**2. Calcium transport and homeostasis in** *S. cerevisiae*

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 [3]. Among many others studies, *S. cerevisiae* was used as a model to investigate the Ca2+-mediated responses to a variety of stimuli: hypotonic stress [4–6], hypertonic and salt stress [7], cold stress [8], high ethanol [9], β-phenylethylamine [10], glucose [11, 12], high pH [13–15], amidarone and antifungal drugs [16, 17], oxidative stress [18], 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

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 [25]. 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 [26–28]. 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 [29], inducing serious illnesses in animals and humans [30–34].

**Figure 1.** Schematic representation depicting the sources of heavy metal pollution and the impact on the environment

and organisms.

understand the role of calcium in cell response to heavy metal exposure.

24 Calcium and Signal Transduction

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 [48–50]. 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 [50]. The HACS consists of two proteins, Cch1p and Mid1p, which are expressed and colocalize 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 voltagegated Ca2+ channels (VGCCs) [53], while Mid1p is as a stretch-activated Ca2 +−permeable cation channel homologous to α2δ subunit of animal VGCCs [54]. 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 [55].

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 [4–14, 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<sup>+</sup> 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

the environmental changes or to cell death [71]. 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 [72–74]. 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 pros-

Calcium and Cell Response to Heavy Metals: Can Yeast Provide an Answer?

coelenteramide relaxes to the ground state, blue light (λmax 469 nm) is emitted and can be

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 [76]. 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 [78]. 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 *S. cerevisiae*, the reconstituted aequorin is used primarily to detect the Ca2+ fluctuations in the cytosol [76]; 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+ [63]. 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) [18] or of lanthanide (Ln3+) ions, which are efficient blockers if ion channels due to size similarity between Ca2+ and Ln3+ [80]. 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

(**Figure 3A**). As the excited

http://dx.doi.org/10.5772/intechopen.78941

27

thetic group, coelenterazine, into excited coelenteramide and CO<sup>2</sup>

easily detected with a luminometer [75].

in yeast, due to their size [79].

**Figure 2.** The mechanisms by which yeast cell regulate cell calcium. Under external stresses, the plasma membrane Ca2+ influx systems HACS (high-affinity Ca2+ influx system) and to a lesser extent LACS (low-affinity Ca2+ influx system) are activated, resulting in a rapid influx of Ca2+ into the cytosol. Transient increases in intracellular Ca2+ concentrations may also be due to release from internal compartments, mainly the vacuole, via Yvc1p. Unlike mammalian cells, where the main Ca2+ stores reside in the endoplasmic reticulum (ER), in yeast the intracellular stores are situated in the vacuole compartment. The increased cytosolic Ca2+ concentrations ([Ca2+] cyt) are sensed by calmodulin, activating calcineurin. Activated calcineurin acts on its downstream target Crz1p, inducing its translocation from cytoplasm to nucleus to further induces the expression of a set of Ca2+/calcineurin-dependent target genes, including *PMC1* and *PMR1*. Calcineurin also regulates Vcx1p at post-transcriptional level. Subsequently, the [Ca2+]cyt concentration is reduced to basal levels via uptake by organelles, especially vacuole (by means of Pmc1p and Vcx1p) and Golgi (by means of Pmr1p).

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 [66]. 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) [67–70].
