytotoxic assays and analyses of Cd2+ this website content in single and double mutants for these proteins. Additionally, we analyzed the expression of yeast genes coding intracellular Ca2+-transporters (PMR1, PMC1, VCX1, YVC1, COD1) after Cd2+ exposure. The strains of S. cerevisiae used in this work are isogenic with wild-type (WT) BY4741 ( Table 1). They were routineraly maintained in YEPD (1% yeast extract, 2% peptone, 2% glucose) and pre-inoculated in SC complete medium ( Burke et al., 2000) before experimental procedures. The estimated Ca2+ concentration of SC medium is about 0.9 mM ( Difco™ & BBL™ Manual, 2nd Edition). The Escherichia coli strain XL1-Blue ( Table 1) was used as a recipient for cloning procedures and was grown in LB medium (1% tryptone, 0.5% yeast extract, 1% NaCl).