Membrane transporters and protein traffic networks differentially affecting metal tolerance: a genomic phenotyping study in yeast
© Ruotolo et al.; licensee BioMed Central Ltd. 2008
Received: 29 December 2007
Accepted: 7 April 2008
Published: 07 April 2008
The cellular mechanisms that underlie metal toxicity and detoxification are rather variegated and incompletely understood. Genomic phenotyping was used to assess the roles played by all nonessential Saccharomyces cerevisiae proteins in modulating cell viability after exposure to cadmium, nickel, and other metals.
A number of novel genes and pathways that affect multimetal as well as metal-specific tolerance were discovered. Although the vacuole emerged as a major hot spot for metal detoxification, we also identified a number of pathways that play a more general, less direct role in promoting cell survival under stress conditions (for example, mRNA decay, nucleocytoplasmic transport, and iron acquisition) as well as proteins that are more proximally related to metal damage prevention or repair. Most prominent among the latter are various nutrient transporters previously not associated with metal toxicity. A strikingly differential effect was observed for a large set of deletions, the majority of which centered on the ESCRT (endosomal sorting complexes required for transport) and retromer complexes, which - by affecting transporter downregulation and intracellular protein traffic - cause cadmium sensitivity but nickel resistance.
The data show that a previously underestimated variety of pathways are involved in cadmium and nickel tolerance in eukaryotic cells. As revealed by comparison with five additional metals, there is a good correlation between the chemical properties and the cellular toxicity signatures of various metals. However, many conserved pathways centered on membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity.
Metals, especially the nonessential ones, are a major environmental and human health hazard. The molecular bases of their toxicity as well as the mechanisms that cells have evolved to cope with them are rather variegated and incompletely understood. The soft acid cadmium and the borderline acid nickel are nonessential transition metals of great environmental concern. Although redox inactive, cadmium and nickel cause oxidative damage indirectly  and they both have carcinogenic effects [2, 3], albeit with reportedly different mechanisms [1, 4–6].
The cellular effects of cadmium are far more studied than those of nickel. Instrumental to the elucidation of some of the basic mechanisms that underlie cadmium toxicity has been the model eukaryote Saccharomyces cerevisiae . It was studies conducted in this organism, for example, that yielded the first demonstration of the indirect nature of cadmium's genotoxic effects, which leads to genome instability by inhibiting DNA mismatch repair  and other DNA repair systems . Similarly, lipid peroxidation as a major mechanism of cadmium toxicity  as well as the central roles played by thioredoxin and reduced glutathione (GSH) , and vacuolar transport systems such as Ycf1 , in cadmium detoxification were first documented in yeast. Some of the above components were shown to be upregulated at both the mRNA [11, 12] and protein [12, 13] levels in cadmium-stressed yeast cells. Predominant among these expression changes was the upregulation of the sulfur amino acid biosynthetic pathway and the induction of isozymes with a markedly reduced sulfur amino acid content as a way to spare sulfur for GSH synthesis . A number of additional cadmium-responsive genes without any obvious relationship to sulfur sparing or cadmium stress were also identified, however. Curiously, only a small subset of the most cadmium-responsive genes produce a metal-sensitive phenotype when deleted , thus reinforcing the notion that transcriptional modulation per se is not a general predictor of the pathways influencing stress tolerance [14, 15]. For example, deletion of genes coding for two major organic peroxide-scavenging enzymes (GPX3 and AHP1; the latter encoding a cadmium-induced alkyl hydroperoxide reductase) did not impair cadmium tolerance .
By comparison, only a few studies have dealt with nickel toxicity in yeast. Interestingly, they showed that unprogrammed gene silencing, which is a major mechanism of nickel toxicity and carcinogenicity in humans [16, 17], also operates in S. cerevisiae. This further emphasizes the high degree of conservation of various aspects of metal toxicity as well as the usefulness of S. cerevisiae as a model organism for elucidating the corresponding pathways in humans. They also suggest, however, that a broad and as yet largely unexplored range of cellular pathways may be involved in alleviating the toxic effects of metals. What is currently missing, in particular, is a global view of such pathways at the phenotype level and a genome-wide comparison of different metals as well as other stressors.
We have addressed these issues by examining the fitness of a genome-wide collection of yeast deletion mutant strains [18, 19] exposed to two chemically diverse metals, namely cadmium and nickel, each of which is a known carcinogen [2, 3, 20]. This allowed us to assess the role of all nonessential proteins in modulating the cellular toxicity (sensitivity or resistance) of these two metals. The results of this screen were integrated with interactome data and compared with the genomic phenotyping profiles of other stressors. To gain further insight into the cytotoxicity signatures of different metals, the entire set of 388 mutants exhibiting an altered viability after exposure to cadmium and nickel was challenged with four additional metals (mercury, zinc, cobalt and iron) plus the metalloid AsO2-. Although overall there is good correlation between the chemical properties and the cellular toxicity signatures of various metals, many conserved pathways centered on (but not limited to) membrane transporters and protein traffic affect cell viability with a surprisingly high degree of metal specificity.
Results and discussion
Genomic phenotyping of cadmium and nickel toxicity
Sublethal concentrations of 50 μmol/l cadmium and 2.5 mmol/l nickel (see 'Materials and methods', below, for details) were used for multireplicate screening of the yeast haploid deletion mutant collection (five replicates for each metal), which was performed by manually pinning ordered sets of 384 strains onto metal-containing yeast extract-peptone-dextrose (YPD)-agar plates (Additional data file 1 [Figure S1A]). After culture and colony size inspection, strains scored as metal sensitive or resistant in at least three screens were individually verified by spotting serial dilutions onto metal-containing plates. Mutant strains exhibiting various levels of metal sensitivity (high sensitivity [HS], medium sensitivity [MS], and low sensitivity [LS]) and a single class of metal resistant mutant strains were recognized (Additional data file 1 [Figures S1B and S1C]).
As revealed by the GO analysis summarized in Figure 1b, a wide range of cellular processes is engaged in the modulation of cadmium and nickel toxicity. At variance with cadmium resistant mutants, which are scattered throughout various GO categories, nickel-resistant as well as cadmium/nickel-sensitive mutant strains were found to be enriched in specific functional categories. Some of the top responsive genes identified by previous expression profiling studies (for example, genes involved in GSH and reduced sulfur metabolism [11, 13]) were found to be among deletion mutants specifically sensitive to cadmium, especially within the 'response to stress' category. As expected for cells treated with agents that are actively internalized by and sequestered into vacuoles, a number of the most significant GO categories are related to 'transport', particularly to the vacuole, and to the biogenesis and functioning (for example, acidification) of this organelle. Several processes not so obviously associated with metal tolerance were also identified. For example, 'nucleocytoplasmic transport' (including nuclear pore complex formation, and functionality) emerged as a process that is specifically impaired in nickel-sensitive mutants. Other processes centered on vesicle-mediated transport also profoundly influence cadmium and nickel tolerance in different, often contrasting ways. For example, many 'Golgi-to-vacuole transport' mutants appear to be sensitive to both cadmium and nickel, whereas defects in 'endosome transport' and 'retrograde transport endosome-to-Golgi' render cells sensitive to cadmium but resistant to nickel (see below).
Importantly, mutants with metal sensitivity phenotypes of varying severity (Additional data files 1 and 2) are present within different mutant classes as well as functional categories. This discounts the possibility that only highly sensitive mutant strains or particular classes of genes are relevant to cadmium/nickel tolerance, and suggests that a suite of pathways, much broader than previously thought, modulates metal tolerance in eukaryotic cells.
Mutations impairing cadmium and nickel tolerance
In accordance with the tight relationship between metal tolerance and vacuole functionality highlighted by GO analysis, the most populated subnetwork (subnetwork 1; P-value < 1.5 × 10-18) comprises a large set of subunits, assembly factors, and regulators of V-ATPase, which is the enzyme responsible for generating the electrochemical potential that drives the active accumulation of various ions within the vacuole . Also related to V-ATPase functionality (although not included in subnetwork 1) is Cys4, which is the first enzyme of cysteine biosynthesis, whose disruption indirectly interferes with vacuolar H+-ATPase activity . Another highly populated subnetwork (subnetwork 2; P-value < 2 × 10-5) contains eight additional vacuole-related genes belonging to either class B or C 'vacuolar protein sorting' (vps) mutants, whose deletion respectively causes a fragmented vacuole morphology or lack of any vacuole-like structure [27, 28]. This indicates that defects in specific aspects of vacuole functionality as well as in late steps of vesicle transport to, and fusion with, the vacuole cause sensitivity to both metal ions. In keeping with this view, three additional proteins (Fab1, Fig4, and Vac14), which also cause cadmium/nickel sensitivity when disrupted, control trafficking to the vacuolar lumen [29, 30]. The role played by the vacuole in metal toxicity modulation may entail both metal sequestration within this organelle as well as the clearance of metal-damaged macromolecules.
Connected with these vacuole-related hot spots, which include a number of genes previously associated with cadmium (but not nickel) tolerance , are five additional subnetworks. One of them (subnetwork 3; P-value < 7 × 10-2) comprises the master regulator Rpn4, which is required for proteasome biogenesis, and three ubiquitin-related proteasomal components (Qri8, Shp1, and Ubp3), thus reinforcing the notion that abnormal protein degradation plays an important role in toxic metal tolerance [31–33]. Other components previously associated with tolerance to cadmium and to other stressors include three subunits of the chromatin remodeling complex SWI/SNF (SWItch/Sucrose NonFermenting; subnetwork 4; P-value < 0.1)  and a group of regulators of the cell wall integrity/mitogen-activated protein kinase signaling pathway (subnetwork 5; P-value < 3.4 × 10-6) [35, 36]. These are functionally linked to the second largest subnetwork (subnetwork 6; P-value < 9.1 × 10-5), which is centered on Ccr4 and its associated proteins. Ccr4 is a multifunctional mRNA deadenylase that can be part of mRNA decay as well as transcriptional regulatory complexes in association with the NOT factors . None of the NOT deletion mutants was identified as metal sensitive, whereas a few other transcriptional regulators interacting with Ccr4 (for example, Dbf2 and Rtf1) cause cadmium/nickel sensitivity when disrupted. Pop2, another major deadenylase in S. cerevisiae , along with three additional RNA processing enzymes (Kem1, Lsm7, and Pat1), were also found among cadmium/nickel sensitive mutants. Previously known to be involved in the response to DNA damaging agents , these proteins thus appear to play a role also in metal tolerance, which might be aimed at ensuring proper translational/metabolic reprogramming under stress conditions. This finding, along with the identification of cadmium/nickel-sensitive mutations affecting three nuclear pore complex subunits (subnetwork 7; P-value < 7.3 × 10-4) and a mRNA export factor (Npl3), points to mRNA decay and trafficking (particularly nuclear export) as a novel hot spot of metal toxicity.
The last two subnetworks pertain to ergosterol biosynthesis (subnetwork 8; P-value < 9.8 × 10-4), which critically influences the structural and functional integrity of the plasma membrane (Additional data file 1 [Figure S1B] shows a representative phenotype), and to essential ion homeostasis (subnetwork 9; P-value < 0.12). The latter includes the endoplasmic reticulum exit protein Pho86, which is required for plasma membrane translocation of the Pho84 phosphate transporter, the high-affinity iron transport complex Ftr1/Fet3, and a transcription factor (encoded by the solitary gene AFT1) that positively regulates FTR1/FET3 expression. All these genes cause cadmium/nickel sensitivity when mutated. A possible explanation for this finding is that toxic metals can make iron, and other essential ions, limiting for cell growth (see below). In fact, one copper transporter (Ctr1) and a copper uptake-related transcription factor (Mac1) were also found among the cadmium/nickel-sensitive mutants in our screen.
Metal-specific sensitive mutants
Subnetwork organization of gene products whose disruption specifically affects nickel or cadmium tolerance
Interacting gene products
Functionally linked gene productsb
Interacting gene products3
Functionally linked gene productsb,c
V-ATPase assembly/regulation (sn. 1)
Rav1, Vma16, Vph1
Proteasome (sn. 3)
Bre5, Cdc26, Doa1, Hlj1, Sel1, Ubi4, Ubp6, Ump1
Chromatin assembly/remodelling (sn. 4)
SAGA complex (Ada2, Chd1, Gcn5, Hfi1, Ngg1, Spt7*, Spt20); Ino80 complex (Arp5, Arp8, Taf14); COMPASS complex (Bre2, Sdc1); Asf1, Ard1, Eaf7*, Esc2, Hda1*, Hmo1, Ioc2
Cell wall integrity pathway (sn. 5)
Bem2, Dom34, Ecm33, Kcs1, Pin4, Pog1, Rvs161, Rvs167, Sic1, Sit4*, Sur7, Swi4, Swi6, Whi2
CCR4 and other mRNA processing enzymes (sn. 6)
Nuclear pore complex (sn. 7)
Nup84, Sac3, Thp1
Essential ion homeostasis (sn. 9)
AP-3 complex (sn. 10)
Apl5, Apl6, Apm3, Aps3
General transcription (sn. 11)
Mft1, Rpb9, Rtt103, Thp2
Mediator complexes (Gal11, Med2, Pgd1, Spt21, Srb8*, Srb10); Cad1, Elp4, Tup1, Yap1
DNA repair (sn. 12)
Ctf4, Him1, Met18, Mms22, Mre11, Pol32, Rad6, Rad27, Xrs2
Antioxidant defense (sn. 13)
Atx2, Ccs1, Sod1, Sod2
Cad1, Glr1, Gsh1, Gsh2, Yap1, Zwf1
Hog1 pathway (sn. 14)
Fps1, Hog1, Pbs2, Rck2, Ste11
Vesicle targeting to, from or within Golgi (sn. 15)
Erv41, Erv46, Get2, Sac1, Sec22, Sec66; Vps13; Cog5, Cog8; Pep7, Tlg2, Vps3, Vps9, Vps21, Vps45; Arl1, Arl3, Ent3, Gga2, Nhx1*, Rgp1, Ric1, Sys1, Yil039w*, Vps51, Vps54, Ypt6; Vam10*, Vps1*, Vps8*; Pep8*, Vps5*, Vps17*, Vps29*, Vps30*, Vps35*, Vps38*
Ubiquitin-dependent sorting to the multivesicular body pathway (sn. 16)
Vps27*; ESCRT I complex (Vps28*, Mvb12*, Srn2*, Stp22*); ESCRT II complex (Snf8*, Vps25*, Vps36*); ESCRT-III complex (Did4*, Snf7*, Vps20*, Vps24*); Bro1*, Did2*, Doa4*, Vps4*
Bsd2*, Bul1*, Nhx1*, Tre1*
Only one of the new subnetworks (subnetwork 10; P-value < 1.6 × 10-3) was found to be specifically associated with nickel sensitivity (Table 1 and Additional data file 3). This includes various components of a multiprotein complex (Adaptor Protein complex AP-3) that is involved in the alkaline phosphatase (ALP) pathway for protein transport from the Golgi to the vacuole. At variance with the other Golgi-to-vacuole transport route (the so-called 'carboxypeptidase Y' [CPY] pathway), which proceeds through an endosome intermediate and includes a number of components that when disrupted cause cadmium sensitivity (see subnetwork 15 in Table 1), the ALP pathway directly targets its cargo proteins to the vacuole. Different metals and/or different metal-specific detoxifying proteins thus appear to be differentially trafficked through the Golgi-vacuole network. A similar differential toxicity effect was recently reported for iron and copper . Also notable in this regard is the observation that mutants impaired in the retrieval of receptors from the endosome to the Golgi (subnetwork 15; P-value < 2.4 × 10-3) and in endosome-to-vacuole transport (subnetwork 16; P-value < 1.6 × 10-8) are specifically sensitive to cadmium but resistant to nickel (see below).
The other cadmium-specific subnetworks are 'DNA repair' (subnetwork 12; P-value < 0.16), which includes the ubiquitin-conjugating DNA repair enzyme RAD6; 'antioxidant defence' (subnetwork 13; P-value < 5.8 × 10-2) and other functionally related components (Table 1 and Additional data file 4); and the Hog1 kinase cascade (subnetwork 14; P-value < 3.7 × 10-2), which was previously shown to be involved in cadmium tolerance . The latter, along with the upstream-acting kinase Pbs2, controls a number of cell wall integrity-related genes. Other genes that when mutated cause cadmium or nickel sensitivity encode plasma membrane (Mal31 and Smf1) and intracellular (Ccc2, Pho88, Pho89, Smf3, Ybt1, and Ycf1) transporters (or transport-related proteins), for most of which involvement in toxic metal mobilization (especially export or reduced uptake) has not previously been reported (see below).
A previously underestimated variety of cellular processes, operating in different subcellular compartments (vacuole, Golgi, and endosome, but also cytosol, nucleus, and plasma membrane), thus appears to be involved in metal tolerance in yeast. Perhaps the most significant among the novel metal toxicity-related processes revealed by our screen are mRNA decay and nucleocytoplasmic transport, and the different protein trafficking (particularly vacuole-to-Golgi) pathways that differentially affect cadmium and/or nickel tolerance when disrupted.
Cadmium and nickel interfere with iron homeostasis through different mechanisms
Also apparent in Figure 4 is the different degree of cadmium/nickel sensitivity of the fet3Δ mutant (same for ftr1Δ), which is only moderately sensitive to cadmium (LS phenotype) but highly sensitive to nickel (HS phenotype). Other distinguishing features of the iron-related phenotypes of cadmium and nickel originate from the low-affinity/low-specificity transporters encoded by the FET4 and SMF1 genes [46, 52]. These transporters become major entry sites for iron under iron overload or fet3/ftr1Δ conditions [53, 54] as well in the absence of the transcription factor Aft1, which positively regulates FET3 and FTR1, whose deletion causes a HS phenotype for both cadmium and nickel (Additional data file 1 [Figure S1B] shows a representative phenotype). In addition to iron, Fet4 and Smf1 internalize other metals such as manganese, copper and cadmium [52, 55, 56], whereas no conclusive data on nickel have thus far been reported. In keeping with this notion, we find that fet4 and smf1 deletion mutants are cadmium (but not nickel) resistant, whereas disruption of Rox1 - a negative regulator of FET4 - makes cells selectively sensitive to cadmium (Additional data file 5). Conversely, over-expression of Smf1 causes cadmium (but not nickel) sensitivity (see Figures 7 and 8, below, for representative phenotypes). Therefore, even though cadmium and nickel toxicity is exacerbated at alkaline pH and both interfere with iron homeostasis, they probably do so with different mechanisms.
Cadmium, but not nickel, is internalized by broad-range transporters such as Fet4, which accumulate under iron-limiting conditions as a way to cope with iron deficiency . Two nonmutually exclusive mechanisms may thus explain the alleviating effect of iron supplementation on cadmium toxicity, in both WT and fet3Δ cells: competition between the two metals at the level of cellular uptake; and downregulation of promiscuous (iron/cadmium) transporters under conditions of iron overload [54, 57]. Competitition between iron and cadmium at the level of cellular uptake may account, for instance, for the anti-cadmium effect of iron that has been described in rats fed with a iron-supplemented diet . Nickel, instead, interferes with iron homeostasis via an as yet unidentified mechanism, which does not appear to rely on direct competition with iron at the level of cellular uptake. An alternative possibility is nickel competition at the level of iron-regulated enzymes, as reported for various Fe-S (for example, aconitase and succinate dehydrogenase) and other iron-dependent enzymes in mammalian cells .
Other iron-related genes whose mutation makes cells specifically sensitive to nickel or cadmium are Ccc2 (a P-ATPase responsible for copper loading of the Fe [II] oxidoreductase Fet3) and Smf3 (a divalent metal transporter that mobilizes iron ions from the vacuole to the cytosol under conditions of iron deficiency). Mutations affecting the human orthologs of these genes respectively cause Wilson disease (characterized by abnormal copper accumulation in liver)  and microcytic anemia with hepatic iron overload  (Additional data file 2).
Other transporter mutants exhibiting cadmium (but not nickel) resistance include smf2Δ, an intracellular manganese transporter  (see also Figure 7), and zrt3Δ, which is a transporter that mobilizes zinc ions from the vacuole to the cytoplasm . Additional mutants of this kind are disrupted in the vacuolar transporter chaperones Vtc4 (nickel/cadmium resistant) and Vtc1 (nickel resistant), both of which have previously been reported to cause manganese resistance when deleted . Also notable among the genes that when deleted cause cadmium and/or nickel resistance are Sif2, a subunit of the Set3C histone deacetylase complex whose disruption increases telomeric silencing, the cell cycle regulators Cln3 and Sap190, and the mitogen-activating protein kinase cascade regulator Sis2.
Mutations in the ESCRT and in the endosome-to-Golgi retromer complexes differentially affect cadmium and nickel tolerance
A different mode of action probably applies to the expanded set of retromer-related mutants that we also identified as nickel resistant (see pathway II in Figure 6a). One of these mutants (vps5Δ) was previously reported to have a nickel uptake capacity similar to that of WT in intact cells, but a threefold higher uptake rate after plasma membrane permeabilization . Based on these observations, it was proposed that in this particular vps mutant an unidentified late Golgi Mg2+/H+ exchanger could be missorted to the vacuole, where it would promote enhanced nickel accumulation (and detoxification). At variance with this hypothesis, we found that only a small fraction of cells mutated in various retromer-related components were able to accumulate nickel (as revealed by Newport Green fluorescence), whereas most cells were not fluorescent and thus apparently unable to accumulate nickel ions (Figure 8c). Whether this is due to a reduced uptake or to an enhanced export of nickel is not known at present. It should be noted, however, that defects in this particular traffic network can cause protein missorting to the vacuole, but also to the plasma membrane [81, 87–89]. It is thus conceivable that avoidance and/or extrusion of nickel by a divalent cation transporter (or exchanger) mislocalized to the plasma membrane might be responsible for the increased nickel tolerance of these mutants. The opposite situation holds for two plasma membrane located uracil and nicotinic acid transporters, Fur4 and Tna1, which when deleted cause nickel resistance along with reduced intracellular nickel accumulation, and for which we propose a promiscuous role in nickel internalization (Additional data file 6).
Other cadmium-sensitive/nickel-resistant strains are mutated in amino acid metabolism enzymes (for example, Aat2 and Aro2) and nuclear components (for example, Mog1, Nnf2, Spt7, and Srb8), including the putative catalytic subunit of a class II histone deacetylase (Hda1), as well as in the uncharacterized ORF YIL039W. Also noteworthy are mitochondrion defective mutants, one of which (mam3Δ) was previously reported to be cadmium sensitive, but resistant to cobalt and zinc .
Cellular toxicity signatures of other metals
The closest resemblance with cadmium was observed for mercury, a highly toxic, thiophilic group IIb metal with a nearly identical atomic radius. This is followed by arsenite, which despite its chemical dissimilarity is known to bind thiols (especially dithiols ) and to share various cellular toxicity similarities with cadmium . The most prominent divergence between cadmium and arsenite pertains to the Rim101 pathway (which when disrupted causes AsO2- sensitivity but resistance to cadmium, nickel and zinc) and to a few mutants (which exhibited the opposite phenotypic response, such as erv41Δ and erv46Δ). Interestingly, the same metal discrimination capacity applies to the Rim101/Nrg1-regulated plasma membrane transporter Tat1, whose disruption leads to resistance to cadmium, nickel and zinc, but not arsenite.
The phenotypic overlap between nickel and cobalt was not as high as one might have expected based on their chemical similarity. Especially noteworthy is the increased sensitivity to cobalt and to all the other tested metals, except nickel, exhibited by ESCRT pathway mutants, and the only partial overlap between nickel and cobalt observed for retromer mutants. Also worthy of note are the different metal tolerance phenotypes associated with the Fur4 and Tna1 transporters, whose deletion causes sensitivity to cadmium and to other metals, but nickel resistance (see Additional data file 6 for representative phenotypes). One of them (Tna1) causes resistance to both nickel and cobalt when disrupted, whereas deletion of the other transporter (Fur4) makes cells resistant to nickel and zinc but not cobalt. Conversely, disruption of the Smf1 transporter as well as disruption of various components of the Adaptor Protein complex AP-3 involved in the ALP pathway (Table 1 and Additional data file 3) causes nickel but not cobalt sensitivity.
As predicted by the protective effect exerted on both WT and mutant cells (Figure 4) and by its 'hard' Lewis acid nature, Fe(III) was the most divergent of the metals investigated. Also apparent in Figure 9 is that under conditions of iron sufficiency, mutations in genes belonging to the iron regulon cause increased sensitivity to all of the examined metals except iron itself. This suggests that, albeit with different mechanisms, toxic metal-induced iron depletion may be a common feature of many (if not all) toxic metals. Zinc and Fe(III), both of which are essential metal ions, clustered together despite their chemical dissimilarity. On the whole, however, we find a fairly close overlap between the chemical properties and the cellular toxicity signatures of the various metals. For example, clustering based on the phenotypic profiles of a selected subset of mutants fits with chemical properties such as the ability of the various metals to form insoluble sulfides in a strongly acidic environment, which is shared by cadmium, mercury and As(III), but not nickel, cobalt, zinc, or Fe(III). As further shown in Figure 9, multimetal phenotypic profiling also allowed to annotate various ORFs, some of which are homologous to disease-related human genes. For instance, YCR045C and YCR051W are homologous, respectively, to Pcsk9 and Tnks2. Pcsk9 is an endoplasmic reticulum serine protease that is involved in an autosomal dominant form of hypercholesterolemia , a disease that is also induced by dietary metal imbalance ; Tnks2 is a cytosolic member of the poly(ADP)ribose polymerase family, whose over-expression affords cytoprotection by preventing excessive poly(ADP)ribose polymerase activation and NAD depletion after exposure to DNA-damaging agents .
As revealed by this study, which interrogated all nonessential genes of S. cerevisiae for their role in modulating metal toxicity, 16 functional subnetworks (comprised of 207 genes, at least half of which had never been implicated in metal tolerance previously) negatively influence cadmium and/or nickel tolerance when disrupted. Core genes influencing cadmium and nickel tolerance were mapped to nine subnetworks, a subset of which (for example, V-ATPase, vacuole fusion, and the ERG pathway) cause enhanced co-sensitivity to mercury, arsenite, cobalt, zinc and iron, thus pointing to the occurrence of multimetal defense systems in yeast. Seven of these subnetworks were expanded to include additional mutations specifically associated with cadmium and/or nickel sensitivity, along with six additional subnetworks causing metal-specific (especially cadmium-specific) sensitivity (Table 1). Only one of the latter subnetworks (with a bearing on the ALP branch of the Golgi-to-vacuole traffic pathway) was found to be specifically involved in nickel tolerance, as opposed to five subnetworks causing cadmium-specific sensitivity when disrupted. Thus, cadmium is not only more toxic than nickel, but it also has a broader spectrum of cellular processes that directly or indirectly contribute to its detoxification. Most prominent among these processes are those related to vesicular protein traffic (including the endocytotic pathway and a different branch of the Golgi-to-vacuole traffic), antioxidant defense, and DNA repair. The latter, in particular, further strengthens the causal relationship between cadmium genotoxicity and DNA repair . In fact, although cadmium and nickel have both been recognized as human carcinogens [2, 3], mutagenic activity appears to be a distinguishing feature of cadmium [1, 4]. Nickel, instead, is a weak mutagen with a marked nuclear tropism, whose carcinogenicity is thought to primarily rely on unprogrammed chromatin modification . It is interesting to note in this regard that the nuclear pore complex is one of the few core subnetworks enriched in nickel-specific sensitive mutants. Also interesting is that three out of eight mutants specifically resistant to nickel (but unrelated to vesicular traffic; Additional data file 7) are deleted in genes encoding distinct chromatin modification enzymes (HDA1, EAF7, and SPT7) and one is deleted in a Ran homolog of the Ras GTPase family (MOG1) that is involved in protein traffic through the nuclear pore.
Many metal toxicity-modulating pathways are related to metal damage prevention or repair, whereas others appear to play a more general (and indirect) role in promoting cell survival/recovery under stress conditions. Especially noteworthy among the latter are mRNA decay and nucleocytoplasmic transport, two processes that to our knowledge have not previously been implicated in metal tolerance and that might explain the variety of putative target genes previously identified as cadmium stress responsive . Their identification among metal sensitivity-conferring mutations suggests that not only the clearance of damaged (or unwanted) proteins by the proteasome and transcriptional regulation, but also mRNA turnover and relocalization are important for translational/metabolic reprogramming under conditions of metal stress. Interestingly, coordinate downregulation of iron-related proteins mediated by mRNA degradation under iron starvation conditions  as well as mRNA mistranslation after chromium exposure  have recently been described in yeast. The fact that structurally diverse yet functionally related gene products cause metal sensitivity when disrupted provides strong evidence that the cellular processes represented in specific subnetworks play an important role in preventing or repairing metal-induced cell damage. However, this does not exclude the possibility that a subset of the mutant strains that we have identified as metal sensitive are due to chemical-genetic synthetic lethality resulting from direct attack (and inactivation) of a functionally related protein target by the metal. Mutations associated with this kind of metal-induced lethality are likely to be enriched in the genes we classified as 'solitary'.
Among the unrelated stressors we examined, alkaline pH emerged as the most closely related to cadmium/nickel stress. This genomic phenotyping resemblance was traced back to iron deficiency, which - albeit with different mechanisms - is caused by both cadmium and nickel and appears to be a fairly general effect of metal toxicity (Figure 9). Broad-range transporters were identified as the most proximal effectors of iron deficiency-related and other kinds of altered metal tolerance. The latter include Tat1, which is a low-affinity Trp/His transporter negatively regulated by Nrg1 , which emerged from this study as one of the downstream effectors of the multimetal resistance caused by disruption of the Rim101 pathway. Toxic metal internalization (or abnormal intracellular mobilization) thus appears to be one of the most general and detrimental effects caused by transporter promiscuity, a trait that has probably evolved as a way to deal with multiple nutritional deficiencies under nutrient limiting (but toxic metal-free) conditions. This provides novel mechanistic support to the notion that nutrient limitation (especially iron and copper, but also amino acids and vitamins) may aggravate metal toxicity in malnourished human populations. Another outcome of this study was the identification of 24 uncharacterized ORFs that are involved in metal tolerance, which lend themselves as novel candidate genes that are worthy of further investigation.
Systematic comparison of the cellular toxicity signatures of cadmium and nickel with those of five additional metals revealed significant overlap between their chemical and cellular toxicity properties. However, it also uncovered an unexpected degree of metal specificity, especially regarding mutations that cause resistance to nickel but sensitivity to most other metals. The hot spots for such mutations were mapped to the ESCRT and the retromer complexes, thus pointing to the ability of these pathways to discriminate between otherwise similar metals and to the potential use of selected toxic metals (for example, cadmium and nickel) as chemical probes of intracellular traffic functionality.
Materials and methods
Yeast strains and culture conditions
The strains used in this study derive from the S. cerevisiae Genome Deletion Project . They were purchased from Open Biosystems (Huntsville, AL, USA) and converted into a 384-well plate format by manual multipinning. Deletion of individual nonessential genes (or ORFs) were in the MATα BY4742 background, except for 79 strains with a MATα BY4739 parental background. Untagged deletion strains utilized for phenotype verification were in the parent MATα BY4741 background and were obtained from EUROSCARF (Frankfurt, Germany). Cells were grown at 30°C on yeast extract-peptone-dextrose (YPD) or synthetic dextrose medium (supplemented with leucine, lysine, and histidine, but without uracil), as indicated. For some experiments, YPD was supplemented with FeCl3 or adjusted to pH values ranging from 6 to 8.5 with the addition of 50 mmol/l MOPS (3- [4-morpholino]propane sulfonic acid) or TAPS (N- [tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid), as specified in the text.
Metal tolerance screening
A total of 4,688 single gene deletion mutants, not including 90 strains that failed quality control and 48 slow-growth strains previously shown to exhibit a high false-positive rate , were utilized for genomic phenotyping. Metal titrations coupled with serial dilution spot assays (starting from cultures pregrown in YPD at an optical density at 600 nm [OD600] of 1.0 and diluted up to 10,000 fold in tenfold increments before spotting; see below) were initially carried out in the parent WT strains to determine metal concentrations, allowing for about 90% of control ('minus metal') growth after 48 hours. These concentrations ranged from 40 to 60 μmol/l and from 2.5 to 4.5 mmol/l for cadmium and nickel, respectively. Metal concentrations for genomic phenotyping were further refined in pilot experiments carried out with an arrayed test set of 384 strains, including the WT strains plus two known cadmium-sensitive mutants (yap1Δ  and ycf1Δ ) and one nickel-sensitive mutant (pep5Δ ) as positive controls. Optimal concentrations of 50 μmol/l cadmium and 2.5 mmol/l nickel were thus determined and utilized for the screening.
To this end, individual plates from the deletion strain collection (384-well format master plates with eight empty wells as contamination controls) were inoculated into 150 μl liquid YPD plus 200 μg/ml G418 (GIBCO-Invitrogen, Carlsbad, CA, USA) using a 384-pin tool (VP 384F; V&P Scientific Inc., San Diego, CA, USA). After 2 days at 30°C, cells were replica inoculated onto YPD-agar without G418, supplemented or not supplemented with the appropriate metal. This was done with the use of a library copier (VP 381; V&P Scientific Inc.) by touching the bottom of the wells and then raising and lowering the multipin replicator three times, in order to mix the cells and obtain a properly diluted inoculum (about 500 cells/pin). After 2 to 3 days at 30°C, plates were examined for metal-sensitive and metal-resistant strains according to relative colony size, followed by digital image recording. A positive result was scored when colony size under metal-supplemented conditions was diminished (no growth or slow growth phenotype in the case of metal sensitivity) or augmented (overgrowth phenotype in the case of metal resistance) compared with neighboring (unaffected) strains and with the colony size of the corresponding strains grown on the 'minus metal' plate (Additional data file 1 [Figure S1A]). Five replicate screens starting from fresh liquid cultures were run for each metal (Additional data file 2 provides details on the cumulative outcome of this multireplicate screening), followed by verification of strains that were scored as sensitive or resistant in at least three screens by serial dilution spot assays (see below).
Validation and multimetal assays
Strains that were deemed as positive (sensitive or resistant) in the primary screen as well as strains consistently exhibiting an overgrowth or slow-growth phenotype in control ('minus metal') plates were individually verified by serial dilution spot assays. Mutant strains of interest were recovered from the original 96-well plates, assembled into a new plate, and cultured in YPD medium as above. After 24 hours at 30°C, the OD600 of individual cultures was determined with a microplate reader, adjusted with YPD medium to an OD600 value of 1.0 and serially diluted in tenfold increments. Aliquots (4 μl) of each dilution were spotted onto YPD-agar plates in the presence or absence of appropriate metal concentrations (40 μmol/l CdCl2 and 2.5 mmol/l NiCl2 for sensitive strains, and 50 μmol/l CdCl2 and 3.5 mmol/l NiCl2 for strains scored as resistant) and growth was examined after incubation at 30°C for 2 to 3 days. Mutant strains exhibiting a reduction in growth at the first, second, or third (or fourth) dilution were classified as having a 'high', 'medium', or 'low' metal sensitivity (HS, MS, and LS, respectively); only one type of metal-resistant phenotype was recorded (Additional data file 1 [Figures S1B and S1C]). The rate of validation of the phenotypes determined in primary screens was 85% for cadmium and 81% for nickel. Appropriately lower metal concentrations (15 to 20 μmol/l cadmium and 1 to 1.2 mmol/l nickel for sensitivity and resistance, respectively) were used for assays carried out in synthetic dextrose medium (see below). Eighteen strains identified as metal-sensitive in our screen correspond to 'dubious' ORFs  overlapping 'bona fide' ORFs that were also found to be metal sensitive. The physical and phenotypic overlapping of this subset of ORFs is annotated in Additional data file 2, from which all redundant ORFs were removed. An additional four strains deleted in 'dubious' ORFs overlapping the 5'-end of ORFs not present in the mutant collection were replaced with the latter ORFs and included in the final dataset. Identical screening and validation assay conditions were applied to the 388 cadmium/nickel sensitive or resistant strains that were tested with four additional metal, plus the metalloid AsO2-. The following concentrations, determined by metal titrations coupled with serial dilution spot assays carried out on WT cells (as described above for cadmium and nickel), were utilized: 190 μmol/l HgCl2, 1.5 mmol/l NaAsO2, 2 mmol/l CoCl2, 18 mmol/l ZnCl2, and 15 mmol/l FeCl3.
Oligonucleotide primers used for DNA amplification
Individual amplicons were cloned into a modified (CpoI restriction site-containing) pYX212 vector. Following sequence verification, individual constructs were utilized for yeast transformation using the lithium acetate procedure .
Newport Green staining
Yeast cells were grown at 30°C to saturation, diluted to an OD600 of 0.3 and exposed to 1 mmol/l NiCl2 for 18 hours. Cells were then washed three times with phosphate-buffered saline (PBS) before being incubated for 30 minutes at 37°C in 1 ml PBS containing 7 μmol/l Newport Green DCF and 0.2% F-127 pluronic acid (Invitrogen-Molecular Probes, Eugene, OR, USA), followed by a further 30 minutes of incubation at room temperature. After an additional wash with PBS, cells were visualized by fluorescence microscopy using a Zeiss fluorescent microscope (argon laser; 488 nm). After visualization and cell counting, the fraction of fluorescent cells was determined in selected mutant strains (specified in the text) and in control WT cells.
Putative human homologs were identified with BLASTP searches and through the Princeton Protein Orthology Database . Information on human disease-related homologs was retrieved from the Online Mendelian Inheritance in Man database , the Saccharomcyes Genome Database , and by manual curation. Biologic processes associated with metal toxicity-modulating genes were identified and evaluated for statistical significance (P-value) with the GO Term Finder program . Enrichment ratios were calculated by comparing the representation of each GO term within individual sets of metal tolerance-modulating genes with their representation in the yeast genome. Interactions between metal tolerance-modulating genes were identified computationally using the Network Visualization System Osprey  and visualized as specified in the BioGRID database (version 2.035 release)  and in the Osprey reference manual. Subnetworks were defined as a minimum of three interacting gene products sharing at least one GO biologic process annotation and connected by at least two physical (two-hybrid, affinity capture-western, affinity capture-MS, or reconstituted complex) or genetic (synthetic lethality, synthetic growth defect, synthetic rescue, dosage rescue, phenotypic suppression, or phenotypic enhancement) interactions. P-values for individual subnetworks were determined by a one-tailed test based on the hypergeometric distribution, using the lowest possible 'child term' (the one yielding the lowest P-value) allowed by the present GO categorization. Random samplings of N proteins (where N is the number of toxicity-modulating gene products in the three sets of metal sensitivity-conferring mutations [cadmium/nickel, nickel, and cadmium]) were performed 10,000 times using a script written in Perl and the above-mentioned criteria, leaving out essential genes. They respectively yielded averages of 1.2, 2.1 and 9.5 subnetworks, as compared with the 9, 11 and 16 subnetworks identified with the cadmium/nickel (79 proteins), nickel (118 proteins), and cadmium (303 proteins) sets.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 provides representative primary screen data and serial dilution growth assays. Additional data file 2 shows detailed phenotypes and related information on the genes whose disruption affects metal tolerance. Additional data file 3 shows interaction subnetworks for gene products whose disruption causes nickel specific sensitivity. Additional data file 4 shows interaction subnetworks involved in cadmium-specific sensitivity Additional data file 5 illustrates broad-range metal uptake system mutants that affect cadmium tolerance. Additional data file 6 shows enhanced nickel tolerance conferred by disruption of the Tna1 and Fur4 transporters. Additional data file 7 provides the multimetal screen database.
endosomal sorting complexes required for transport
optical density at 600 nm
open reading frame
We thank our colleagues at the Department of Biochemistry and Molecular Biology, University of Parma, for help with statistical analysis (Riccardo Percudani), advice on metal chemistry (Angelo Merli), and for critical reading of the manuscript (Giorgio Dieci and Barbara Montanini). We are also grateful to Dan Burke (Department of Biochemistry and Molecular Genetics, University of Virginia) for advice on the use of the 384-multipinner replicator; to Roberto Tirindelli (Department of Neurosciences, University of Parma), and to Erasmo Neviani and Benedetta Bottari (Department of Genetics, University of Parma) for sharing their fluorescence microscopy instrumentation; and to Claudia Donnini (Department of Genetics, University of Parma) for the gift of the EUROSCARF control strains. This work was supported by the Regione Emilia-Romagna (PRRIITT Program SIQUAL), by the Ministry of University and Research (PRIN), and by a grant from the Fondazione Cariparma.
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