Role of the yeast multidrug transporter Qdr2 in cation homeostasis and the oxidative stress response

1 1 2 Role of the yeast multidrug transporter Qdr2 in cation homeostasis and the oxidative 3 stress response. 4 5 Gabino RíosÁ, Marc Cabedo, Baltasar Rull, Lynne Yenush, Ramón Serrano and José M. 6 Mulet. 7 8 Instituto de Biología Molecular y Celular de Plantas (IBMCP), Universitat Politècnica 9 de València-CSIC, ES-46022 Valencia, Spain. 10 ÁPresent address: Instituto Valenciano de Investigaciones Agrarias (IVIA), carretera 11 Moncada-Náquera km 4.5, ES-46113 Moncada, Valencia, Spain. 12 13 14 Correspondence: José M. Mulet, 15 IBMCP 16 Universitat Politècnica de València, 17 Camino de Vera S/N 18 46022 Valencia (Spain) 19 Tel.:+34 96 3877775; fax: +34 96 3877859; 20 e-mail: [email protected] 21 22 Keywords: Ion transport, copper homeostasis, oxidative stress. 23 24 Running title: Role of Qdr2 in copper homeostasis and oxidative stress. 25 2 1 Abstract: 2 3 We have identified QDR2 in a screening for genes able to confer tolerance to sodium 4 and/or lithium stress upon overexpression. Qdr2 is a multidrug transporter of the major 5 facilitator superfamily, originally described for its ability to transport the antimalarial 6 drug quinidine and the herbicide barban. In order to identify its physiological substrate, 7 we have screened for phenotypes dependent on QDR2 and found that Qdr2 is able to 8 transport monovalent and divalent cations with poor selectivity, as shown by growth 9 tests and the determination of internal cation content. Moreover, strains overexpressing 10 or lacking QDR2 also exhibit phenotypes when reactive oxygen species producing 11 agents, such as hydrogen peroxide or menadione, were added to the growth medium. 12 We have also found that the presence of copper and hydrogen peroxide repress the 13 expression of QDR2. In addition, the copper uptake of a qdr2 mutant strain is similar to 14 a wild type, but the extrusion is clearly impaired. Based on our results, we propose that 15 free divalent copper is the main physiological substrate of Qdr2. As copper is a 16 substrate for several redox reactions that occur within the cytoplasm, this function in 17 copper homeostasis explains its role in the oxidative stress response. 18 19 3 Introduction: 1 2 The yeast overexpression approach has been a powerful technique to identify the genes 3 defining the molecular mechanisms underlying ion homeostasis in yeast (reviewed in 4 Arino et al., 2010). This technique has allowed the identification of the HAL genes, that 5 comprise regulators of potassium transport such as HAL1 (Gaxiola et al ., 1992) and 6 HAL3 (Ferrando et al., 1995), the HAL4 and HAL5 protein kinases (Mulet et al., 1999), 7 targets of ion toxicity as HAL2 (Murguía et al., 1995) and the HAL6-10 transcription 8 factors (Mendizabal et al., 1998), among them the calcineurin dependent transcription 9 factor CRZ1/HAL8/TCN1 (Matheos et al., 1997; Stathopoulos and Cyert, 1997). This 10 technique has proven to be very powerful to identify genes encoding for soluble 11 proteins, but has been less successful in identifying genes encoding transporters or 12 membrane proteins in general. This could be due to some technical problems as genes 13 encoding membrane proteins are usually under-represented in cDNA or genomic 14 libraries (our unpublished observations). The main transporters determining ion 15 homeostasis in Sa c c h a r o my c es c er ev i si a e are the proton pump ATPase Pma1 (Serrano 16 et al., 1986), responsible for the creation of the proton gradient, and the high affinity 17 potassium transport system encoded by the TRK1 and TRK2 genes (Gaber et al., 1988). 18 This system is responsible of maintaining the internal content of potassium around 10019 200 mM independently of the potassium concentration in the medium, and therefore is 20 the main consumer of the membrane potential generated by Pma1 (Madrid et al., 1998). 21 Sodium and lithium are toxic for Sa c c h a r o my c es c er ev i si a e. The main protein 22 responsible for extrusion of these toxic cations from the cytoplasm is Ena1 (Haro et al., 23 1991). In addition, the plasma membrane sodium/proton antiporter Nha1 (Prior et al., 24 1996; Kinclova-Zimmermannova et al. 2006) participates in sodium extrusion at acidic 25 pH and the sodium/proton antiporter Nhx1 localized in the prevacuolar compartment is 26 the major transporter involved in sodium compartimentalization (Nass and Rao, 1998). 27 28 Even with this apparent negative selection against membrane proteins, in a screening for 29 yeast genes able to confer salt tolerance upon overexpression, we have identified QDR2, 30 a multidrug resistance gene belonging to the major facilitator superfamily (MFS) 31 (Goffeau et al., 1997). We isolated QDR2 in our screening based on its ability to 32 improve growth under sodium stress. MFS transporters are ubiquitously present in 33 eukaryote and bacterial genomes, and can function as proton-gradient coupled 34 antiporters, uniporters or symporters (Pao et al., 1998). In most cases the multidrug 35 resistance family encodes transport systems which drive the extrusion of hydrophobic 36 molecules, most of them not present in the natural environment of the organism. The 37 Qdr2 protein is localized in the plasma membrane and sequence prediction indicates that 38 it contains 12 transmembrane segments. The QDR2 gene belongs to the DHA1 family 39 and is not conserved in related yeasts such as Ashbya gossipii or Kluyveromices lactis 40 (Gbelska et al., 2006). Qdr2 was originally identified for its ability to confer tolerance 41 to the antimalarial drug quinidine and the herbicide barban (Vargas et al., 2004). A later 42 report indicated that Qdr2 can also transport the anticancer agents cisplatin and 43 bleomicin (Tenreiro et al., 2005). None of these molecules are present in the 44 environment, so the physiological function of Qdr2 remains to be determined. It has 45 been proposed that MFS transporters could also participate in ion homeostasis. 46 Specifically, it has been proposed that some MFS proteins may contribute to sodium 47 extrusion (Krulwich et al., 2005). Qdr2 has also been proposed to have a role in 48 potassium homeostasis (Vargas et al., 2007). In addition, the four identified substrates 49 for Qdr2 are positively charged at physiological pH, suggesting that the physiological 50 4 role of Qdr2 may be related to cation homeostasis. In these report, we present evidence 1 that Qdr2 is able to transport monovalent and divalent cations, including transition 2 metals, among them, copper. In the environment copper is usually found as Cu2+, owing 3 mainly to the fact that Cu+ is very insoluble and is oxidized by O2, and thus, its 4 bioavalibility is low. Extracellular copper is reduced by the Ftr1/2 iron reductase 5 system, then Cu+ is transported to the cytoplasm by Ctr1 (Puig and Thiele, 2002). 6 Copper is an essential micronutrient for yeast, as it is incorporated in the metallic core 7 of antioxidant enzymes, such as Sod1, and is also present in some subunits of the 8 mitochondrial cytochrome c oxidase (reviewed in Bleackley and MacGillivray, 2011). 9 Another feature of copper is that the redox pair of Cu+ and Cu2+, ranging from +0,2 to 10 +0,8 is extremely useful for biological reactions (Frausto da Silva and Williams, 2001) 11 but, on the other hand, these redox reactions can lead to the formation of hydroxyl 12 radicals through the Fenton reaction (Valko et al., 2005). Copper homeostasis should be 13 tightly controlled, as it can be very toxic due to unspecific binding to sulphur, oxygen 14 and imidazole ligands (Culotta, 2010). Our data indicates that Qdr2 extrudes divalent 15 copper. This is, to date, the first description of a yeast protein able to extrude copper. 16 Previous reports have shown that Qdr2 is able to transport non-physiological substrates, 17 or potassium under very particular conditions. Here we propose that copper is the main 18 physiological substrate of Qdr2. As copper is a substrate for some deleterious redox 19 reactions that can occur inside the cell, this role in copper homeostasis also relates Qdr2 20 to redox homeostasis. 21 5 1 Materials and Methods: 2 3 Yeast strains and culture conditions: 4 Standard methods for yeast culture and manipulation were used (Guthrie and Fink, 5 1991). The BY4741 strains lacking QDR2 or QDR1 were obtained from the Euroscarf 6 collection (Frankfurt, Germany). YPD medium contained 2% glucose, 2% peptone, and 7 1% yeast extract. SD medium (synthetic minimal medium) contained 2% glucose, 0.7% 8 yeast nitrogen base (Difco) without amino acids, 50 mM succinic acid adjusted to pH 9 5.5 with Tris, and the amino acids, purine and pyrimidine bases required by the strains. 10 Growth assays were performed on solid media by spotting serial dilutions of saturated 11 cultures onto plates with the indicated composition. The indicated salts were added at 12 the indicated concentration in each case, with the exception of H2O2, and menadione, 13 that were added after autoclaving. 14 15 Isolation of QDR2 and plasmid construction: 16 The screen for tolerance to sodium and lithium has been described previously (Mulet et 17 al., 1999). QDR2 was isolated from the genomic clone PM54 as a Bgl II fragment that 18 contained the full ORF YIL121w, comprising 1107 bp before the start codon and 299 19 bp after the stop codon, and subcloned into the Bam HI site of YEp351 (2 µm origin, 20 LEU2 marker) (Hill et al., 1986), provisionally named HAL11, but renamed QDR2 after 21 the publication of (Vargas et al., 2005). 22 23 For analysis of QDR2 expression using the Lac-Z reporter gene, we amplified 611 bp of 24 the promoter region of QDR2 with Primer Prom QDR2D XSVWUHDP¶-CTC AAG CTT 25 TCC CAC ATG ACG TGC AG; Hind III site underlined) and Primer Prom QDR2R 26 GRZQVWUHDP¶-CCC AAG CTT GCC ATC GTT GCA GTAC; EcoR I site underlined), 27 digested and ligated into the Hind III site of plasmid pYIp355 (ampicillin resistance in 28 bacteria and URA3 complementation in yeast; Myers et al, 1986). The resulting plasmid 29 was named JM214. 30 31 Measurement of Intracellular cation concentrations: 32 Cells were grown in YPD to an absorbance at 660 nm of 0.6 to 0.7, centrifuged for 5 33 min at 1.900 X g, resuspended at the same concentration in YPD containing the 34 indicated chemical at the indicated concentration and incubated at 30 ºC for 90 minutes. 35 Aliquots were taken, centrifuged in plastic tubes for 5 min at 2.000 rpm and 4 ºC and 36 washed twice with 10 ml of ice cold solution of 20 mM MgCl2. The cell pellets were 37 resuspended in 0.5 ml of 20 mM MgCl2. Ions were extracted by heating the cells for 15 38 min at 95 ºC. After centrifugation, aliquots of the supernatant were analyzed with an 39 atomic absorption spectrometer (SensAA) in flame emission mode. For the copper 40 extrusion assays strains were incubated with the indicated amounts of copper for 120 41 minutes. At that point aliquots were taken to determine the copper content at time 0 and 42 the rest of the culture was washed twice with 20 mM MgCl2 and transferred to fresh 43 YPD medium. Aliquots were taken at the indicated times and treated as explained 44 previously. Copper was measured in a plasma emission spectrophotometer (Shimadzu). 45 46 47 ȕ-Galactosidase assays 48 Plasmid JM214, digested with Nco I, was integrated by homologous recombination in 49 the URA3 locus of the BY4741 yeast strain. Three independent colonies were used for 50 6 analysis. Cultures were incubated for 1.5 h after addition of the mentioned chemical. ȕ - 1 Galactosidase activity was measured in permeated cells as described previously (Rios et 2 al.,1997). Units of activity were normalized to cell density. 3 4 5 6 7 7 1 Results: 2 3 Qdr2 confers tolerance to sodium and lithium 4 5 We have screened for yeast genes able to confer tolerance upon overexpression. In the 6 past, this strategy has been useful to identify determinants for ion homeostasis, such as 7 the HAL genes (Arino et al., 2010, and references within). This technique has also been 8 useful to screen for genes from other organisms, such as plants (Mulet et al., 2004; 9 Serrano et al., 2003). Despite the amount of published data, some identified genes 10 remain uncharacterized. We screened 200.000 independent colonies of yeast 11 transformed with an episomal plasmid containing Sa c c h a r omy c es c er ev i si a e genomic 12 fragments. A fragment containing QDR2 was isolated from four independent clones for 13 its ability to confer tolerance to lithium and sodium. Only QDR2 (YIL121w) was 14 complete in the 4 different clones. This gene shares 70% homology with QDR1 15 (YIL120w), which was also present in some of the isolated clones. Therefore, we 16 subcloned both and compared their ability to confer tolerance to sodium or lithium upon 17 overexpression. Only QDR2 was responsible for the salt tolerance phenotype, as 18 overexpression of QDR1 did not confer sodium or lithium tolerance (Fig. 1). The 19 original screening was performed in the RS16 genetic background (Gaxiola et al., 20 1992). In order to assess whether the phenotype was reproducible in different genetic 21 backgrounds, we transformed different yeast strains with the plasmid overexpressing 22 QDR2. We could reproduce the observed tolerance to sodium and lithium in W303-1A 23 (data not shown) and in BY4741 (Brachmann et al., 1998) (Fig. 2). The P-type ATPase 24 ENA1 is the main transporter responsible for sodium and lithium extrusion from the 25 cytoplasm in S. c er ev i si a e (Haro et al., 1991). This gene belongs to a family composed 26 by three or four members (depending on the strain) located in tandem in the yeast 27 genome. In order to determine whether the observed sodium and lithium tolerance could 28 be due to an indirect effect on ENA1, we transformed a SKY697 strain (Ferrando et al., 29 1995) which has a complete deletion of the four ENA genes. We could also observe 30 tolerance in this genetic background (data not shown). In addition overexpression of 31 QDR2 had no effect on ENA1 expression under normal conditions or after induction 32 with sodium or lithium (data not shown), so the sodium and lithium phenotype is 33 independent of the main extrusion pump for sodium and lithium, Ena1. 34 35 Qdr2 transports lithium, but is not essential for monovalent cation homeostasis. 36 37 After confirming that the salt tolerance phenotype was reproducible in different strains 38 and independent of ENA1, we investigated whether QDR2 function was essential for 39 tolerance to monovalent toxic cations by investigating the phenotypes of a qdr2 strain. 40 A qdr2 strain showed a very weak sensitivity phenotype when grown in the presence of 41 monovalent toxic cations (Fig. 2A). We also analyzed the qdr1 mutant strain under the 42 same conditions, but growth was similar to the wild type control strain in all conditions 43 assayed (data not shown). We also tried to understand the mechanism of tolerance 44 determined by QDR2. The most obvious explanation for the observed tolerance is that 45 Qdr2 is transporting toxic cations outside the cell. We grew different strains in medium 46 containing LiCl and our results indicate that cells lacking QDR2 accumulate more 47 lithium and cells overexpressing QDR2 accumulate less than control cells, indicating 48 that Qdr2 is transporting lithium (Fig. 2B). 49 50 8 Qdr2 has a role in divalent cation homeostasis. 1 2 In order to investigate the spectrum of cations transported by Qdr2, we tested other 3 toxic cations and we found phenotypes related to transition metals such as nickel, 4 manganese and copper. Overexpression of QDR2 confers tolerance to Ni2+ and Mn2+ 5 (Fig. 3A), although we could not observe any sensitivity in the mutant strain. Ion 6 content analysis showed small differences (data not shown). We could not observe any 7 clear phenotype upon overexpression of QDR2 in copper containing medium, but the 8 qdr2 mutant strain was very sensitive to this cation. This result suggests that Qdr2 has a 9 role in divalent cation extrusion (Fig. 3A). We also investigated copper content after a 10 ¶LQFXEDWLRQ,QWHUQDOFRQWHQWEHWZHHQZLOGW\SHDQGWKHVWUDLQRYHUH[SUHVVLQJ QDR2 11 was similar, confirming the observed phenotype that overexpression of QDR2 does not 12 confer tolerance, but the mutant strain accumulated about 50% more than copper than 13 the wild type (Fig. 2B). 14 15 16 We further investigated whether Qdr2 could contribute to homeostasis of essential 17 divalent cations, such as calcium or magnesium. We did not observe any difference in 18 growth in the presence of excess magnesium or calcium, or differences in internal 19 content (data not shown). These results do not discard that Qdr2 could have a role in 20 conditions with limiting calcium or magnesium. For this purpose, we compared the 21 growth of different strains in the presence of the divalent cation chelators Ethylene 22 diamine tetra-acetic acid (EDTA) or ethylene glycol tetra-acetic acid (EGTA). Under 23 these conditions the mutant strains showed better growth that wild type or strains 24 overexpressing QDR2, suggesting that Qdr2 could take part in calcium or magnesium 25 extrusion (Fig 3C). 26 27 Qdr2 can transport divalent cations inside the cell. 28 29 Interestingly, when we investigated the spectrum of divalent cations transported by 30 Qdr2, we found that some transition metals produced different results. We found that 31 strains defective for qdr2 were slightly tolerant to cadmium and cobalt. We did not 32 observe any phenotype upon overexpression of QDR2 (Fig. 4A). We performed most of 33 our experiments in rich media (YPD) that does not select for the plasmid. Under normal 34 conditions YEp351, a 2 micron derivative yeast episomal plasmid used in this study is 35 very stable (Hill et al., 1986). However, when this plasmid contains a gene whose 36 expression has some deleterious effect, a negative selection can occur, such that strains 37 that have lost the plasmid or express less of the inserted gene are selected. To test 38 whether the lack of phenotype in strains overexpressing QDR2 was due to a negative 39 selection, we used minimal SD media without leucine, to prevent the growth of yeast 40 colonies without plasmid. Under these conditions strains overexpressing QDR2 grow 41 less than control strains in the presence of cobalt, indicating that QDR2 overexpression 42 is deleterious under these growth conditions (Fig. 4B). To asses whether this effect 43 could be related to transport or whether it is an indirect effect, we measured the 44 accumulation of this cation in cells grown in the presence of cobalt. We observed that 45 the qdr2 mutant accumulates less cobalt that wild type control cells (Fig. 4C). 46 47 48 9 QDR2 expr ession is repressed by copper and by hydrogen peroxide 1 2 In order to investigate the regulation of QDR2, we constructed a plasmid containing the 3 Lac-Z reporter gene (Myers et al., 1986) expressed under the control of the QDR2 4 promoter. We have shown that Qdr2 is involved in monovalent and divalent cation 5 homeostasis, with poor selectivity. So first we tested changes in expression after 6 treatments with different toxic cations, but we did not observe any significant 7 differences, with the exception of copper, where we could observe an approximately 10 8 fold repression (Fig. 5B). This observation suggests that the physiological role of QDR2 9 is deleterious in the presence of copper, an apparent discrepancy with the fact that a 10 qdr2 mutant strain is very sensitive to copper. Monovalent copper is insoluble, so 11 copper is present in the medium as a divalent cation. Divalent copper is reduced in the 12 extracellular matrix by the Cu-Fe reductase Fre1. Monovalent copper is then transported 13 inside the cell by the high affinity transporters Ctr1 and Ctr2 (Dancis et al., 1994). 14 Besides being a micronutrient, intracellular Cu+ pools must be tightly controlled, as an 15 excess of this cation can lead to toxicity through the formation of oxygen radicals via de 16 Fenton reaction (Valko et al., 2005). Specifically, Cu+ can react with hydrogen peroxide 17 to produce the hydroxyl radical and Cu2+. Alternatively, Cu2+ could react with the 18 superoxide anion via the Haber-Weiss reaction to form molecular oxygen and Cu+. The 19 involvement of copper cations in these classical bioinorganic chemistry reactions could 20 provide a hint to understand the physiological role of Qdr2. In the presence of hydrogen 21 peroxide Cu+ will produce hydroxyl radicals, deleterious for the cell, and thus 22 compromising H2O2 detoxification by catalases or glutathione peroxidases. If Qdr2 is 23 extruding Cu2+ from the cell, this could increase the rate of Fenton reaction by 24 eliminating one of the products. If this hypothesis is correct, we would predict that Qdr2 25 would be deleterious in the presence of H2O2. As indicated in Fig. 5A, qdr2 strain 26 grows better than the wild type control strain and QDR2 expression is repressed in the 27 presence of H2O2 (Fig. 5B). Intracellular Cu2+ can also induce the formation of 28 molecular oxygen via the Haber-Weiss reaction, using the superoxide anion as a 29 substrate. If Qdr2 is extruding Cu2+ from the cytoplasm, the deleterious effect of Haber30 Weiss reaction will be diminished, as Qdr2 will eliminate the substrate from the 31 cytoplasm. We used menadione as a superoxide generator (Castro et al., 2008) and 32 found that overexpression of QDR2 confers tolerance to menadione (Fig. 5A), and we 33 did not observe a significant decrease in expression of QDR2 upon a treatment with 34 menadione (Fig. 5B). 35 36 37 Extrusion of copper depends on QDR2 38 39 We have found that copper is the only cation that regulates QDR2 expression and that a 40 qdr2 mutant strain is sensitive to copper. These results suggest that cytoplasmic Cu2+ 41 should be the most relevant physiological substrate of Qdr2. To confirm this hypothesis 42 we have measured copper uptake and copper extrusion in qdr2 mutants. A qdr2 mutant 43 accumulates more copper than its parental wild type, but uptake at short times is 44 undistinguishable, suggesting that uptake rate is similar and the difference is the 45 extrusion rate (Fig. 6A). To confirm this hypothesis we evaluated the copper extrusion 46 in qdr2 cells. 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