The PacC-family protein Rim101 prevents selenite toxicity in Saccharomyces cerevisiae by controlling vacuolar acidification

Fungal Genetics and Biology 71 (2014) 76–85

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The PacC-family protein Rim101 prevents selenite toxicity in Saccharomyces cerevisiae by controlling vacuolar acidification Maria Pérez-Sampietro, Enrique Herrero ⇑ Departament de Ciències Mèdiques Bàsiques, Universitat de Lleida, IRBLleida, Edifici Biomedicina I, Rovira Roure 198, 25198-Lleida, Spain

a r t i c l e

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Article history: Received 5 August 2014 Accepted 6 September 2014 Available online 17 September 2014 Keywords: ESCRT machinery Oxidative stress PacC family Rim101 Selenite Vacuole

a b s t r a c t Saccharomyces cerevisiae Rim101 is a member of the fungal PacC family of transcription factors involved in the response to alkaline pH stress. Further studies have also implicated Rim101 in the responses to other stresses, and have shown its genetic interaction with the iron deprivation-responsive factor Aft1. The present study shows that the absence of Rim101 leads to hypersensitivity to oxidants such as t-butyl hydroperoxide and diamide, and also to the prooxidant agent selenite. The protective role of Rim101 against selenite requires the sensing complex component Rim8, the ESCRT-I/II/III complexes and the Rim13 protease involved in proteolytic activation of Rim101. The Nrg1 transcriptional repressor is a downstream effector of Rim101 in this response to selenite, as occurs in the responses to alkaline pH, Na+ and Li+ stresses. Deletion of RIM101 causes downregulation of the vacuolar ATPase genes VMA2 and VMA4, which becomes accentuated compared to wild type cells upon selenite stress, and activation of the Rim101 protein prevents inhibition of vacuolar acidification caused by selenite. These observations therefore support a role of Rim101 in modulation of vacuolar acidity necessary for selenite detoxification. In addition, a parallel Rim101-independent pathway requiring the complete ESCRT machinery (including the ESCRT-0 complex) also participates in protection against selenite. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Responses of Saccharomyces cerevisiae yeast cells to environmental challenges are mediated by a network of interacting signalling pathways (Granek et al., 2011). A specific regulator carrying out a central function in a particular response pathway may also participate in additional pathways modulating other responses. Thus, Rim101, a member of the fungal PacC family of C2H2 zinc finger transcriptional regulators, modulates the response of yeast cells to alkaline pH (Lamb et al., 2001; Peñalva et al., 2008). In addition, it is involved in sporulation and invasive growth (Li and Mitchell, 1997), protection against Na+ and Li+ toxicity (Lamb and Mitchell, 2003), cell wall assembly (Gómez et al., 2009), protection against weak organic acids (Mira et al., 2009) and regulation of calcium homeostasis (Zhao et al., 2013). In S. cerevisiae Rim101 acts as a transcriptional repressor that inhibits expression of other transcriptional repressor genes such as NRG1 (in alkaline pH protection and ion homeostasis) or SMP1 (in sporulation and invasive Abbreviations: CCA, concanamycin A; GSH, reduced glutathione; NAC, N-acetyl cysteine; t-BOOH, tert-butyl hydroperoxide. ⇑ Corresponding author. E-mail address: [email protected] (E. Herrero). http://dx.doi.org/10.1016/j.fgb.2014.09.001 1087-1845/Ó 2014 Elsevier Inc. All rights reserved.

growth), eventually acting as a positive regulator of such downstream functions (Lamb and Mitchell, 2003). Activation of Rim101 (or its fungal PacC homologues) requires the removal of its C-terminal domain, which in S. cerevisiae is done by the calpain-like protease Rim13 with the participation of other upstream signal transducers such as the b-arrestin homologue Rim8 (component of the plasma membrane-associated sensing complex) and the scaffold protein Rim20 (Peñalva et al., 2008; Maeda, 2012). Proteolytic activation of Rim101 is functionally and physically connected with endocytic vesicles, requiring the participation of the ESCRT-I, -II and -III complexes (Xu et al., 2004; Subramanian et al., 2012). These complexes, together with the ESCRT-0 complex, were originally characterized as involved in the formation of multivesicular bodies for delivering ubiquitinated proteins to be degraded at the vacuole or lysosome, although later studies demonstrated the role of the ESCRT machinery in additional cellular processes (Henne et al., 2011; Rusten et al., 2011). In the case of yeast, these include Rim101 signalling. In the transcriptional regulation of some genes in response to alkaline pH, Rim101 acts in parallel to the calcineurin/Crz1 pathway (essentially involved in the response to high levels of calcium) and the Snf1 pathway (implicated in metabolic shift under glucose depletion conditions) (Viladevall et al., 2004; Platara et al., 2006;

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M. Pérez-Sampietro, E. Herrero / Fungal Genetics and Biology 71 (2014) 76–85 Table 1 Strains employed in this study. Strain

Genotype

Source and comments

W303-1A W303-1B MML348 MML1740 MML1758 MML1781 MML1783 MML1785 MML1787 MML1872 MML1874 MML1876 MML1879 MML1881 MML1925 MML1928 MML1929 MML1931 MML1932 MML1934 MML1935 MML1937 MML1938 MML1942 MML1962 MML1973 AMP1591

MATa ura3-1ade2-1 leu2-3,112 trp1-1 his3-11,15 can1-1 MATa ura3-1ade2-1 leu2-3,112 trp1-1 his3-11,15 can1-1 W303-1A aft1-D5::URA3 W303-1A rim101::natMX4 W303-1A aft1-D5::URA3 rim101::natMX4 W303-1A nrg1::kanMX4 W303-1A smp1::kanMX4 W303-1A rim101::natMX4 smp1::kanMX4 W303-1A rim101::natMX4 nrg1::kanMX4 W303-1A vma2::natMX4 W303-1A rim8::natMX4 W303-1A rim13::natMX4 W303-1A rim101::natMX4 rim13::natMX4 W303-1A rim13::natMX4 nrg1::kanMX4 W303-1B rim101::natMX4 rim8::natMX4 W303-1A snf7::kanMX4 W303-1A rim101::kanMX4 snf7::kanMX4 W303-1A vps27::kanMX4 W303-1A rim101::natMX4 vps27::kanMX4 W303-1A vps25::kanMX4 W303-1A rim101::natMX4 vps25::kanMX4 W303-1A vps28::kanMX4 W303-1A rim101::natMX4 vps28::kanMX4 W303-1B rim101::natMX4 snf7::kanMX4 nrg1::kanMX4 W303-1A vps27::kanMX4 vma2::natMX4 W303-1A vps28::kanMX4 vma2::natMX4 MATa RIM101-HA2 IME2

Wild type Wild type From our laboratory This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work This work From A. Mitchell (Li and Mitchell, 1997)

Ruiz et al., 2008). This transcriptional response involves upregulation of the expression, among others, of some genes of the iron regulon (Lamb et al., 2001; Viladevall et al., 2004). The genes of this regulon are under the positive control of the Aft1 transcriptional factor in iron-starvation conditions, and regulate uptake of external iron across the cell envelope (in the soluble ferrous form) and redistribution of intracellular iron stores in such conditions (Kaplan and Kaplan, 2009). Upregulation of the iron regulon at alkaline pH would be an indicator that alkalinisation of the medium may disturb cellular iron homeostasis, in accordance with the fact that null mutants in the AFT1 and FET3 genes (the last one coding for a plasma membrane high-affinity iron cotransporter) are hypersensitive to alkaline pH conditions (Serrano et al., 2004). Upregulation of some iron regulon genes in alkaline conditions is abrogated in a rim101 mutant, while expression of other genes of this regulon is upregulated in the absence of Rim101 (Lamb et al., 2001; Lamb and Mitchell, 2003; Barwell et al., 2005; Berthelet et al., 2010), indicating a complex relationship between Rim101 and transcription of the iron regulon at alkaline pH. A genetic approach has demonstrated the interaction between the Rim101-mediated and the iron regulons even during growth in mild acidic to neutral pH medium (Berthelet et al., 2010). Thus, a double Drim101Daft1 mutant is synthetically sick for growth and this phenotype is rescued by additional supplementation of iron to the medium. On the other hand, in addition to iron starvation or alkaline pH, the Aft1-mediated regulon is upregulated in other environmental conditions such DNA replication stress by hydroxyurea (Dubacq et al., 2006), heavy metal stress by zinc (Pagani et al., 2007), DNA damage by cisplatin (Kimura et al., 2007), or oxidant treatment (Castells-Roca et al., 2011). Selenite is toxic in S. cerevisiae cells after its conversion to selenide (Tarze et al., 2007), consequently promoting double-strand breaks, high mutagenicity rate, cell cycle arrest, depletion of reduced glutathione (GSH) and extensive protein oxidation (Pinson et al., 2000; Letavayová et al., 2008; Izquierdo et al., 2010; Mániková et al., 2012; Peyroche et al., 2012; Pérez-Sampietro et al., 2013). In yeast cells, heavy metals detoxification generally occurs by internalization of metal-GSH adducts into the vacuole in a process mediated by the vacuolar membrane ABC transporter

Ycf1 (Wysocki and Tamas, 2010). However, in the case of selenium forms, the absence of Ycf1 causes cell resistance to selenite (Pinson et al., 2000) while overexpression of the transporter leads to hypersensitivity to that compound (Lazard et al., 2011), in this last condition probably due to depletion of cytosolic GSH and consequent redox imbalance. On the other hand, a genome-wide screen of yeast mutants for selenite hypersensitivity has shown that individual mutants in different subunits of the vacuolar H+-ATPase (V-ATPase) complex have reduced viability in the presence of this agent (Mániková et al., 2012), pointing to an essential role of vacuole acidification in selenite detoxification. Transcriptomic studies (Salin et al., 2008) have demonstrated that selenite treatment causes upregulation of genes of the iron regulon in addition to genes participating in stress and protein degradation responses. This, together with (i) the fact that Daft1 mutant cells are hypersensitive to selenite (Pérez-Sampietro et al., 2013), and (ii) the above mentioned interactions between Aft1 and Rim101, prompted us to analyse a possible role of the Rim101-mediated pathway in protection against selenite stress. In the present study we show that Rim101 in fact is involved in protection against selenite together with the ESCRT machinery, and that this protection involves regulation of vacuolar acidification, therefore relating Rim101 with vacuolar functions.

2. Materials and methods 2.1. Strains, plasmids and growth conditions The strains employed in this study (W303 genetic background unless otherwise indicated) are listed in Table 1. Plasmid pRS315-Rim101(1-531) (a gift from Olivier Vincent, Instituto de Investigaciones Biomédicas, CSIC) derives from vector pRS315 and expresses a constitutively active truncated version of Rim101 (aa 1–531) from the own gene promoter. Plasmid pMM1101 was obtained by cloning the GLR1 ORF plus promoter and terminator sequences in the polylinker of the multicopy plasmid YEplac181 (Gietz and Sugino, 1988). YPD (1% yeast extract, 2% peptone, 2% glucose) or SC medium (Sherman, 2002) were usually employed for S. cerevisiae cell

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growth. Media were solidified with 2% agar. Sodium selenite (Sigma) or other compounds were added at the concentrations indicated in each case. To examine calcium sensitivity, plates of solid modified SD medium with the desired CaCl2 concentration were prepared as described in Demaegd et al. (2013). Cells were grown at 30 °C, with shaking in the case of liquid cultures. 2.2. Genetic methods Standard protocols were used for DNA manipulations and transformation of yeast cells. Single null mutants were generated using the short-flanking homology approach after PCR amplification of the natMX4 (Goldstein and McCusker, 1999) or the kanMX4 cassettes (Wach et al., 1994) and selection for nourseothricin or geneticin resistance respectively. Disruptions were confirmed by PCR analysis. Multiple mutants were obtained by crossing the parental mutant strains, followed by diploid sporulation, tetrad analysis, and selection of the mutant combinations (Sherman, 2002). 2.3. Determination of growth sensitivities Sensitivity to selenite or other agents was determined in plate growth assays by spotting serial 1:5 dilutions of exponential cultures onto plates with solid medium containing the respective agent, and recording growth after 2–4 days of incubation at 30 °C. Growth of several strains in liquid medium under parallel separate treatments was automatically recorded (optical density at 600 nm) at 1-h intervals, using shaken microtiter plates sealed with oxygen-permeable plastic sheets, in a PowerWave XS (Biotek) apparatus at controlled temperature. 2.4. Northern blot analyses RNA isolation and electrophoresis, probe labelling with digoxigenin, hybridization, and signal detection were done as described previously (Bellí et al., 1998). Gene probes were generated by PCR from genomic DNA, using oligonucleotides designed to amplify internal open reading frame sequences. SNR19 mRNA was employed as loading control. Blot signals were quantified with the ChemiDoc™ MP Imaging System (Bio-Rad) software, and background levels were processed as in Castells-Roca et al. (2011). 2.5. Immunoblot analysis of Rim101 Cells of strain AMP1591 carrying a Rim101-HA version of the protein (Li and Mitchell, 1997) were grown exponentially in synthetic SC medium with 0.1 M HEPES titrated to pH 3.5. Selenite or NaCl stresses were applied by adding the adequate amount of the respective agent. For immediate shifting to pH 7.0 KOH was added to 70 mM final concentration. Samples were taken when required and subjected to rapid boiling through the heat inactivation/alkaline treatment method (Orlova et al., 2008). Protein extracts were separated by SDS–PAGE and analysed by immunoblotting with monoclonal anti-HA antibodies (Roche) at a 1:5000 dilution. Chemoluminiscent Clarity™ Western ECL Substrate (BioRad) was employed for signal detection in the ChemiDoc™ MP Imaging System equipment.

containing 100 mM HEPES buffer pH 7.6, and freshly prepared 400 lM quinacrine (Sigma). The suspension was incubated with mild rotation during 8 min at 30 °C, followed by 5 min on ice. After centrifugation at 4 °C, the cell pellet was resuspended in 100 mM HEPES buffer pH 7.6 plus 2% glucose, washed twice with the same cold buffer and resuspended in 0.1 ml of the same solution. After mixing identical volumes of the processed cell suspension and a 1% low-melting agarose solution, cells were observed with an Olimpus BZ51 fluorescence microscope, using the U-MNUA3 filter. 3. Results 3.1. Rim101 is required for protection against selenite toxicity The hypersensitivity of Daft1 cells to selenite is rescued by addition of iron to the medium (Pérez-Sampietro et al., 2013). Given the interactions between the Aft1- and Rim101-mediated regulons (see Section 1) and the induction of iron regulon genes by selenite (Salin et al., 2008), we determined the sensitivity of Drim101 cells to this agent in normal and high-iron conditions. As occurs with Daft1 cells, the Drim101 mutant was also hypersensitive to selenite, but in this case such phenotype was not substantially rescued by supplementation of additional iron to the medium (Fig. 1A), pointing to different roles of Aft1 and Rim101 in protection against selenite toxicity. As already described (Berthelet et al., 2010), the double Drim101Daft1 mutant was synthetically sick, and displayed an extreme sensitivity to selenite that was only very moderately counteracted by iron supplementation to the medium (Fig. 1A). In summary, these results indicate that Rim101 exerts a protective role against selenite that, contrary to the role of Aft1, is independent of iron homeostasis. Next, we tried to relate the previous growth observations under selenite treatment with the transcriptional activation of genes of the iron regulon in the same treatment conditions. With this objective, we analysed expression of several genes of the regulon by Northern blot, in selenite-treated cells for longer times compared with the previous transcriptomic study (Salin et al., 2008). Three of the analysed genes (FIT3, CTH2 and ARN2) were in fact significantly induced over basal levels during the 6 h of treatment in the wild type strain background (Fig. 1B). On the contrary, the two genes for the plasma membrane high-affinity iron transporter complex (FET3 and FTR1) were moderately downregulated upon treatment. Therefore, only a subset of the iron regulon genes becomes induced in selenite-treated cells, contrary to the situation under iron depletion (Kaplan and Kaplan, 2009). While induction of ARN2 and basal expression of FET3 and FTR1 depended on Aft1, selenite induction of FIT3 and CTH2 was independent of this factor (Fig. 1B). With respect to Rim101, this factor seemed to carry out a repressor role on basal expression of FIT3 and CTH2, but these two genes (as well as ARN2) were still upregulated under selenite treatment in a Drim101 mutant (Fig. 1B). These results point to other uncharacterized regulators of some iron regulon genes in conditions other than iron starvation, and confirm that the transcriptional role of Rim101 under selenite treatment differs from that of Aft1.

2.6. Vacuole staining with quinacrine

3.2. The Rim101-mediated protective response against selenite shares upstream and downstream components with the alkaline pH response

To visualize vacuoles using the fluorescent probe quinacrine, the protocol described in Perzov et al. (2002) was followed, with some modifications. Basically, samples of the corresponding culture at a concentration of about 3  107 cells/ml were kept on ice for 5 min, then 1 ml of cell suspension was centrifuged (9000g, 2 min) and the cell pellet was resuspended in 0.1 ml of SC medium

We next investigated whether the protective function of Rim101 against selenite depends on its repressor role on NRG1 or SMP1 expression. Neither the single Dnrg1 or Dsmp1 mutants nor the Drim101Dsmp1 mutant displayed additional sensitivity to selenite compared to wild type cells. However, deletion of NRG1 in a Drim101 mutant rescued the selenite sensitivity phenotype of the

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Fig. 1. Rim101 protects against selenite toxicity in an iron-independent manner. (A) Exponential cultures of the following strains in YPD medium were serially diluted and spotted on YPD plates containing selenite: wild type (W303-1A), Daft1 (MML348), Drim101 (MML1740) and Daft1Drim101 (MML1758). Plates contained no additional iron (upper panels) or were added with 90 lM bathophenanthroline sulphonic acid plus 100 lM ferrous sulphate (lower panels). Growth was recorded after 2 days at 30 °C. (B) Northern blot analysis of expression of the indicated genes in wild type, Drim101 and Daft1 cells growing exponentially in YPD liquid medium and then treated with 2 mM selenite for different time periods. SNR19 was employed as loading control.

latter (Fig. 2A), as occurs with sensitivity to alkaline pH or to NaCl and LiCl (Lamb and Mitchell, 2003). This supports that the transcriptional repressor role of Nrg1 (although not that or Smp1) mediates the function of Rim101 in protection against selenite. To determine whether the sensing complex and the Rim13 protease, which are required for Rim101 activation in response to alkaline pH (Maeda, 2012), are also operating in the response to selenite stress, we determined the growth phenotype of the Drim8 and Drim13 mutants in the presence of this agent. Both mutants were hypersensitive to selenite (Fig. 2B), supporting that the complex involving Rim8 as well as the Rim13 protease participate in selenite sensing and Rim101 processing. While NRG1 deletion abolishes the sensitivity of Drim13 cells, cells lacking Rim101 plus Rim8 or Rim13 show additive sensitivity to the agent compared to the respective single mutants (Fig. 2B). This therefore points to some roles of Rim8 and Rim13 in protection against selenite that are independent of Rim101. We then determined whether such additive effect was also observed in the case of the reported protective role of Rim101 against NaCl stress (Lamb and Mitchell,

2003), and this was also the case. In fact, as expected the single Drim101, Drim8 and Drim13 mutants are more sensitive to 0.6 M NaCl than wild type cells, but the double Drim101Drim8 and Drim101Drim8 mutants display additivity (Fig. 2C). Once we determined that the Rim13 protease is required for protection against selenite, we then analysed whether this agent induces proteolytic processing of Rim101. Cells growing at pH 3.5 were treated with selenite or NaCl, or alternatively shifted to pH 7.0 as control. While the last treatment caused rapid and complete processing of Rim101 as expected, neither selenite nor NaCl caused additional proteolysis of Rim101 over the constitutive one, even after 2 h of treatment (Fig. 2D). Therefore, Rim13-mediated constitutive processing of Rim101 is sufficient to protect yeast cells against selenite or NaCl at high concentrations. 3.3. The ESCRT machinery is required for protection against selenite The ESCRT-I, -II and -III complexes are necessary to protect S. cerevisiae cells against alkaline pH and stress by calcium excess,

Fig. 2. Components of the Rim101 pathway participating in protection against selenite toxicity. (A) Exponential cultures of the following strains in YPD medium were serially diluted and spotted on YPD plates containing selenite: wild type (W303-1A), Drim101 (MML1740), Dsmp1 (MML1783), Dnrg1 (MML1781), Drim101Dsmp1 (MML1785) and Drim101Dnrg1 (MML1787). Growth was recorded after 3 days of incubation at 30 °C. (B) As in part (A) with the following strains: wild type, Drim101, Drim8 (MML1874), Drim101Drim8 (MML1925), Drim13 (MML1876), Drim101Drim13 (MML1879) and Drim13Dnrg1 (MML1881). (C) Cultures of the indicated strains were spotted on YPD plates containing NaCl. (D) Exponential cultures of AMP1591 cells (with the Rim101-HA construct) in SC medium with 0.1 M HEPES at pH 3.5 were treated with selenite or NaCl, or shifted to pH 7.0. At the indicated times, samples were taken for western blot analysis with anti-HA antibodies.

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and this protection involves the participation in the activation of the machinery for Rim101 proteolytic processing (Xu et al., 2004; Maeda, 2012; Subramanian et al., 2012; Zhao et al., 2013). We therefore examined whether strains with deletions in genes encoding components of ESCRT-I (Vps28), ESCRT-II (Vps25) or ESCRT-III (Snf7) were hypersensitive to selenite, extending the analysis also to ESCRT-0 (Vps27). In fact, this was the case, and the selenite sensitivity of the single mutants in any of the four ESCRT complexes was higher than that of the Drim101 mutant (Fig. 3A, note that the concentration employed is lower than in Fig. 2A and B), pointing to additional Rim101-independent roles of the ESCRT machinery in selenite detoxification. Such additional roles would also require the function of ESCRT-0. In accordance with those observations, double mutants carrying the Drim101 deletion plus a deletion in any of the four ESCRT genes showed an additive hypersensitivity phenotype. Additional deletion of NRG1 only very moderately rescued the hypersensitivity of a Drim101Dsnf7 mutant (Fig. 3A), allowing us to conclude that the Nrg1 repressor is not a significant effector of the putative Rim101-independent ESCRT-dependent protective functions. To confirm that Rim101 and the ESCRT machinery play both dependent and independent roles in selenite protection, a constitutively-active truncated form of Rim101 consisting of aa 1–513 (Xu and Mitchell, 2001) was expressed in the respective Drim101, ESCRT-0 and ESCRT-III mutants. Such Rim101 form bypasses the need of the ESCRT-I, -II and -III complexes for the activation of

the pathway. When it was expressed in the Dvps27 and Dsnf7 mutants, Rim101(1-531) restored only in part the wild type phenotype for selenite sensitivity (Fig. 3B), confirming the existence of Rim101-independent ESCRT-dependent functions acting in selenite protection. The Rim101-specific protection was more significant at low (1 mM) selenite concentration than at higher concentrations. ESCRT-0 functions have been discarded as participating in the Rim101 pathway against alkaline pH (Xu et al., 2004), in contrast with our results with selenite stress. We therefore extended this study to Na+ stress. In this case, the ESCRT-0 mutant displayed a wild type phenotype for Na+ sensitivity, and the Dnrg1 deletion completely rescued the hypersensitivity of a mutant defective in Rim101 and the ESCRT-III component Snf7 (Fig. 3A). Therefore, the same linear pathway as in the response to alkaline pH seems to be operating in the response to Na+ toxicity, which involves ESCRT-I to-III complexes, Rim101 and Nrg1. However, the Drim101 mutation is additive with a mutation in any of the ESCRT complexes, including ESCRT-0, with respect to sensitivity to Na+ (Fig. 3A). This points to a situation in which in the absence of the Rim101 pathway, an alternative Na+-detoxifying pathway that requires the complete ESCRT machinery is operating with partial efficiency. This pattern of sensitivity is similar to that of calcium stress, the defence against which requires ESCRT-I to III components (Zhao et al., 2013) but also ESCRT-0 when Rim101 is absent (Fig. S1).

Fig. 3. Additive role of Rim101 and the ESCRT machinery in protection against selenite toxicity. (A) Exponential cultures of the following strains in YPD liquid medium were serially diluted and spotted on YPD plates containing the indicated agents: wild type (W303-1A), Drim101 (MML1740), Dvps27 (MML1931), Dvps28 (MML1937), Dvps25 (MML1934), Dsnf7 (MML1928), Drim101Dvps27 (MML1932), Drim101Dvps28 (MML1938), Drim101Dvps25 (MML1935), Drim101Dsnf7 (MML1929) and Drim101Dsnf7Dnrg1 (MML1942). Growth was recorded after 2 days of incubation at 30 °C. (B) Exponential cultures in SC liquid medium of the indicated strains transformed with vector pRS315 or its derivative pRS315-Rim101(1-531) (Rim101⁄) were serially diluted and spotted on SC plates with selenite. Growth was recorded after 2 days of incubation at 30 °C.

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3.4. The role of Rim101 in protection against selenite is related to vacuolar functions Our next goal was to explain the complementary functions of Rim101 and ESCRT components in protection against selenite toxicity. Original observations suggesting that selenite was detoxified in yeast cells through vacuolar internalization (Pinson et al., 2000) were reinforced by the demonstration that S. cerevisiae cells mutated in subunits of the vacuolar V-ATPase were selenite-hypersensitive (Mániková et al., 2012). We confirmed that the treatment of wild type cells with concanamycin A (CCA), an inhibitor of the V-ATPase (Bowman et al., 2004), greatly increased sensitivity to selenite (Fig. 4A). On the other hand, the ESCRT-0 to III complexes are required for targeting V-ATPase proteins to the vacuolar membrane and consequent acidification of the organelle lumen (Raymond et al., 1992), and also for turnover of the vacuole (Michaillat and Mayer, 2013). This could explain the hypersensitivity of ESCRT-0 to III mutants to selenite. To confirm this hypothesis through genetic approaches, we tried to separately construct double mutants lacking Vma2 (V-ATPase subunit B) plus a protein of one of the ESCRT complexes. Tetrad analyses demonstrated synthetic lethality between the Dvma2 mutation on one hand and Dvps25 (ESCRT-II) or Dsnf7 (ESCRT-III) mutations on the other. On the contrary, the Dvma2Dvps27 (ESCRT-0) and Dvma2Dvps28 (ESCRT-I) mutants were viable, although especially in the case of

Fig. 4. The ESCRT machinery and vacuolar V-ATPase activity participate in selenite detoxification. (A) Growth of wild type (W303-1A), Drim101 (MML1740) and Dvma2 (MML1872) cells in YPD liquid medium without or with CCA (150 nM) and/ or selenite (2 mM) was automatically recorded. For each condition, optical density (600 nm) values after 20 h are represented, once normalised by the value corresponding to untreated wild type cells (mean ± s.d., three independent experiments). (B) Exponential cultures of the following strains in YPD liquid medium were serially diluted and spotted on YPD plates containing the indicated agents: wild type, Dvps27 (MML1931), Dvps28 (MML1937), Dvma2 (MML1872), Dvps27Dvma2 (MML1962) and Dvps28Dvma2 (MML1973). Growth was recorded after 2 and 4 days of incubation at 30 °C.

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Dvma2Dvps28 cells, they grew very slowly in ordinary YPD medium (Fig. 4B). Sensitivity of these two double mutants to selenite was tested. Disruption of the ESCRT-0 or -I complexes in cells lacking Vma2 did not increase the sensitivity to the agent compared with the single Dvma2 mutant (Fig. 4B), supporting that the ESCRT machinery participates in selenite detoxification through maintenance of vacuolar integrity and functions. Focusing on Rim101 functions in selenite detoxification, growth of Rim101-defective cells was compromised by CCA at a higher extent than in wild type cells, suggesting some constitutive defects in vacuolar functions in the mutant. In addition, growth levels of Drim101 cells in the presence of selenite (independently of the presence or not of CCA) were about the same as those of selenite-treated Dvma2 cells (Fig. 4A). Those results support that the hypersensitivity of Rim101-minus cells to selenite is mostly based on vacuolar function defects. Next, we analysed expression levels of several genes encoding for subunits of the V-ATPase (VMA1, VMA2, VMA4, VMA5, VMA8, VMA9, VPH1) in wild type and Drim101 cells before and after selenite treatment. In the case of VMA2 and VMA4, their expression was already downregulated in basal

Fig. 5. Expression of several V-ATPase genes is altered in the absence of Rim101. (A) Northern blot analysis of expression of the indicated genes in wild type (W303-1A) and Drim101 (MML1740) cells growing exponentially in YPD liquid medium, and then treated with 3 mM selenite for different times. SNR19 was employed as loading control. (B) Quantification of expression of VMA2 and VMA4 from Northern blot analyses, from exponential cultures in YPD medium treated with 3 mM selenite for the indicated times. The following strains were employed: wild type, Drim101 and Drim101Dnrg1 (MML1787). Expression of each gene was normalised by the loading control (SNR19), and then compared to the respective expression in untreated wild type cells, which was given the unit value. Bars correspond to the mean of four independent experiments (±s.d.).

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Fig. 6. Rim101 activity prevents vacuolar dysfunction upon selenite treatment. (A) Fluorescence microscopy of quinacrine-stained cells from cultures in SC medium of the MML1929 strain (Drim101Dsnf7) transformed with vector pRS315 or with the pRS315-Rim101(1-531) plasmid (RIM101⁄). Cultures were treated with 1 mM selenite before quinacrine staining. Untreated cells were employed as control. Left and right panels correspond to fluorescence and phase contrast images respectively. (B) Percentage of cells displaying quinacrine fluorescence concentrated in vacuoles or cytosol or dispersed over the entire cell. At least 200 stained cells were counted in each of three independent experiments done in the conditions described in part (A). Bars correspond to the mean of the three experiments (±s.d.).

conditions in Drim101 cells compared to wild type cells, and this downregulation was increased at advanced times after selenite addition (Fig. 5A and B). These results pointed to a regulatory role of Rim101 on expression of at least these two VMA genes in untreated and treated cells. To determine whether Nrg1 also participates in such regulatory role, we quantified expression of VMA2 and VMA4 in Drim101Dnrg1 cells compared to wild type and Drim101 controls. Although constitutive levels of expression of both genes in the double mutant were similar to those in Drim101 cells, downregulation of expression of VMA2 and VMA4 upon selenite treatment was not observed in Drim101Dnrg1 cells, contrary to cells lacking only RIM101 (Fig. 5B). Expression levels of both VMA genes after long selenite exposure times (6 h) were similar in wild type and Drim101Dnrg1 cells. This observation parallels the rescuing effect of the Dnrg1 mutation on the phenotype of growth hypersensitivity caused by the Drim101 mutation (Fig. 2A), and supports a transcriptional role of Nrg1 upon selenite treatment. In order to specifically analyse the possible function of Rim101 in vacuolar acidification and consequent selenite detoxification and discriminate it from the ESCRT roles, we treated a Drim101Dsnf7 mutant and its derivative expressing the constitutively active Rim101(1-531) form with low concentration of selenite. In these conditions activated Rim101 plays the central protective role (Fig. 3B). To analyse vacuolar acidification quinacrine was employed. This is a fluorescent dye that becomes sequestered into yeast vacuoles when an acidic environment is created at the organelle lumen by the V-ATPase, and is therefore employed as a reporter for vacuolar functionality (Umemoto et al., 1990; Perzov et al., 2002; Sambade et al., 2005). In the absence of selenite, both strains localised the fluorescence at the vacuole, and an intense fluorescence was also observed in prevacuolar endosome-like compartments adjacent to the vacuole (Fig. 6A). This fluorescence pattern is characteristic of ‘class E’ vacuolar mutants such as those defective in the Snf7 function (Raymond et al., 1992). Selenite treatment provoked fluorescence delocalisation in Drim101Dsnf7 cells, which was

rescued by expression of Rim101(1-531) (Fig. 6A). In the absence of Rim101 activity, only 10% of selenite-treated cells displayed exclusively vacuolar/prevacuolar quinacrine localisation, while the cell percentage rose to 60% when active Rim101 was expressed (Fig. 6B). Cytosolic fluorescence in quinacrine-stained cells has been reported before in several vma mutants (Umemoto et al., 1990), and we have also observed it in Dvma2 cells (Fig. S2). It may be related to acidification of the cytosol in vma mutant cells (Martínez-Muñoz and Kane, 2008). The above observations confirm that in selenitetreated cells Rim101 activity is important for the maintenance of vacuolar acidification. 3.5. Rim101 protects against disturbance of vacuolar acidification upon oxidative stress Selenite causes oxidative stress and depletion of reduced glutathione in yeast cells (Pinson et al., 2000; Lazard et al., 2011; Pérez-Sampietro et al., 2013). The reductant N-acetyl cysteine (NAC) rescued the hypersensitivity of Drim101 mutant cells to selenite (Fig. 7A), pointing to a relationship between the Rim101 protective role and selenite-induced oxidative stress. We confirmed that cells lacking Rim101 are also moderately hypersensitive to the oxidants tert-butyl hydroperoxide (t-BOOH) and diamide, and that this hypersensitivity of the mutant is rescued when a Dnrg1 mutation is additionally introduced (Fig. 7B). Also, alteration of the glutathione redox homeostasis towards a more reducing state due to overexpression of the glutathione reductase gene GLR1 rescues the selenite hypersensitivity of Drim101 cells (Fig. 7C), confirming the relationship of this phenotype with the prooxidant effect of selenite. Next, we determined if in the case of sensitivity to oxidants there exists an additive effect between mutations in Rim101 and in the ESCRT machinery, as occurs in selenite stress conditions. The experiments demonstrated that hypersensitivity to diamide of the Drim101 cells is totally reverted by the Rim101 truncated form, that the double Drim101Dsnf7 mutant is not more

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Fig. 7. Absence of Rim101 causes hypersensitivity upon oxidative stress. (A) Exponential cultures of wild type (W303-1A) and Drim101 (MML1740) cells in YPD liquid medium were serially diluted and spotted on YPD plates containing selenite and NAC. Growth was recorded after 2 days of incubation at 30 °C. (B) The strains tested in part (A) plus Dnrg1 (MML1781) and Drim101Dnrg1 (MML1787) strains were serially diluted and spotted on YPD plates containing t-BOOH or diamide. (C) Exponential cultures of wild type or Drim101 cells transformed with the YEplac181 vector or the pMM1101 plasmid overexpressing GLR1 were serially diluted and spotted on SC plates containing selenite. Growth was recorded after 3 days at 30 °C. (D) Exponential cultures of the following strains [transformed with vector pRS315 or its derivative pRS315-Rim101(1531) (Rim101⁄)] in SC liquid medium were serially diluted and spotted on SC plates containing the indicated agents: wild type, Drim101, Dsnf7 (MML1928) and rim101Dsnf7 (MML1929). Growth was recorded after 2 days of incubation at 30 °C. (E) Fluorescence microscopy of quinacrine-stained cells from cultures in SC medium of the MML1929 strain (Drim101Dsnf7) transformed with vector pRS315 or with the pRS315-Rim101(1-531) plasmid (RIM101⁄). Cultures were treated with 0.3 mM t-BOOH (8 h) before quinacrine staining. Untreated cells were employed as control. The percentage of cells displaying quinacrine fluorescence concentrated in vacuoles or cytosol or dispersed over the entire cell is shown. At least 200 stained cells were counted in each of three independent experiments. Bars correspond to the mean (±s.d.). (F) Quantification of the expression of VMA2 in Northern blot analyses of samples from wild type and Drim101 cells growing exponentially in YPD liquid medium, untreated or treated with 0.1 mM tBOOH. VMA2 expression was normalised by the loading control (SNR19), and then compared to the expression in untreated wild type cells, which was given the unit value. Bars correspond to the mean of four independent experiments (±s.d.).

sensitive to diamide than the single Drim101 mutant and that expression of Rim101(1-531) in this double mutant results in similar growth pattern in the presence of selenite as in wild type cells (Fig. 7D). That is, the role of Snf7 in protection against diamide (and probably also against other oxidants) seems to be circumscribed to its participation in the Rim101 pathway. Mutants in V-ATPase genes are hypersensitive to oxidative stress (Outten et al., 2005; Milgrom et al., 2007). Given our above observations, we employed quinacrine to analyse vacuolar acidification upon oxidative stress using the same strains as with selenite treatment in Fig. 6B. In the Rim101-minus strain, most of the cells in the population displayed delocalised cytosolic and vacuolar fluorescence upon treatment with t-BOOH, while expression of the Rim101(1-531) form allowed exclusive-vacuolar localisation of the fluorescence in about half of the cells (Fig. 7E). These results confirmed that Rim101 is required for maintaining appropriate vacuolar acidification upon oxidative stress, probably by regulating

expression of some V-ATPase genes. To test this hypothesis, we analysed by Northern blot the expression of the VMA2 gene, and in fact this was downregulated in the Drim101 mutant compared to wild type cells both in basal conditions and upon treatment with t-BOOH (Fig. 7F). 4. Discussion Yeast vma mutants chronically overaccumulate reactive oxygen species and are hypersensitive to external oxidants such as peroxides and diamide, indicating the importance of V-ATPase functions for protection against oxidants (Milgrom et al., 2007). However, the cellular bases of this phenomenon are not fully understood. Our observations that Rim101 regulates expression of the VMA2 gene, this expression being dramatically downregulated upon oxidative stress in Drim101 cells, and consequently, that vacuolar acidification is severely compromised in the absence of Rim101

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Fig. 8. Scheme of the involvement of Rim101 and the ESCRT complexes in protection of S. cerevisiae cells against selenite. See the text for details.

activity may explain the hypersensitivity of Drim101 cells to external oxidants. The constitutively active form of Rim101 completely bypasses the requirement of ESCRT-III for displaying wild type levels of sensitivity to diamide, supporting that this oxidant does not directly require morphologically normal and functional vacuoles for its detoxification. Also, transcriptome studies with the Drim101 mutant have not revealed a constitutive role of this transcription factor in regulating the expression of antioxidant genes (Lamb et al., 2001), which points toward an exclusive protecting effect of Rim101 through regulation of vacuolar functionality. Selenite generates reactive oxygen species and depletes intracellular levels of GSH (Pinson et al., 2000; Lazard et al., 2011; Pérez-Sampietro et al., 2013), thus provoking oxidative stress. This is therefore in accordance with the requirement of Rim101 activity for protection against selenite as described in this work. As occurs with other oxidants, selenite alters vacuolar acidification in the absence of active Rim101. Since V-ATPase mediated vacuole acidification is necessary for detoxification of selenite and heavy metals (Pinson et al., 2000; Li and Kane, 2009; Wysocki and Tamas, 2010; Lazard et al., 2011), this would explain the protective role of Rim101 against selenite. We thus propose that the prooxidant action of selenite downregulates expression of VMA genes in a Rim101-dependent manner, then interferes with vacuolar functions and consequently avoids its own detoxification. However, the above scheme becomes more complex when considering the participation of components of the Rim101 pathway. Thus, the sensitivity of mutants in the different ESCRT-0 to -III complexes is higher than that of the single Drim101 mutant and there is an additive effect between the absence of components of the ESCRT complexes and that of Rim101. Consequently, an ESCRT-independent constitutively active form of Rim101 almost totally restores the selenite sensitivity of ESCRT mutants to wild type levels at low concentrations of the agent but not at high ones. In contrast, and also consequent with the prooxidant role of selenite to explain its relationship with Rim101-mediated protection, the constitutively active form of Rim101 totally rescues the hypersensitivity of an ESCRT-III mutant to diamide, indicating that the exclusive function of the ESCRT machinery in protection against this oxidant is to activate Rim101. Thus, we propose a model (Fig. 8) in which, at moderate levels of selenite, activation of Rim101 is sufficient to protect against the agent. This protection

requires the canonical Rim101 pathway involving the sensing complex, the ESCRT-I to III complexes (but not ESCRT-0) and the Rim13 protease for Rim101 proteolysis. Background levels of the active Rim101 form seem to be sufficient for protection, as selenite does not induce further accumulation of the proteolysed active form of Rim101. The phenotypes of the respective mutants indicate that the Nrg1 repressor is the downstream effector of active Rim101, regulating expression of V-ATPase genes needed for selenite detoxification. This Rim101-Nrg1 pathway and its dependence on ESCRT components is similar to its involvement in yeast cells for protection against alkaline pH and Na+, Li+ and Ca2+ stresses (Lamb and Mitchell, 2003; Zhao et al., 2013). Interestingly, in the case of both selenite and Na+ stresses, we have observed additivity on selenite sensitivity between the absence of Rim101 and that of a component of the sensing complex (Rim8) or the Rim13 protease, which points to uncharacterised Rim101-independent functions of Rim8 and Rim13 in such protection. At higher concentrations of selenite (Fig. 8), cell protection would not be as dependent on the Rim101 pathway, but would still require the complete ESCRT machinery (in this case including ESCRT-0), probably for adequate vacuole biogenesis and acidification as a prerequisite for vacuole-mediated detoxification of selenite. The requirement also of the ESCRT-0 complex at this level is in accordance with its participation in formation of mature vacuoles (Raymond et al., 1992). Interestingly, an ESCRT-0 mutant displays wild type levels of sensitivity to NaCl, congruently with the fact that the vacuole does not seem to play a role in detoxification of this salt. Although genetic interactions have been shown between the Aft1 and Rim101 regulons (Berthelet et al., 2010) and both Daft1 and Drim101 mutants are hypersensitive to selenite, they seem to act separately in protection against this agent. In the case of Aft1, this would become activated probably as a consequence of some metabolic depletion of iron more or less directly caused by selenite. This would explain why iron addition to the medium suppresses the selenite hypersensitivity of the Daft1 mutant, supporting the competition between iron and selenite at some metabolic level in the cell. Surprisingly, activation of two genes of the Aft1 regulon (FIT3 and CTH2) upon selenite treatment is not Aft1dependent, pointing to more complex regulatory networks on these regulon genes. With respect to the physiological functions of Rim101 as transcriptional factor member of the PacC family, its originally characterized function in the response to alkaline pH has been extended to responses against other stresses (see Section 1). In some of these responses, Rim101 acts coordinately with other pathways regulating transcriptional programmes such as the calcineurin pathway responding to high Ca2+ and Na+ stress (Viladevall et al., 2004; Platara et al., 2006; Zhao et al., 2013). In the present work we have extended the number of physiological functions influenced by Rim101 to protection against oxidants and consequently against selenite. In this protection Rim101mediated maintenance of vacuolar acidity through the newly characterized regulation of expression of some VMA gens seems to play an important role. It remains to be determined how the core components of the Rim101 pathway discriminate between different environmental stress signals to switch-on different transcriptional programmes. Acknowledgments We acknowledge Aaron P. Mitchell and Olivier Vincent for providing biological materials. The excellent technical assistance of Silvia Porras and Meritxell Martín is appreciated. M.P.-S. was the recipient of a predoctoral Grant from Generalitat de Catalunya. This work was supported by Grants BFU2010-17656 (from Ministerio de Economía y Competitividad, Spain) and 2009/SGR/196 (from Generalitat de Catalunya).

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