**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 [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 prosthetic group, coelenterazine, into excited coelenteramide and CO<sup>2</sup> (**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 [75].

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 yeast, due to their size [79].

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

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+-

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).

**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

cyt) are sensed by calmodulin, activating calcineurin.

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

**3. Aequorin, a transgenic molecular tool for detecting [Ca2+]cyt**

induced Ca2+ release (CICR) [67–70].

compartment. The increased cytosolic Ca2+ concentrations ([Ca2+]

**changes in** *S. cerevisiae*

26 Calcium and Signal Transduction

wall and reach the membrane to exert their toxic effect by disrupting the lipid bilayer or by

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, 38–47]. 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

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

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

**Figure 4.** Toxicity of heavy metal exposure. A. Schematic representation of transporters involved in the uptake of essential metals under normal conditions. B. Under high surplus of heavy metals, the transporters will carry the excess cations into the cell, where they bind non-specifically to biomolecules, altering their structure and functionality [91].

surplus metals (**Figure 4B**) when cells are exposed to contaminated environments [91].

, Mn2+ and Zn2+, respectively [84–86]. Low-affinity plasma

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

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

29

assaulting the membrane transporters.

in the high-affinity uptake of Cu<sup>+</sup>

**Figure 3.** Transgenic aequorin as a tool for measuring intracellular Ca2+. A. Schematic representation of aequorin bioluminescence [72–74]. Cells expressing apo-aequorin are first incubated with the cell-permeant coelenterazine to produce functional aequorin. When Ca2+ binds to aequorin, the protein undergoes a conformational change leading to the destabilization of the peroxide group (-O-O-), linking apoaequorin to coelenterazine, decomposing it to to coelenteramide and CO<sup>2</sup> ; the coelenteramide, which is in an excited state, generates blue light (λmax = 469 nm). B. Schematic representation of Ca2+-induced bioluminescence of yeast cells expressing reconstituted aequorin in the cytosol. When cells are exposed to an insult (e. g., environmental stress) the secondary messenger Ca2+ ions enter the cytosol and bind to aequorin, rendering the cell luminescent. Luminescence traces indicate the intensity and the duration of the [Ca2+] cyt wave [75, 76].

cytosol with the exception of La3+ (lanthanum) and to a lesser extent, Pr3+ and Nd3+ [81]. 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 [82].
