Identification of arsenite and cadmium sensitive yeast mutants
We individually exposed ~4700 haploid Saccharomyces cerevisiae gene deletion mutants (the entire set of nonessential genes in this yeast) to arsenite and cadmium. The screen was limited to the trivalent form of arsenic (arsenite) since arsenite is more toxic than arsenate and is the form that is principally responsible for the biological effects of arsenic in medical therapy. Cells were exposed to increasing concentrations of each metal (3 doses) on solid medium and their growth was monitored by digital imaging and computational analysis as described in the Methods section. Deletion strains were considered sensitive if they showed a significant growth reduction in the presence of metal compared to its growth in the absence of metal. We also screened the homozygous diploid strain collection to ensure that phenotypes manifested only in haploid or diploid strain backgrounds were identified. In this case, homozygous deletion strains that exhibited reduced growth relative to the wild-type in liquid medium containing metal were identified as sensitive. Sensitivity of these strains was confirmed by re-screening. The combined set of deletion strains from the two screens (haploid and homozygous diploid) comprised 306 arsenite-sensitive and 382 cadmium-sensitive mutants. 106 mutants (18%) were sensitive to both metals (Fig 1A). For a list of all As and Cd sensitive mutants, see Additional file 1.
Cellular functions contributing to both arsenite and cadmium tolerance
To pin-point cellular functions that protect cells from metal toxicity, we searched for functional categories (according to FunCat, Munich Information Center for Protein Sequences (MIPS) [20]) that were significantly enriched (p-value < 0.005) in the sensitive gene set as compared to the whole genome. As was the case with individual genes (see above), this analysis revealed common as well as metal-specific tolerance functions (Fig 1). Prominent among those required for both As and Cd tolerance, were functions related to methionine and sulphur metabolism, mRNA synthesis and transcriptional regulation, environmental sensing, and vacuolar/endosomal transport and sorting. The p-values and fold enrichment factors of genes within individual functional categories are available in Fig 1C and Additional file 1.
Methionine and sulphur metabolism
Cells respond to As or Cd exposure by inducing expression of genes and enzymes in the sulphur assimilation and glutathione biosynthesis pathways. Moreover, cells also channel a large part of assimilated sulphur into biosynthesis of the low molecular weight thiol molecule glutathione [21–24]. Glutathione is important for metal tolerance since it may protect cells by: 1) metal chelation and sequestration; 2) protection against metal-induced oxidation since glutathione is considered the main redox buffer of the cell; and 3) binding to reactive sulphhydryl groups on proteins (glutathionylation) to shield them from irreversible metal binding and/or oxidative damage [25, 26]. The fact that we identified several metal sensitive mutants having functions in the sulphur assimilation and glutathione biosynthesis pathways as well as transcriptional regulators controlling these pathways, underscores the importance of the sulphur/glutathione biosynthesis pathways for tolerance acquisition. Moreover, the ycf1 Δ mutant lacking an ABC-type transporter responsible for sequestration of metal-glutathione conjugates into vacuoles [27–29] was also As and Cd sensitive. The enzymes in the sulphur assimilation/glutathione biosynthesis pathways are transcriptionally regulated by the transcription factors Yap1p and Met4p, and cells lacking any of these factors display As and Cd sensitivity [21, 22]. Met4p is recruited to target promoters by the DNA-binding proteins Met31p, Met32p and Cbf1p forming the complexes Met4p-Met31p/Met32p and Met4p-Cbf1p. An additional protein, Met28p stabilizes the Met4p-containing complexes [30]. Our data confirms the importance of Yap1p for metal tolerance and suggests that different Met4p-complexes may be important during As and Cd exposure; mutants lacking MET28 and CBF1 were As-sensitive whereas cells lacking MET32 and MET28 were Cd-sensitive. Taken together, these findings are consistent with the central role of sulphur metabolism and glutathione biosynthesis for As and Cd tolerance.
mRNA synthesis and transcriptional regulation
As or Cd exposure results in altered expression of a large number of genes [21, 22, 31–33]. Accordingly, mutants defective in mRNA synthesis and transcriptional control were sensitized to both metals (Fig 1). Among the gene-specific regulators that confer metal tolerance was Yap1p (regulates expression of genes with functions in oxidative stress defence and sulphur/glutathione metabolism), Met28p, Cbf1p, Met32p (sulphur/glutathione metabolism) and Rpn4p (protein degradation). Transcriptional activators may stimulate gene-specific expression by recruiting the co-regulator complexes Mediator, SAGA and/or Swi/Snf as well as RNA polymerase II to promoters. Indeed, several metal-sensitive mutants lacked subunits of RNA polymerase II (rpb4 Δ, rpb9 Δ) or components of the Mediator (pgd1 Δ, srb8Δ), SAGA (gcn5 Δ, ada2 Δ, spt7 Δ), and Swi/Snf (swi3 Δ, snf2 Δ) co-regulator complexes. Although sensitivity of these mutants could be a result of a general impairment of transcriptional activity, both Yap1p and Met4p interact physically with components of these co-regulator complexes [34, 35].
Interaction with the environment
Cells defective in functions related to environmental sensing and signalling were metal sensitive (Fig 1). The limited overlap between the As and Cd sensitive mutants within this category suggests that cells may use distinct sensing/signalling pathways in response to these metals. For instance, cells appear to use distinct MAP kinase pathways for sensing/responding to As and Cd since mutants defective in the cell integrity pathway (slg1 Δ, bck1 Δ, slt2 Δ) were preferentially Cd sensitive whereas cells defective in the osmosensing HOG pathway (ssk1 Δ, ssk2 Δ, pbs2 Δ, hog1 Δ) were preferentially As sensitive. We note that cells lacking the HOG pathway components Ssk1p and Hog1p were also Cd sensitive. The As and Cd-sensitivity of the latter mutants may reflect different functions/targets of the corresponding proteins under the two stress conditions. Hog1p is phosphorylated in response to As and contributes to tolerance by at least two mechanisms; first, Hog1p restricts arsenite influx through the aquaglyceroporin Fps1p by phosphorylating this protein (deletion of FPS1 results in arsenite resistance) [36, 37]. Secondly, Hog1p regulates the exit from As-induced G1 arrest [38]. Hog1p is also phosphorylated in response to Cd [39] but the target(s) and mechanism(s) through which Hog1p mediates Cd tolerance are unknown. Interestingly, Hog1p has a role in the cell integrity pathway and the cellular response to cell wall damage involves both the Hog1p and Slt2p/cell integrity MAP kinase pathways [40]. The fact that cells lacking HOG1 or components of the cell integrity pathway are Cd sensitive, suggests that the role of Hog1p under Cd exposure may be linked to cell wall damage.
Vacuolar/endosomal transport and sorting
Many functions throughout the secretory pathway appear important for metal tolerance since cells defective in vacuolar acidification, endocytosis, exocytosis, vesicular transport, and vacuolar transport displayed enhanced As and Cd sensitivity (Fig 1). The fact that this category was more enriched in the Cd-sensitive gene set indicates that the secretory pathway is more severely affected by Cd than by As. Interestingly, mutants that lack individual components of various protein complexes functioning in protein sorting were metal sensitive; such mutants included components of the HOPS (ho motypic fusion and vacuole p rotein s orting) complex (Vps16p, Vps33p), ESCRT (e ndosomal s orting c omplex r equired for t ransport) I (Stp22p, Srn2p), ESCRT II (Snf8p, Vps36p, Vps25p), ESCRT III (Snf7p, Vps20p) and GARP (G olgi-a ssociated r etrograde p rotein) complex (Vps51p-52p-53p-54p) (Fig 2A).
The secretory pathway is implicated in many important processes such as lipid biosynthesis, protein targeting and secretion, as well as the unfolded protein response. Consequently, an intact secretory pathway is crucial for the cell to withstand many different environmental conditions. Indeed, mutations impairing proper functioning of the secretory pathway cause sensitivity to a broad range of treatments and growth conditions [41, 42]. Metals may impact one or more aspects of the secretory pathway; first, metals may enter the endomembrane system and disturb various processes by interacting with proteins and/or lipids. Secondly, failure to maintain appropriate secretory pathway function may cause metal sensitivity due to defects in delivery or targeting of transmembrane transporters, sensors or signalling receptors. Thirdly, since vacuolar sequestration is important for tolerance acquisition, a decreased capacity to do so is likely to contribute to the observed sensitivity of secretory pathway mutants.
Arsenite-specific defence functions
Arsenite-specific defence functions that were significantly enriched (p-value < 0.005) in our data set were related to cell cycle regulation, lipid and fatty acid metabolism, mitochondrial biogenesis, and the cytoskeleton. The p-values and fold enrichment factors are available in Fig 1C and Additional file 1.
Cell cycle
Deletion of genes encoding cell cycle related functions produced As-sensitivity; those genes encompassed functions involved in regulation of cell cycle progression (HOG1, PHO85, PIN4, DBF2, CLB2), as well as spindle body and microtubule formation (BIK1, BIM1, CNM67, JNM1, CIN8, CIK1, CSM1, BNI1, CIN1, CIN2, PAC2, TUB3, SPC72). This indicates that cell cycle progression may be targeted by As, confirming our recent finding that As exposure triggers transient G1 and G2 delays in yeast and that cells defective in Hog1p signalling cannot exit the arsenite-induced G1 checkpoint arrest [38]. Similarly, the mechanisms of As action in cancer cells include cell cycle arrest at G1 and G2 phases, mitotic arrest and subsequent apoptosis. Arsenite can interrupt mitosis by interfering with tubulin polymerization or by disrupting mitotic spindles [12, 43]. Elucidating the molecular details of arsenite-induced cell cycle regulation and how this relates to As toxicity and carcinogenicity is important for understanding the potential of As-containing drugs in medical therapy.
Mitochondrion
Many mitochondrial proteins are known to be targeted by arsenicals [44]. Indeed, we found several As-sensitive mutants defective in mitochondrial function and biogenesis, including mitochondrial protein biosynthesis and genome maintenance. This observation is consistent with the notion that arsenic toxicity targets mitochondrial processes and that impaired oxidative phosphorylation causes As sensitivity [12, 45, 46].
Lipid and fatty acid metabolism
Deletion of genes encoding functions in lipid and fatty acid metabolism impaired As tolerance; these genes were related to ergosterol (ERG24, ERG2, ERG3, ERG4, ERG6), phospholipid (OPI1, OPI3, ARV1) and sphingolipid (DPL1, CSG2, SUR4, DEP1) biosynthesis. Trace elements and arsenicals appear to affect the fluidity and the thermotropic properties of cell membranes [47, 48]. Hence, As may have a direct impact on the lipid bilayer. Alternatively, the lipid composition of cell membranes might be altered in the mutants listed above, thereby changing the properties of the membrane and impacting on the activities of membrane-embedded transporters, sensors or signalling receptors [49]. Interestingly, the activity of the aquaglyceroporin Fps1p, through which arsenite enters cells [36, 37] is affected by the plasma membrane ergosterol content [50]. Moreover, an fps1 Δ mutant has reduced whole-cell and plasma membrane ergosterol levels compared to wild-type cells [50]. Hence, altered plasma membrane ergosterol levels may change Fps1p activity to facilitate arsenite entry.
Cytoskeleton
Loss of genes encoding cytoskeletal proteins caused As sensitivity. In particular, mutations affecting the function of the heterohexameric GIM (G enes I nvolved in M icrotuble biogenesis)/prefoldin protein complex, which is required for formation of functional tubulin, sensitized cells to As. These genes include YKE2 (GIM1), PAC10 (GIM2), GIM3, GIM4, GIM5, and PFD1 (GIM6). Analysis of the entire set of As-sensitive mutants for known protein-protein interactions indicated that the proteins encoded by these genes form a highly interacted molecular machine in cells and that disruption of any one of the components of this complex rendered cells sensitive to As (Fig 2B). The cytoskeleton is a known target of As; As triggers actin reorganization in various mammalian cell lines [51, 52], and directly interacts with actin as well as with the α and β subunits of tubulin [53, 54].
Other functions promoting arsenite tolerance
Several other As-specific defence functions were pin-pointed by our analysis (see Additional File 1). Most notable among those is the arsenic resistance gene cluster ACR3-ACR2-YAP8 [55]. ACR3 (also called ARR3) encodes an arsenite export protein [56], ACR2 (ARR2) an arsenate reductase [57] and YAP8 (ACR1/ARR1) encodes an arsenic-responsive transcription factor that controls expression of the former two genes [58–60].
Cadmium-specific defence functions
Cadmium-specific defence functions that were significantly enriched (p-value < 0.005) in our data set were mainly related to sugar/carbohydrate metabolism, and metal-ion homeostasis and transport. The p-values and fold enrichment factors are available in Fig 1C and Additional file 1.
Ion homeostasis and transport
Genes encoding functions in metal-ion homeostasis were enriched in the Cd-specific data set including the ion transporter genes PCA1 (Cd exporting ATPase), TRK1 (K+ transporter) and SPF1 (ER-localized Ca2+ ATPase). Interestingly, the majority of the genes in this category encode functions related to iron homeostasis including the iron regulated transcription factors AFT2 and AFT1/RCS1, the multicopper oxidases FET3 and FET5 (involved in iron uptake), FRE6 (ferric reductase), FRE8 (iron/copper reductase) and ISA2 (required for maturation of mitochondrial and cytosolic Fe-S proteins). Importantly, while this paper was being written, Ottonello and co-workers reported that iron addition rescues Cd-sensitivity of certain yeast mutants indicating that Cd interferes with iron homeostasis [61]. This is similar to the situation in plants where Cd exposure was shown to provoke iron deficiency [62]. Indeed, expression of several genes related to iron and heme metabolism (e.g. FET3, ATX1, FRE5, ARN1) is stimulated by Cd [22, 32] and during iron deprivation [63, 64] providing further support to the notion that Cd-exposed cells experience iron deficiency. Since Fe-S cluster homeostasis regulates transcription of the iron homeostasis systems [65], our data indicate that Cd may affect one or more aspects relating to Fe-S clusters, such as their biosynthesis and/or maturation.
Sugar/carbohydrate metabolism
Another Cd-specific defence function appears to involve the glucose sensing Snf1p-pathway since cells lacking SNF1 (homologous to mammalian AMP-activated protein kinases), SNF4, REG1, SIP5 (regulators of Snf1p), and MIG2 (transcriptional repressor targeted by Snf1p) were Cd sensitive (see Additional file 1). Snf1p plays a central role in modulating carbon metabolism under glucose limiting and environmental stress conditions. Snf1p also regulates proteins involved in metabolism of reserve carbohydrates such as glycogen and trehalose [66]. Interestingly, we noted that cells lacking either phosphofructokinase (PFK1 or PFK2) were Cd sensitive. Moreover, deletion of FBP1, whose product acts in the reverse step from that catalysed by Pfk1p and Pfk2p also led to Cd sensitivity. Finally, Cd stimulated expression of many genes with functions in glucose metabolism (GLK1, PDC6, ALD4, VID22, FBP1, FBP26) [32]. Hence, appropriate functioning of glycolysis may be crucial for Cd tolerance. Cd could impact the cell in such a way that flux through the glycolytic pathway is altered. For example, Cd could have a direct effect on glucose uptake systems [67], on glycolysis itself or on other systems that result in an extra demand on the glycolytic pathway.
Metal stress tolerance vs. oxidative stress tolerance
The toxicity of As and Cd has in part been attributed to their potential to induce ROS formation and hence to cause oxidative damage in cells [9, 68, 69]. To address to what extent As, Cd and various oxidants have overlapping toxicity profiles, we compared our gene sets with a set of genes previously reported to mediate tolerance to a number of ROS-generating agents including hydrogen peroxide, menadione, cumene hydroperoxide, diamide, and linoleic acid 13-hydroperoxide [70]. Hierarchical cluster analysis indicated that the genes that confer tolerance to As, Cd and the oxidative stress agents above are to a large extent distinct (Fig 3). Nevertheless, there was a cluster of genes whose absence resulted in sensitivity to metals and oxidative stress. The genes in the metal and oxidative stress cluster were significantly (p-value < 0.005) enriched in functions related to mRNA synthesis and transcriptional control, protein synthesis, ribosomal proteins and biogenesis, protein sorting and vacuolar transport, and lipid and fatty acid metabolism. Taken together, some of the genes and cellular functions required for As and Cd tolerance are also necessary for oxidative stress tolerance. However, from the sensitivity profiles it is not possible to draw any conclusions about the source and type of ROS that As and Cd generate and that in turn contribute to their toxicity. Instead, the data indicates that cells employ largely distinct protective mechanisms in different environmental conditions.
Expression vs. tolerance
In agreement with other studies [71], we found little overlap between genes necessary for As or Cd tolerance and genes whose expression is induced by As or Cd. However, for some defence systems there appears to be such a correlation. For instance, most genes (and enzymes) of the sulphur assimilation and glutathione biosynthesis pathways were strongly up-regulated in response to As and Cd [21, 22, 32], and many mutants in this pathway were metal sensitive. Another example is genes involved in iron homeostasis: expression of several such genes was induced by Cd whereas their absence resulted in Cd sensitivity. Similarly, expression of the arsenic-resistance genes ACR2 and ACR3 was stimulated by As and their presence was required for As tolerance (see Additional file 1).
We next asked whether the gene expression profiles of As- and Cd-treated cells would be more similar to each other than the As and Cd sensitivity profiles. As stimulated expression of 334 genes (0.2 mM arsenite, 1 hour) [21] whereas Cd exposure increased expression of 305 genes (0.3 mM Cd, 2 hours) [32] of which 80 genes were common (see Additional file 1). The overlap in those gene sets was about 14% which is similar to the overlap in the sensitive gene sets (18%). Interestingly, the genes induced by both treatments mainly encode functions in sulphur assimilation and glutathione biosynthesis pathways as well as in (oxidative) stress responses and detoxification, and many are transcriptionally regulated by Met4p and Yap1p. Hence, several processes that contribute to both As and Cd tolerance are transcriptionally stimulated by these metals. We also checked the overlap between the common As/Cd sensitivity gene set (106 genes) and the common As/Cd-induced gene set (80 genes). Curiously, these gene sets only had three genes in common (MET16, MET17, CYS3). Hence, although the two gene sets (phenotype and expression) are enriched for genes encoding related functions, the identities of those genes are largely distinct.
Human orthologues of yeast genes mediating arsenite and cadmium tolerance
We next asked to what extent the genes in our As- and Cd-sensitive gene set have human homologues. Using the HomoloGene database [72] we found that 43 of the 106 genes in the common As/Cd sensitive gene set have at least one human homologue (41%) (see Additional file 1). These proteins are involved in diverse functions in humans including signalling, transcription, chromatin modification, and vacuolar protein sorting. Moreover, these genes were significantly (p-value < 0.005) enriched for functions related to carbohydrate metabolism as well as protein targeting and sorting. We conclude that similar biological processes may act in yeast and humans to prevent metal toxicity and carcinogenesis.
Comparison of this study to other genome-wide screens
While preparing this manuscript, several genome-wide phenotypic screens were published that included arsenite and cadmium sensitivity data [31, 33, 61, 73, 74]. Haugen and co-workers [31] found 214 As-sensitive mutants of which 53 (11%) were present also in our data set, whilst Jin et al [33] identified 65 As-sensitive mutants, 40 (12%) of which were common to our study. 303 Cd-sensitive mutants were found by Ruotolo and colleagues [61], 73 by Serero and co-workers [74], and 276 by Jin et al [33]. Of those, 106 (18%), 39 (9%) and 94 (17%) respectively were present in our gene set whereas the overlap between the Ruotolo and Serero gene sets was 19%. Hence, the overlap in terms of mutants is roughly 10–20% and this relatively poor overlap is clearly visible in the heat maps in Figures 4 and 5. A reason for this discrepancy could be that none of the screens is saturated. Moreover, the corresponding screens were performed in dissimilar conditions e.g. on solid vs. liquid medium or screening mutants individually vs. in a mixture using a TAG array approach. Another factor likely to affect the outcome of a screen is the way sensitive genes are identified e.g. by visual inspection, TAG array hybridization, continuous measurements of optical density or by scoring growth after a specific time has elapsed. These factors probably influence the number and identity of mutants that are scored to affect growth. Despite the discrepancy, we reasoned that the gene sets probably contain relevant biological information. We therefore looked for mutants that were consistently identified as sensitive in at least two of the studies; these core-sets contained 89 As-sensitive and 209 Cd-sensitive mutants (see Additional file 1). The As core-set was enriched in functions related to sulphur metabolism, environmental sensing and signalling, transcription, and the cytoskeleton whereas the Cd core-set was enriched in functions related to sulphur and glutathione biosynthesis, transcription, intracellular transport and sorting, regulation of carbon metabolism, and metal-ion homeostasis and transport (Figs 4 and 5). Overall, these categories were similar to those found by us and others (Fig 1; [31, 33, 61, 74]). As-defence genes identified in all studies included YAP8, ACR3 and YCF1 whilst Cd-defence genes found in all studies included GSH1, GSH2, YCF1, YAP1 (glutathione biosynthesis), BCK1, SLT2 (MAP kinase signalling), as well as genes related to transcription and intracellular transport. Importantly, hierarchical cluster analysis of functional categories that were significantly (p-value < 0.005) enriched for metal sensitive mutants revealed a much better correlation than on the gene level (Figs 4 and 5). Hence, although these screens identified largely non-overlapping gene sets, they did uncover similar biological functions. In several cases, the studies above identified different mutants encoding proteins in the same pathway. One such example is the sulphur assimilation and glutathione biosynthesis pathways in the As-sensitive gene sets; 5 genes were common to at least two of the screens whereas 9 were found by only one study (Fig 4B). Of those, 14 were identified by us, four by Haugen and coworkers [31] and one by Jin et al [33]. Similarly, all six components of the GIM complex were found by us, four by Jin et al [33] and none by Haugen and co-workers [31]. Taken together, since the functional categories affected by metals found in each study clustered well, we conclude that genome-wide phenotypic screens provide biologically relevant insight into cellular systems mediating tolerance. However, a single screen is unlikely to uncover all genes and systems conferring tolerance.
Molecular clues to arsenite and cadmium toxicity and tolerance mechanisms
Next, we wanted to exploit the information in our data sets to discover toxicity and tolerance mechanisms at a molecular level. To do so, we explored the role of two cellular systems identified above in more detail, one related to arsenite and one related to cadmium.
The cytoskeleton is targeted by arsenite
Loss of any component of the heterohexameric GIM/prefoldin complex sensitized cells to As. The GIM complex is involved in actin and tubulin folding, and GIM mutants are defective in actin and tubulin organization, and are cold- and osmosensitive [75–77]. Interestingly, whereas GIM mutants are sensitive only at relatively high osmolarity, they are sensitive already at very low As concentrations; these mutants grew poorly at 50 μM arsenite as evident from the small colonies formed (Fig 6A). To gain further insight into the role of the GIM complex, we analysed all synthetic lethal (SL) interactions between GIM genes and the genome (see Additional file 1). Of the 107 genes showing SL interactions with at least four of the GIM genes, 29 were also As-sensitive (Fig 6B) indicating a significant enrichment (Fischer's test: 9.25 E-10) in As-sensitivity among GIM-interacting genes. These 29 genes encoded cytoskeletal proteins and functions that involve the cytoskeleton such as the cell cycle, budding and cell polarity. This data can be interpreted in two ways; As might target the GIM complex directly and that is the reason why the GIM mutants are sensitive. Alternatively, As might target the SL interacting gene-products and the cells cannot cope with defects in the GIM complex at the same time. Given that most of the interacting genes perform functions that require a functional GIM complex argues that the complex itself is directly targeted by As.
To further explore how As affects the cytoskeleton, we monitored α-tubulin organization by following GFP-tagged Tub1p and Tub3p. In the absence of As, extended fluorescence arrays were visible in most cells. In contrast, the microtubule arrays decreased dramatically in response to As exposure indicating disassembly of cytoplasmic microtubules (Fig 6C). Also the actin cytoskeleton was strongly affected by As. In untreated wild-type cells, actin patches were predominantly localized to the buds whereas As exposure triggered a temporary depolarization of the actin cytoskeleton with an even distribution of patches in the bud and the mother cell (Fig 6D). After 4 hours, the actin cytoskeleton recovered its polar distribution in nearly all cells and this recovery coincided with growth resumption (Fig 6E). Ssk2p is a kinase that activates the MAP kinase Hog1p in response to osmotic stress. In addition, Ssk2p binds to actin and promotes actin cytoskeleton recovery after osmotic stress [78]. We previously demonstrated that ssk2 Δ cells are As sensitive and that this sensitivity is unrelated to Hog1p phosphorylation [36]. Instead, Ssk2p might have a role in actin recovery also during As-exposure; actin remained depolarized in As(III)-treated ssk2 Δ cells (Fig 6D) and the mutant could not resume growth (Fig 6E). Taken together, these results provide strong support for the cytoskeleton being a direct target of As toxicity and that As inhibits the activity of the GIM complex in vivo. Hence, arsenite has a dual effect on the cytoskeleton; it binds to and disrupts the actin and tubulin cytoskeleton, and it inhibits the GIM complex that is required for folding of de novo synthesised actin and tubulin monomers.
Snf1p phosphorylation is required for Cd tolerance
The phenotypic data from our screen indicated a role of the glucose sensing Snf1p pathway for Cd tolerance. The Snf1p kinase is a heterotrimeric complex consisting of the catalytic α-subunit Snf1p, the regulatory γ-subunit Snf4p, and one of the three β-subunits (Sip1p, Sip2p, Gal83p). Snf1p activation involves Snf4p and any of the redundant upstream kinases Sak1p, Tos3p and Elm1p that phosphorylate Snf1p on threonine 210 (T210) [66]. We found that snf1 Δ, snf4 Δ and the sak1 Δ tos3 Δ elm1 Δ triple mutant were Cd sensitive (Fig 7A) suggesting that Snf1p phosphorylation is important for Cd tolerance. Indeed, the unphosphorylatable SNF1-T210A allele was unable to rescue growth of snf1 Δ in the presence of Cd (Fig 7B). To monitor Snf1p phosphorylation directly, we treated cells with cadmium (or low glucose as a control), prepared cell extracts and performed Western blot analysis using an anti-phospho-Snf1p antibody (Fig 7C). Basal Snf1p phosphorylation did not increase upon Cd-treatment whereas low glucose triggered strong Snf1p phosphorylation. No phosphorylation was detected in cells expressing the SNF1-T210A allele. We also monitored phosphorylation of the transcriptional repressor Mig1p, which is targeted by Snf1p under low glucose conditions. Cd did not trigger Mig1p phosphorylation whilst Mig1p was strongly phosphorylated in low glucose conditions (Fig 7D). Together with the fact that mig1 Δ is not Cd sensitive (Fig 6A), these results indicate that Snf1p action in Cd tolerance is different from that under low glucose conditions. Nevertheless, our data clearly shows that a low level of Snf1p kinase activity is necessary and sufficient to confer significant Cd tolerance to yeast cells. In line with this observation, low level of Snf1p kinase activity is sufficient to confer tolerance to other toxic agents like lithium and hydroxyurea [79, 80] although the mechanisms are not well understood. To conclude, high level of Snf1p kinase activity is required for growth in the absence of glucose while a low level of kinase activity is sufficient to confer tolerance to toxic agents when glucose is present.