Despite constant efforts to maintain a clean environment, heavy metal pollution continues to raise challenges to the industrialized world. Exposure to heavy metals is detrimental to living organisms, and it is of utmost importance that cells find rapid and efficient ways to respond to and eventually adapt to surplus metals for survival under severe stress. This chapter focuses on the attempts done so far to elucidate the calcium-mediated response to heavy metal stress using the model organism Saccharomyces cerevisiae. The possibilities to record the transient elevations of calcium within yeast cells concomitantly with the heavy metal exposure are presented, and the limitations imposed by interference between calcium and heavy metals are discussed.
- heavy metal
- stress adaptation
- Saccharomyces cerevisiae
Responding to environmental stimuli is a prerequisite for cell adaptation to the ever-changing conditions in the cell surroundings. Stress conditions such as sudden changes of temperature, pH, irradiation, or elevations in various chemicals concentration need to be sensed by the cell in order to respond and adapt to these changes. Calcium ions are one of the most widespread second messengers in the eukaryotic cell, being responsible for triggering many responses to external stress conditions . Various biotic and abiotic stresses induce an increase in cytosolic calcium ions ([Ca2+]cyt), which in turn activate many proteins involved in signaling pathways, from yeast to humans . Thanks to easy manipulation, rapid growth, genetic amenability and with many genes bearing resemblance with higher eukaryotic genes, the yeast Saccharomyces cerevisiae is one of the widely used model organisms which helped in elucidating a wide variety of molecular mechanisms conserved along evolution, related to cell cycle and cell proliferation, homeostasis, adaptation and survival . Among many others studies, S. cerevisiae was used as a model to investigate the Ca2+-mediated responses to a variety of stimuli: hypotonic stress [4, 5, 6], hypertonic and salt stress , cold stress , high ethanol , β-phenylethylamine , glucose [11, 12], high pH [13, 14, 15], amidarone and antifungal drugs [16, 17], oxidative stress , eugenol [19, 20], essential oils [21, 22], or heavy metals [23, 24]. This chapter focuses on the studies made on S. cerevisiae cells in the effort to understand the role of calcium in cell response to heavy metal exposure.
Heavy metals represent a constant threat to clean environments as they are constantly released in the course of various anthropogenic activities (Figure 1), both industrial (mining, electroplating, smelting, metallurgical processes, nanoparticles, unsafe agricultural practices) and domestic (sewage and waste, metal corrosion), all in the context of rapid industrialization and urbanization . Heavy metals as contaminants are included in the category of persistent pollutants, because they cannot be destroyed or degraded. Being natural components of the earth crust, the environmental contamination becomes serious when heavy metals have the possibility to leach into surface or underground water, or undergo atmospheric deposition and metal evaporation from the water resources [26, 27, 28]. The ultimate threat imposed by the spread of heavy metals into the environment is their accumulation in the living organisms (Figure 1) via the food chain , inducing serious illnesses in animals and humans [30, 31, 32, 33, 34].
Some heavy metals (Co2+, Cu2+, Fe2+, Mn2+, Ni2+, Mo2+, and Zn2+) are essential for life, contributing to various biochemical and physiological functions in the living organisms. The nutritional requirements of these elements are generally low and they must be present in food in trace concentrations . However, excessive exposure to higher concentrations is deleterious, representing a threat to living organisms . Other heavy metals (Ag+, Cd2+, Pb2+, Hg2+) are not essential for life and have no established biological roles, but they are highly toxic because they compete with the essential metals for their biological targets or they simply bind nonspecifically to biomolecules; these metals are able to induce toxicity at low doses . Essential or not, the hazardous heavy metals such as Cd2+, Co2+, Cu2+, Mn2+, Ni2+, Pb2+, Zn2+ are known to be major threats to the environment . The molecular mechanisms involved in heavy metal transport and homeostasis have been intensively studied in S. cerevisiae , along with many aspects regarding their toxicity, tolerance, accumulation, or extrusion [38, 39, 40, 41, 42, 43, 44, 45, 46, 47]. Some of the relevant studies performed in S. cerevisiae correlating heavy metal exposure to calcium-related mechanisms are presented in the following section.
2. Calcium transport and homeostasis in S. cerevisiae
Intracellular calcium ions are important second messengers in all organisms, including yeast. The mechanisms involved in calcium transport and homeostasis in S. cerevisiae cells have been extensively studied [48, 49, 50]. Under normal conditions, the [Ca2+]cyt is maintained very low (50–200 nM) at external Ca2+ concentrations ranging from <1 μM to >100 mM [51, 52]. Abrupt changes in the environment can be transduced inside the yeast cells by sudden elevations in [Ca2+]cyt which can be the result of Ca2+ influx from outside the cell, Ca2+ release from internal stores (usually vacuole), or both (Figure 2). The yeast plasma membranes contain at least two different Ca2+ influx systems, the high-affinity Ca2+ influx system (HACS) and the low-affinity Ca2+ influx system (LACS), the former being responsible for Ca2+ influx under stress conditions . The HACS consists of two proteins, Cch1p and Mid1p, which are expressed and co-localize to the plasma membrane. These two subunits form a stable complex that is activated in response to sudden stimulation, boosting the influx of Ca2+ from the extracellular space. In S. cerevisiae, Cch1p is similar to the pore-forming α1 subunit of mammalian L-type voltage-gated Ca2+ channels (VGCCs) , while Mid1p is as a stretch-activated Ca2 +−permeable cation channel homologous to α2δ subunit of animal VGCCs . HACS is regulated by Ecm7p, a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes the λ subunits of VGCCs. Ecm7p is stabilized by Mid1p, and Mid1p is stabilized by Cch1p under non-signaling conditions .
Changes in the cell environment are signaled by a sudden increase in [Ca2+]cyt which can be a consequence of either external Ca2+ influx via the Cch1p/Mid1p channel on the plasma membrane [4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 56], release of vacuolar Ca2+ into the cytosol through the vacuole-located Ca2+ channel Yvc1p [18, 57], or both (Figure 2). After delivering the message, the level of [Ca2+]cyt is restored to the normal very low levels through the action of Ca2+ pumps and exchangers. Thus, the Ca2+-ATPase Pmc1p [58, 59] and a vacuolar Ca2+/H+ exchanger Vcx1p [60, 61] independently transport [Ca2+]cyt into the vacuole, while Pmr1p, the secretory Ca2+-ATPase, pumps [Ca2+]cyt into endoplasmic reticulum (ER) and Golgi along with Ca2+ extrusion from the cell [62, 63]. These responses are mediated by the universal Ca2+ sensor protein calmodulin that can bind and activate calcineurin, which inhibits at the post-transcriptional level the function of Vcx1p [60, 64, 65] and induces the expression of PMC1 and PMR1 genes via activation of the Crz1p transcription factor [64, 65]. The release of Ca2+ from intracellular stores stimulates the extracellular Ca2+ influx, a process known as capacitative calcium entry . Inversely, the release of vacuolar Ca2+ via Yvc1p can be further stimulated by the Ca2+ from outside the cell as well as that released from the vacuole by Yvc1p itself in a positive feedback called Ca2+-induced Ca2+ release (CICR) [67, 68, 69, 70].
3. Aequorin, a transgenic molecular tool for detecting [Ca2+]cyt changes in S. cerevisiae
As a second messenger, Ca2+ triggers a variety of cascade responses by temporarily activating Ca2+-binding components of signaling pathways which can lead either to adaptation to the environmental changes or to cell death . To determine the [Ca2+]cyt fluctuations during cell exposure to environmental changes, it is necessary to have an system capable to detect the sudden and transient elevations in [Ca2+]cyt. This was made possible by the isolation of aequorin, a Ca2+-binding photoprotein, isolated from the luminescent jellyfish, Aequorea victoria. Aequorin consists of two distinct units, the apoprotein apoaequorin (22 kDa) and the prosthetic group, coelenterazine, which reconstitute spontaneously in the presence of molecular oxygen, forming the functional protein [72, 73, 74]. Aequorin has become a useful instrument for the measurement of intracellular Ca2+ levels, since it has binding sites for Ca2+ ions responsible for protein conformational changes that convert through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO2 (Figure 3A). As the excited coelenteramide relaxes to the ground state, blue light (λmax 469 nm) is emitted and can be easily detected with a luminometer .
The expression of cDNA for apoaequorin in yeast cells and subsequent regeneration of apoaequorin into aequorin provide a noninvasive, nontoxic and effective method to detect the transient variations in yeast [Ca2+]cyt . The yeast strains to be analysed must express the A. victoria apoaequorin, and they need to be reconstituted into fully active aequorin by association with coelenterazine (Figure 3B). The latter cannot be synthesized by yeast itself; therefore, the way to achieve reconstitution is to incubate the apoaequorin-expressing cells with coelenterazine, prior to Ca2+ determination. Coelenterazine is a hydrophobic molecule, and therefore, it is easily taken up across yeast cell wall and membrane, making aequorin suitable as a Ca2+ reporter [52, 77]. Aequorin has a number of advantages over other Ca2+ indicators as follows: because the protein is large, it has a low leakage rate from cells compared to lipophilic dyes and it does not undergo intracellular compartmentalization or sequestration. Also, it does not disrupt cell functions, and the light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so problems with auto-fluorescence are eliminated . The primary limitation of aequorin is that the prosthetic group coelenterazine is irreversibly consumed to produce light. Such issues led to developments of other genetically encoded calcium sensors including the calmodulin-based sensor cameleon, which were less successful in yeast, due to their size .
In S. cerevisiae, the reconstituted aequorin is used primarily to detect the Ca2+ fluctuations in the cytosol ; there have been few attempts to obtain apoaequorins targeted to various cell compartment in yeast. One notable example was the construction of a recombinant apoaequorin cDNA whose product localizes in the ER lumen; using this product, a steady state of 10 μM Ca2+ was detected in the ER lumen of wild type cells, and it was possible to demonstrate that the Golgi pump Pmr1p also controls, at least in part, the ER luminal concentration of Ca2+ . Nevertheless, no reports on Ca2+ fluctuation in the ER in response to environmental stress are available in yeast. Surprisingly, no vacuole-targeted aequorin has been reported in yeast, in spite of the fact that the vacuole is the main storage compartment for Ca2+ in yeast; instead, the vacuolar Ca2+ traffic was determined indirectly, using genetic approaches (knockout mutants of various Ca2+ pumps and transporters) [61, 80] or blockers of the Ca2+ influx across the plasma membrane. This latter approach makes use of cell-impermeant Ca2+ chelators such as 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA)  or of lanthanide (Ln3+) ions, which are efficient blockers if ion channels due to size similarity between Ca2+ and Ln3+ . Of all Ln3+, Gd3+ is the most widely used as Ca2+-channel blocker. It was shown that at 1 mM concentration in the medium all the cations from the Ln3+ series block Ca2+ entry into cytosol with the exception of La3+ (lanthanum) and to a lesser extent, Pr3+ and Nd3+ . Care must be taken when using Ln3+ as channel blockers, as it was shown that at low concentrations Ln3+ may leak into the cytosol via the Cch1p/Mid1p system .
4. Correlations between calcium and heavy metal exposure as seen in S. cerevisiae cells
When grown in media contaminated with heavy metals, the yeast cell wall is the first to get in contact with the surplus cations present in the cell surroundings. If the contamination is not excessive, the cations would probably get stuck at this level, due to the mannoproteins that compose the outer layer of the cell wall (alongside of β-glucans and chitin) which are heavily phosphorylated and carboxylated, decorating the cell façade with a negatively charged shield prone to bind to positively charged species, such as the metal cations . Excess metal ions which escape the negatively charged groups on the cell wall surface penetrate the porous cell wall and reach the membrane to exert their toxic effect by disrupting the lipid bilayer or by assaulting the membrane transporters.
Several heavy metals (Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+) are essential for life in their ionic forms, acting mainly as cofactors for a variety of enzymes. They are necessary only in minute amounts inside the cell (hence their denomination as “trace” elements); if their concentration goes beyond the physiological threshold they become toxic by nonspecifically binding to any biomolecule bearing a negative charge or a metal-chelator fragment. The bipolar nature of trace metals determined the development of intricate cellular systems dedicated to their uptake, buffering, sequestration, intracellular trafficking, compartmentalization and excretion. As in many other directions of study, S. cerevisiae brought a considerable contribution to the understanding of the molecular mechanism involved in trace metal transport and homeostasis [3, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47]. Several heavy metal transporters were identified at the plasma membrane level (Figure 4A), with both high and low affinity. For example, Ctr1p, Smf1p and Zrt1p are involved in the high-affinity uptake of Cu+, Mn2+ and Zn2+, respectively [84, 85, 86]. Low-affinity plasma membrane transporters are more numerous and less specific: Fet4p for Fe2+, but also for Cu2+, Cd2+, Mn2+, and Zn2+; Zrt2p for Zn2+, but also for Fe2+, Co2+, Cu2+, Cd2+, Mn2+ [87, 88]. Transporters for phosphate or amino acids were also shown to participate in the low-affinity transport of Cd2+, Co2+, Cu2+, Mn2+, and Ni2+ [89, 90]. All these transporters are likely to be assaulted by surplus metals (Figure 4B) when cells are exposed to contaminated environments .
To have any chance of survival under heavy metal stress, the cell needs to be one step ahead of the “villain” ions and to get prepared for defense by using various strategies. The attempts to understand the role of calcium in preparing the yeast cell to resist the heavy metal attack are summarized in the following sections.
Cd2+ is one of the most studied non-essential heavy metals as it is a global environmental pollutant present in soil, air, water, and food, representing a major hazard to human health . External Cd2+ was shown to unequivocally induce the [Ca2+]cyt elevations in S. cerevisiae, as recorded in aequorin-expressing cells, which responded through a sharp increase in the [Ca2+]cyt, just a few seconds after being exposed to high Cd2+ . Interestingly, the chemically similar Zn2+ and Hg2+ failed to elicit [Ca2+]cyt elevations under the same conditions . The response to high Cd2+ depended mainly on external Ca2+ (transported through the Cch1p/Mid1p channel) and to a lesser extent on the vacuolar Ca2+ (released into the cytosol through the Yvc1p channel). The adaptation to high Cd2+ was influenced by perturbations in Ca2+ homeostasis in that the tolerance to Cd2+ often correlated with sharp Cd2+-induced [Ca2+]cyt pulses (Figure 5A, B), while the Cd2+ sensitivity was accompanied by the incapacity to rapidly restore the low levels of [Ca2+]cyt  (Figure 5C).
It had been suggested that Cd2+ toxicity was a direct consequence of Cd2+ accumulation in the ER and that Cd2+ does not inhibit disulphide bond formation (which could account for the lack of response in the case of Zn2+ and Hg2+) but perturbs calcium metabolism. Cd2+ activates the calcium channel Cch1/Mid1 under low external Ca2+, which also contributes to Cd2+ entry into the cell ; the protective effect of Ca2+ may be the result of competitive uptake between the two cations at the plasma membrane. In this line of evidence, it was shown that excess concentration of extracellular Ca2+ attenuates the Cd2+-induced ER stress . It was determined that divalent Cd2+ and Ca2+ have very similar physical properties, with ionic radii of Ca2+ (0.97 Å) and Cd2+ (0.99 Å) giving similar charge/radius ratios, meaning that these ions are able to exert strong electrostatic forces on biological macromolecules . Under such circumstances, the Cd2+-induced aequorin luminescence observed could also be the result of aequorin binding to Cd2+ instead of Ca2+. This was not the case though: when measuring the Cd2+ accumulation in yeast cells, it was revealed that the Cd2+-induced aequorin luminescence occurred significantly faster than the Cd2+ uptake, indicating that the luminescence produced was the result of increase in [Ca2+]cyt .
Cu2+ is one of the most important essential metals: a variety of enzymes require copper as a cofactor for electron transfer reactions . Nevertheless, when in excess, Cu2+ is very toxic in the free form because of its ability to produce free radicals when cycling between oxidized Cu2+ and reduced Cu+. Studies correlating Ca2+ with Cu2+ toxicity in yeast are scarce, but it had been known that the inhibitory effect of Cu2+ on glucose-dependent H+ efflux from S. cerevisiae could be alleviated by Ca2+ . The role of Ca2+ in mediating the cell response to high concentrations of Cu2+ was investigated in parallel with Cd2+, and it was noted that exposure to high Cu2+ determined broad and prolonged [Ca2+]cyt waves which showed a different pattern from the [Ca2+]cyt pulses induced by high Cd2+ . In contrast to Cd2+, Ca2+ − mediated responses to high Cu2+ depend predominantly on internal Ca2+ stores  (Figure 6A).
It was found that the cell exposure to high Cu2+-induced broad Ca2+ waves into the cytosol which were accompanied by elevations in cytosolic Ca2+ with patterns that were influenced by the Cu2+ concentration but also by the oxidative state of the cell [18, 24]. When Ca2+ channel deletion mutants were used, it was revealed that the main contributor to the cytosolic Ca2+ pool under Cu2+ stress was the vacuolar Ca2+ channel, Yvc1p, also activated by the Cch1p-mediated Ca2+ influx (Figure 6). Using yeast mutants defective in the Cu2+ transport across the plasma membrane, it was found that the Cu2+-dependent Ca2+ elevation could correlate with the accumulated metal, but also with the Cu2+ − induced oxidative stress and the overall oxidative status. Moreover, it was revealed that Cu2+ and H2O2 acted in synergy to induce Ca2+-mediated responses to external stress . Interestingly, other redox active metals such as Mn2+ or Fe2+ were inactive in inducing [Ca2+]cyt waves (, unpublished observations), probably because these metals are less redox-reactive than the Cu2+/Cu+ couple (Figure 6D) under aerobic conditions .
High manganese failed to elicit Ca2+ elevations irrespective of the magnitude of the insult applied (; unpublished observations). The response was monitored over a wide range of concentrations (from the quasi-physiological 0.5 mM to the super lethal 50 mM) and times (up to 60 min of exposure). Of all the cations, Mn2+ is the closest to Ca2+ in terms of ionic radius and charge. This similarity is so relevant that Mn2+ effectively supports yeast cell-cycle progression in place of Ca2+ . This similarity probably renders the cell irresponsive to high concentrations of an otherwise toxic metal. A more subtle Mn2+-Ca2+ interplay exists though, being manifested at several levels . For example, high Mn2+ is controlled by calcineurin/Crz1p-regulated Pmr1p and Pun1p . Importantly, the tolerance of yeast cells to Mn2+ is related to both Pmr1p and Vcx1p [41, 64, 65, 101] two determinants of maintaining low [Ca2+]cyt by transporting the ions to the vacuole and Golgi/ER, respectively [60, 61, 62, 63]. The Ca2+-dependent response to Mn2+ surplus seems to be induced not by external Mn2+, but by the cations accumulated inside the cell. For example, it was found that internal Mn2+ can be redistributed by calcium-stimulated vesicle trafficking .
Fe2+ toxicity can be the result of direct ionic effect, but the indirect effect of catalyzing Fenton reactions, in which highly reactive oxygen species arise, represents the main concern raised by Fe2+ surplus. As in the case of Mn2+, excess Fe2+ did not elicit sudden elevations in [Ca2+]cyt upon exposure . It had been reported that yeast strains lacking the components of the Cch1p/Mid1p plasma membrane channel were hypersensitive to Fe2+. When measuring the relative Ca2+ accumulation, it was noted that iron stress also increased the residual Ca2+ uptake in the cch1Δ mid1Δ double knockout mutant . As the Ca2+ measurements in this study were done radiometrically, there must have been a considerable lag between application of the stimulus and Ca2+ measurement (unlike aequorin determinations, which allow Ca2+ detection simultaneously with stimulus application), and the mutant’s sensitivity towards Fe2+ might have been caused by Ca2+ lingering in the cytosol, as in the case of Cd2+-sensitive mutants .
4.5. Other metals
The surplus of heavy metals such as Ni2+, Co2+, Pb2+, Hg2+, and Ag+ did not have the ability to rapidly induce elevations in [Ca2+]cyt. In some cases, (Ni2+ and Co2+) exogenous Ca2+ alleviated the toxicity of the metal ions, but this effect was rather related to the inhibition of Co2+ or Ni2+ uptake by Ca2+ .
5. Concluding remarks
In this chapter, we attempted to highlight the studies made in S. cerevisiae which correlate the exposure to high concentrations of heavy metals with the Ca2+-mediated cellular responses. S. cerevisiae is a very good model to study the cell response to sudden changes of metal concentration in the environment; such studies were greatly facilitated by the ease of obtaining yeast cells expressing aequorin in the cytosol, thus allowing the real-time detection of [Ca2+]cyt fluctuations. By combining Ca2+ monitoring under metal stress with the genetic approaches that make use of mutants with perturbed heavy metal or Ca2+ homeostasis, important aspects related to cell adaptation or cell death under heavy metal stress have been elucidated. Using yeast cells expressing aequorin in the cytosol provides answers regarding the immediate Ca2+-mediated responses, which are crucial for deciding the cell fate. Nevertheless, to understand the Ca2+-mediated cell responses which occur at later phases, developing sensitive Ca2+ sensors targeted to specific compartments is still a desiderate for future studies.