Manganese Suppresses the Haploinsufficiency of Heterozygous trpy1Δ/TRPY1 Saccharomyces cerevisiae Cells and Stimulates the TRPY1-Dependent Release of Vacuolar Ca²⁺ under H₂O₂ Stress

Abstract

Transient potential receptor (TRP) channels are conserved cation channels found in most eukaryotes, known to sense a variety of chemical, thermal or mechanical stimuli. The Saccharomyces cerevisiae TRPY1 is a TRP channel with vacuolar localization involved in the cellular response to hyperosmotic shock and oxidative stress. In this study, we found that S. cerevisiae diploid cells with heterozygous deletion in TRPY1 gene are haploinsufficient when grown in synthetic media deficient in essential metal ions and that this growth defect is alleviated by non-toxic Mn²⁺ surplus. Using cells expressing the Ca²⁺-sensitive photoprotein aequorin we found that Mn²⁺ augmented the Ca²⁺ flux into the cytosol under oxidative stress, but not under hyperosmotic shock, a trait that was absent in the diploid cells with homozygous deletion of TRPY1 gene. TRPY1 activation under oxidative stress was diminished in cells devoid of Smf1 (the Mn²⁺-high-affinity plasma membrane transporter) but it was clearly augmented in cells lacking Pmr1 (the endoplasmic reticulum (ER)/Golgi located ATPase responsible for Mn²⁺ detoxification via excretory pathway). Taken together, these observations lead to the conclusion that increased levels of intracytosolic Mn²⁺ activate TRPY1 in the response to oxidative stress.

1. Introduction

Living cells are continuously exposed to various changes that threaten the dynamic equilibrium associated with the steady state of homeostatic balance. Such changes—often induced by stress agents—need to be sensed and signaled by cell components which belong to intricate networks responsible for homeostatic regulation. Calcium is a secondary messenger used by all eukaryotes—animal, plants, microorganisms—to connect various stimuli or stresses to their corresponding cellular responders. The budding yeast Saccharomyces cerevisiae has been constantly used as a model eukaryote to study the calcium-dependent response to various types of external stresses, which include salt [1], hypotonic [2,3], hypertonic [1,4,5], salicylate [6], alkaline [7], cold [8], ethanol [9,10], drugs [11] antifungals [12,13,14,15,16], electric [17] oxidative [18,19,20] or heavy metal [8,20,21,22] insults. The S. cerevisiae cells respond to such stresses by a sudden increase in cytosolic Ca²⁺—denoted henceforth [Ca²⁺]_cyt—following the stimulus-dependent opening of Ca²⁺ channels situated in the plasma membrane and/or in internal compartments. Abrupt increase in [Ca²⁺]_cyt represents a versatile and universally used mechanism which triggers either cell survival/adaptation or cell death [23]. In S. cerevisiae the stress-dependent rise in [Ca²⁺]_cyt can be a consequence of Ca²⁺ influx via the Cch1/Mid1 channel on the plasma membrane [1,2] release of vacuolar Ca²⁺ into the cytosol through the vacuole-located Ca²⁺ channel TRPY1 [4,24], or both [19,20]. After delivering the message, the normal very low level of [Ca²⁺]_cyt is restored through the action of Ca²⁺ pumps and exchangers [25]. Thus, the Ca²⁺-ATPase Pmc1 [26] and a vacuolar Ca²⁺/H⁺ exchanger Vcx1 [27,28] independently transport [Ca²⁺]_cyt into the vacuole, while Pmr1, the secretory Ca²⁺-ATPase, pumps [Ca²⁺]_cyt into endoplasmic reticulum (ER) and Golgi along with Ca²⁺ extrusion from the cell [29,30].

In S. cerevisiae, TRPY1 is almost exclusively localized at the vacuolar membrane [4], playing an important role in adaptation to environmental stresses [4,19,20,21]. Initially named Yvc1, TRPY1 is encoded by TRPY1 gene (systematic gene name, YOR087W) and it is the only member of the TRP (Transient Receptor Potential) superfamily of cationic channels expressed in S. cerevisiae [31]. TRP channels are conserved cation channels found in most eukaryotes, known to sense chemical, thermal, or mechanical stimuli in animals [32]. In yeast, TRPY1 is the main channel responsible for of [Ca²⁺]_cyt elevation under hyperosmotic shock [4,31], when calcium accrues predominantly from vacuolar stores [4]. This behavior can be explained by the mechano-sensitive traits of TRPY1: under hypertonic conditions water evacuates passively from the cytoplasm and then from the vacuole causing deformation of the vacuolar membrane and consequently the opening of the TRPY1 channel, with the release of vacuolar Ca²⁺ [5,33]. In contrast, under alkaline stress, the elevated [Ca²⁺]_cyt has its origin exclusively from the cell’s exterior, with the Cch1/Mid1 channel solely responsible for the majority of Ca²⁺ entry, and with no contribution of vacuolar Ca²⁺ [7]. In between these two situations, oxidative stress triggers [Ca²⁺]_cyt waves which pool both external and vacuolar Ca²⁺ [19]. TRPY1 is necessary for attaining a maximum level of [Ca²⁺]_cyt under oxidative stress and TRPY1 depends on [Ca²⁺]_cyt elevation for maximal gating, in a process known as Ca²⁺-induced Ca²⁺ release [34].

TRPY1 gene is not essential for survival and the knockout mutant cells trpy1Δ have no clear growth defects under various stresses. Rather, it was shown that trpy1Δ cells are slightly more resistant to the oxidative stress imposed by exogenous hydrogen peroxide or tert-butylhydroperoxide [19] and Cu²⁺ [20] but also less fit under high Cd²⁺ [21] or tunicamycin-induced ER-stress in Ca²⁺-depleted medium [31]. In contrast, cells overexpressing the TRPY1 gene are hypersensitive to surplus Ca²⁺ [4] or oxidative stress [19]. Also, it was revealed in a wide-scale survey that heterozygous trpy1Δ/TRPY1 diploid cells are less fit under nutrient limiting conditions than the wild-type TRPY1/TRPY1 ([35], Supplementary material). Haploinsufficiency occurs when the heterozygous mutation of a gene in a diploid organism results in a reduction of the corresponding gene product which can be correlated with negative alterations of the wild-type phenotype. In this study, we performed a chemical screen and found that non-toxic concentrations of Mn²⁺ alleviated the trpy1Δ/TRPY1 haploinsufficiency observed by us in minimal growth medium containing half of the recommended amount of essential metal ions, probably by stimulating the TRPY1-mediated Ca²⁺ release into the cytosol.

2. Materials and Methods

2.1. Yeast Strains and Growth Media

The S. cerevisiae diploid strains used in this study were isogenic with the “wild-type” (WT) parental strain BY4743 (MATa/α; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; met15Δ0/MET15; LYS2/lys2Δ0; ura3Δ0/ura3Δ0), a S288C-based yeast strain [36]. The strains harbored either heterozygous (BY4732, orf::kanMX4/ORF) or homozygous (BY4732, orf::kanMX4/orf::kanMX4) knockout mutations of individual gene open reading frames (ORF). The heterozygous knockout mutants are referred to in the text as orfΔ/ORF and were cch1Δ/CCH1, mid1Δ/MID1, pmc1Δ/PMC1, pmr1Δ/PMR1, vcx1Δ/VCX1, trpy1Δ/TRPY1. The homozygous knockout mutants are referred to in the text as orfΔ/orfΔ and were trpy1Δ/trpy1Δ, smf1Δ/smf1Δ, and pmr1Δ/pmr1Δ. The strains were obtained from EUROSCARF (European S. cerevisiae Archive for Functional Analysis, www.euroscarf.de) and were propagated, grown, and maintained in YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) or SD (0.17% yeast nitrogen base without amino acids, 0.5% (NH₄)₂SO₄, 2% glucose, supplemented with the necessary amino acids) [37]. The strains transformed with the plasmids harboring apo-aequorin cDNA [38] were selected and maintained on SD lacking uracil (SD-Ura). Minimal defined media (MM) were prepared adding individual components as described [37] using ultrapure reagents (Merck, Darmstadt, Germany) and contained 1 mM Ca²⁺, 0.25 µM Cu²⁺, 2 µM Mn²⁺, 2 µM Fe³⁺ and 2 µM Zn²⁺. Low-metal minimal defined medium (LMeMM) had 0.5 mM Ca²⁺, 0.1 µM Cu²⁺, 1 µM Mn²⁺, 1 µM Fe³⁺ and 1 µM Zn²⁺, corresponding roughly to half of the amount of essential metals recommended [37]. The concentrations of metals in MM and LMeMM were confirmed by inductively coupled plasma with mass spectrometry (ICP-MS, Perkin-Elmer ELAN DRC-e, Concord, ON, Canada) against Multielement ICP Calibration Standard 3, matrix 5% HNO₃ (Perkin-Elmer Pure Plus, Shelton, CT, USA). All synthetic media had their pH adjusted to 6.5. For solid media, 2% agar was used. For growth improvement, all the synthetic media were supplemented with an extra 20 mg/L leucine [39]. All chemicals, including media reagents were from Merck (Darmstadt, Germany),

2.2. Plasmid and Yeast Transformation

For heterologous expression of aequorin, yeast cells were transformed with the multicopy URA3-based plasmid pYX212-cytAEQ harboring the apo-aequorin cDNA under the control of the strong TPI (triosephosphate isomerase) yeast promoter [40]. Plasmid pYX212-cytAEQ was a generous gift from Martegani and Tisi (University of Milano-Bicocca, Milan, Italy). Yeast transformation [41] was performed using S.c. EasyComp™ Transformation Kit (Invitrogen, Carlsbad, CA, USA) following manufacturer’s indications.

2.3. Yeast Cell Growth Assay

2.3.1. Growth in Liquid Media

Unless otherwise specified, cells were incubated at 30 °C under agitation (200 rpm). Yeast strains were pre-grown overnight in MM then diluted (1/20) in fresh MM and grown for 2 h. Cells were harvested by centrifugation, washed with ice-cold water, and resuspended in liquid LMeMM at density which corresponded to optical density measured at 600 nm (OD₆₀₀) = 0.05. Strain growth in liquid LMeMM was monitored in time by measuring OD₆₀₀ recorded in a plate reader equipped with thermostat and shaker (Varioskan, Thermo Fisher Scientific, Vantaa, Finland). Relative growth was expressed as the ratio between OD₆₀₀ recorded at time t and OD₆₀₀ recorded at time 0. For screening of chemicals against trpy1Δ/TRPY1 haploinsufficiency (HIP), cells shifted to LMeMM (OD₆₀₀ = 0.05) were incubated for 2 h before chemicals were added from concentrated sterile stocks. Cell growth (%) was determined 24 h from chemical addition and calculated relatively to growth (OD₆₀₀) of WT strain, no added chemicals. Chemicals used were CuCl₂, FeCl₂, MnCl₂, ZnCl₂, ascorbate, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), GdCl₃, glutathione (GSH), indole and were all of high-grade purity.

2.3.2. Growth on Solid Media

For dilution plate assay, exponentially growing cells were 10-fold serially diluted in a 48-well microtiter plate and stamped on agar plates using a pin replicator (approximately 4 μL/spot). Plates were photographed after incubation at 30 °C for 3 days.

2.4. TRPY1 Gene Expression by Quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR)

Wild-type cells BY4743 (TRPY1/TRPY1), heterozygous (trpy1Δ/TRPY1), and homozygous (trpy1Δ/trpy1Δ) diploid cells from overnight pre-cultures were inoculated at OD₆₀₀ = 0.1 in MM or LMeMM and grown to OD₆₀₀ = 0.5 before Mn²⁺ was added to final concentration 10 µM, then incubated for 2 additional hours before being harvested for total ribonucleic acid (RNA) isolation. Total RNA was isolated using the RiboPure™ RNA Purification Kit for yeast (Ambion™, Thermo Fischer Scientific, Vilnius, Lithuania) following the manufacturer’s instructions. Approximately 500 ng of total RNA was transcribed into cDNA using GoScript™ Reverse Transcription System (Promega, Madison, WI, USA). Finally, a total of 10 ng cDNA was used for each qRT-PCR done with the GoTaq^® qPCR Master Mix (Promega). Each reaction was performed in triplicate using MyiQ Single-Color Real-Time PCR Detection System (BioRad, Hercules, CA, USA). Expression of TRPY1 mRNA was normalized to the relative expression of ACT1 in each sample. The qRT-PCR cycling conditions were 95 °C for 1 min, and 40 cycles of 95 °C for 10 s, 59 °C for 10 s, 72 °C for 12 s. The primers used for amplification of cDNA were: TRPY1-F: 5′-AGATTCTCAG GGTTACGTTA, TRPY1-R: 5′-CAATATGGAATACCACTCAC; ACT1-F: 5′-GGTTGCTGCTTTGGTTATTG, ACT1-R: 5′-CAATTGGGTAACGTAAAGTC.

2.5. Assay of Cell Mn²⁺

Measurements of cell total manganese content were done on cells grown in LMeMM medium to an OD₆₀₀ of 1.0. Cells were harvested in triplicate samples, washed two times in ice-cold 10 mM 2-(N-morpholino) ethanesulfonic acid (MES)-Tris buffer (pH 6.0). Cells were finally suspended in deionized water (OD₆₀₀ = 10) and used for manganese and cell protein assay. Manganese analysis was done by ICP-MS after digestion of cells with 65% ultrapure HNO₃ (Merck, Darmstadt, Germany). The metal cellular content was normalized to total cellular proteins, as described [42]. Total cellular manganese was expressed as nanomoles of metal per mg cell protein.

2.6. Detection of [Ca²⁺]_cyt by Aequorin Bioluminescence Assay

Cells transformed with pYX212-cytAEQ were maintained on SD-Ura selective medium and prepared for Ca²⁺ dependent luminescence detection as described [43] with slight modifications. Overnight pre-cultures of cells expressing apo-aequorin were washed and suspended (OD₆₀₀ = 0.5) in LMeMM-Ura then incubated to late exponential phase (OD₆₀₀ = 1, 4–6 h). For pre-incubation with Mn²⁺, MnCl₂ was added to the desired concentration and cells were grown for an additional 2 h. Cells were harvested by centrifugation and resuspened (to OD₆₀₀ = 10) in LMeMM-Ura in which the corresponding Mn²⁺ concentration was maintained. To reconstitute functional aequorin, native coelenterazine was added to the cell suspension (from a methanol stock, 20 µM final concentration) and the cells were incubated for 2 h at 30 °C in the dark. The excess coelenterazine was removed by centrifugation. The cells (approximately 10⁷ cells/determination) were finally resuspended in LMeMM-Ura with corresponding Mn²⁺ concentration and transferred to the luminometer tube. A cellular luminescence baseline was determined for each strain by approximately one minute of recordings at 1/s intervals. After ensuring a stable signal, chemicals tested were injected from sterile stocks to give the final concentrations indicated, and the Ca²⁺-dependent light emission was monitored in a single tube luminometer (Turner Biosystems, 20ⁿ/20, Sunnyvale, CA, USA). The light emission was measured at 1 s intervals and expressed as relative luminescence units (RLU). To ensure that the total reconstituted aequorin was not limiting in our assay, at the end of each experiment aequorin activity was checked by lysing cells with 1% Triton X-100 with 5 mM CaCl₂ and only the cells with considerable residual luminescence were considered. Relative luminescence emission was normalized to an aequorin content giving a total light emission of 10⁶ RLUs in 10 min after lysing cells with 1% Triton X-100. The relative luminescence maximum (RLM) was the average of the RLUs flanking the maximum value minus the average luminescence baseline recorded before cells were exposed to the stimulus (10 values on each side), all normalized as described above.

2.7. Statistics

All experiments were repeated, independently, in three biological replicates at least. For each individual experiment, values were expressed as the mean ± standard error of the mean (SEM). For aequorin luminescence determinations, traces represent the mean ± SEM from three independent transformants. The numerical data were examined by Student t test or by analysis of variance with multiple comparisons (ANOVA) using the statistical software Prism version 6.05 for Windows (GraphPad Software, La Jolla, CA, USA). The differences were considered to be significant when p < 0.05. One sample t test was used for the statistical analysis of each strain/condition compared with a strain/condition considered as reference. Asterisks indicate the level of significance: * p < 0.05, ** p < 0.01, and *** p < 0.001.

3. Results

3.1. Haploinsufficiency of Yeast Strain Heterozyous for TRPY1 Is Alleviated by Mn²⁺

To highlight new aspects related to TRPY1 function in yeast cells, the main target of our study was to identify conditions which interfere with TRPY1 activity. In this direction, haploinsufficiency is a genetic trait which can be very useful in the attempts to identify small molecules which influence the behavior of functional proteins [44]. A genome-wide survey had already pinpointed the heterozygous trpy1Δ/TRPY1 as possibly less fit under nutrient limiting conditions ([35], Supplementary material). We noticed that the growth of the heterozygous trpy1Δ/TRPY1 diploid mutant was not significantly different from the growth of the wild-type diploid when the two strains were incubated in YPD, SD (data not shown) or MM medium (Figure 1a), but trpy1Δ/TRPY1 cells exhibited somehow slower growth (p < 0.001) in minimal synthetic medium LMeMM which had approximately half of the amount of essential metals recommended [37] (Figure 1b).

The haploinsufficiency in LMeMM was noted only for TRPY1; no similar phenotype was recorded for heterozygous strains with mutations in the genes which encode the other transporters known to participate in regulating [Ca²⁺]_cyt, e.g., CCH1, MID1, PMC1 or VCX1 (Figure 2a, dark blue bars).

To identify compounds which potentially interact with TRPY1 activity we screened for conditions which may alleviate or augment the haploinsufficient phenotype observed. The tested substances are presented in

Table 1. Substances screened for the capacity to alleviate trpy1Δ/TRPY1 haploinsufficiency in LMeMM.

Substance Tested 1	Concentration Range	Effect on trpy1Δ/TRPY1 Haploinsufficiency
CaCl2	2–10 mM	No
CuCl2	0.5–50 µM	No
FeCl3	1–50 µM	No
MnCl2	1–50 µM	Alleviation
ZnCl2	1–50 µM	No
EGTA	0.1–2 mM	Augmentation
GdCl3	0.1–1 mM	Augmentation
Ascorbate	1–10 mM	No
Glutathione 2	1–10 mM	No
Indole 3	1–10 mM	No

¹ The quantitative results are presented in Supplementary Files, Figure S1. ² Glutathione depletion leads to TRPY1 activation [45]. ³ Indole activates TRPY1 under hyperosmotic stress [46].

. These substances were added to the LMeMM at the point where the heterozygous trpy1Δ/TRPY1 diploid cells were in the early log phase of growth (OD₆₀₀ = 0.1) and the effect on growth was determined 24 h after chemical addition. We tested the effect of adding physiological concentrations of the metals initially depleted in LMeMM (i.e., Ca²⁺, Cu²⁺, Fe³⁺, Mn²⁺, Zn²⁺) but also of glutathione and indole, which had been reported to interact with TRPY1 [45,46]. As glutathione is a universal intracellular antioxidant, we also tested an exogenous antioxidant, i.e., ascorbate. EGTA was chosen as a chelator of Ca²⁺ in the growth medium, while Gd³⁺ was tested as a blocker of the Ca²⁺ channels. The results showing the effect of the tested compounds on trpy1Δ/TRPY1 haploinsufficiency in LMeMM are included in Supplementary Files, Figure S1.

Out of the compounds tested, only Mn²⁺ alleviated the trpy1Δ/TRPY1 haploinsufficiency observed in LMeMM. In contrast, EGTA and Gd²⁺ augmented the LMeMM-associated growth defect (Figure 2a). The level of TRPY1 gene expression was lower in trpy1Δ/TRPY1 compared with wild-type, but this level was not significantly influenced by surplus Mn²⁺ (Figure 2b), suggesting that Mn²⁺ acts—directly or indirectly—by activating the TRPY1 channel. The trpy1Δ/TRPY1 haploinsufficiency was also noted on solid LMeMM. In contrast, the trpy1Δ/trpy1Δ growth was not affected (Figure 2c).

3.2. Mn²⁺ Potentiates the Increase of [Ca²⁺]_cyt under Oxidative Stress in Strain trpy1Δ/TRPY1

The observation that both EGTA (calcium chelator) and Gd³⁺ (inhibitor of Ca²⁺ transport across plasma membrane) augmented the LMeMM-related haploinsufficiency of the trpy1Δ/TRPY1 strain prompted the idea that preventing Ca²⁺ entry into the cell is deleterious to trpy1Δ/TRPY1, while the observed opposite action of Mn²⁺ may be the result of Mn²⁺-related activation of the extant TRPY1 that would compensate the heterozygous loss of TRPY1. To check this possibility, we used cells expressing aequorin, a system suitable for detecting transient modifications in the [Ca²⁺]_cyt [38]. For this purpose, trpy1Δ/TRPY1 cells were transformed with a plasmid harboring the cDNA of the luminescent Ca²⁺ reporter apo-aequorin under the control of a constitutive promoter, which afforded abundant transgenic protein within the cytosol [40]. The cells expressing apo-aequorin were pre-treated with the cofactor coelenterazine to reconstitute the functional aequorin, and then the cells were exposed to various stimuli directly in the luminometer tube. It was noted that while Mn²⁺ alone failed to induce any increase in the luminescence of the reconstituted aequorin (data not shown), cell pre-incubation with 10 µM Mn²⁺ significantly increased the [Ca²⁺]_cyt elevation induced by H₂O₂ exposure (Figure 3a). Remarkably, pre-incubation with Mn²⁺ did not augment the cell luminescence when aequorin-expressing trpy1Δ/TRPY1 cells were exposed to hyperosmotic shock (HOS, Figure 3b,c). Surplus Mn²⁺ reached maximum stimulating activity on trpy1Δ/TRPY1 cells exposed to H₂O₂ at 10 µM (Figure 3c), a non-toxic concentration to both WT and trpy1 mutants.

3.3. Mn²⁺ Stimulates TRPY1 to Release Ca²⁺ into the Cytosol under H₂O₂ Stress

Furthermore, we wondered whether Mn²⁺ influence on elevating [Ca²⁺]_cyt under H₂O₂ was the result of TRPY1 stimulation by Mn²⁺. To test this possibility, we determined the effect of Mn²⁺ on the Ca²⁺-mediated response to H₂O₂ of cells completely lacking TRPY1. It was noticed that in WT cells expressing reconstituted aequorin, the H₂O₂-induced Ca²⁺-dependent luminescence was significantly increased by cell pre-incubation with 10 µM Mn²⁺, indicating that in the case of WT cells too, Mn²⁺ potentiates the Ca²⁺-dependent response to oxidative stress (Figure 4a). In contrast, homozygous knockout mutant trpy1Δ/trpy1Δ exhibited much lower H₂O₂-luminescence (Figure 4b, left), which was not altered by pre-incubation with 10 µM Mn²⁺ (Figure 4b, right). This observation suggested that Mn²⁺ augments the H₂O₂-induced [Ca²⁺]_cyt elevation by activating TRPY1, a phenotype clearly absent in the trpy1Δ/trpy1Δ homozygous knockout mutant (Figure 4b).

If Mn²⁺ were required for TRPY1 activation under oxidative stress, it would be expected that cells with low cytosolic Mn²⁺ would be less responsive in terms of increasing the [Ca²⁺]_cyt under oxidative stress. Mn²⁺ is an essential metal which is carried into the yeast cell by the divalent metal ion transporter Smf1, known to have high affinity for Mn²⁺ [47]. It was noted indeed that the homozygous knockout mutant smf1Δ/smf1Δ expressing reconstituted aequorin exhibited a significantly lower luminescence trace when exposed to H₂O₂ than WT (Figure 4c). In this line of evidence, the pmr1Δ/pmr1Δ cells expressing reconstituted aequorin responded strongly to H₂O₂ (in media not supplemented with Mn²⁺) with a luminescence curve (Figure 4d) which was not significantly different from that obtained from WT cells preincubated with 10 µM Mn²⁺ (Figure 4a, right). PMR1 encodes the major Golgi/ER membrane P-type ATPase ion pump responsible for transporting Ca²⁺ and Mn²⁺ into the Golgi apparatus [48] providing a major route for cellular detoxification of Mn²⁺ via the secretory pathway vesicles [49]. It was shown that cells knockout for PMR1 gene have the intracellular Mn²⁺ levels considerably higher than the WT cells [50], a fact that may account for the stronger response of pmr1Δ/pmr1Δ cells (Figure 4d) compared to WT (Figure 4a, left). In this line of evidence, we found that pmr1Δ/pmr1Δ cells had significantly (p < 0.05) more cellular Mn²⁺ than the WT, while smf1Δ/smf1Δ cell had significantly (p < 0.05) less cellular Mn²⁺ than the WT (

Table 2. Manganese content (pmoles/mg cell protein) of diploid yeast cells grown in LMeMM supplemented or not with Mn²⁺.

Strain	Surplus Mn2+
	0	10 µM	50 µM
WT	0.12 ± 0.12	0.64 ± 0.2	0.92 ± 0.3
trpy1Δ/TRPY1	0.11 ± 0.14	0.72 ± 0.1	0.98 ± 0.2
trpy1Δ/trpy1Δ	0.12 ± 0.2	0.7 ± 0.2	0.84 ± 0.2
smf1Δ/smf1Δ	0.01 ± 0.014	0.1 ± 0.2	0.72 ± 0.2
pmr1Δ/pmr1Δ	0.7 ± 0.22	0.84 ± 0.2	8.4 ± 1.2

).

The influence of Mn²⁺ on the RLM recorded under oxidative stress for various strains which expressed reconstituted aequorin was also determined (Figure 5), revealing that Mn²⁺ significantly increased the RLM of WT cells exposed to H₂O₂. RLM determined for trpy1Δ/trpy1Δ was significantly low and was not augmented by Mn²⁺, indicating the necessity of functional TRPY1 for Mn²⁺ action. RLM for smf1Δ/smf1Δ cells expressing reconstituted aequorin was also low under H₂O₂ exposure, indicating that the lack of the high-affinity Mn²⁺ transporter is associated with cytosolic Mn²⁺ concentration (Table2) which is too low for an efficient activation of TRPY1. In fact, smf1Δ/smf1Δ attained responses similar to WT only at higher Mn²⁺ supplementation (Figure 5, grey line), when Mn²⁺ cell content was high enough (Table 2) for efficient TRPY1 activation. In contrast to smf1Δ/smf1Δ strain, pmr1Δ/pmr1Δ expressing reconstituted aequorin attained high RLM upon H₂O₂ exposure which was not significantly augmented by surplus Mn²⁺, suggesting that the intrinsic high level of cytosolic Mn²⁺ associated with PMR1 knockout [50] is sufficient for attaining efficient activation of TRPY1 (Table2). Moreover, it was noted that when applying Mn²⁺ concentrations higher than 20 µM the maximum response of pmr1Δ/pmr1Δ to H₂O₂ started to decline (Figure 5, yellow line) probably due to the hypersensitivity of this strain to Mn²⁺ [50].

4. Discussion

TRPY1 of S. cerevisiae is a key component in releasing vacuolar Ca²⁺ into the cytosol for the Ca²⁺-dependent activation of mechanisms involved in the cell response to hyperosmotic [4] and oxidative stress [19]. Starting from the observation that Mn²⁺ alleviated the haploinsufficiency exhibited by the heterozygous trpy1Δ/TRPY1 strain in synthetic media deficient in essential metals (LMeMM) we found that Mn²⁺ differentially stimulated TRPY1 to release Ca²⁺ from the vacuole under H₂O₂ exposure, but not under hyperosmotic shock. Mn²⁺ alone does not induce [Ca²⁺]_cyt elevation—neither under low (0.05–1 mM) nor under high (2–10 mM, lethal) surplus [21, unpublished observations]. The Mn²⁺ concentrations found to augment the H₂O₂-induced stimulation of TRPY1 were within the physiological limits (10–50 µM) and far below the concentration that would induce a hyperosmotic shock, explaining why the TRPY1 was not extra stimulated by Mn²⁺ under hyperosmotic stress (Figure 3b). It was shown that the release of vacuolar Ca²⁺ via TRPY1 can be stimulated by Ca²⁺ from outside the cell as well as that released from the vacuole by TRPY1 itself in a positive feedback, a process known as Ca²⁺-induced Ca²⁺ release [34,51]. In this regard, Mn²⁺ surplus could stimulate TRPY1 similarly to Ca²⁺. Mn²⁺ is an essential cation which strongly resembles Ca²⁺ not only in ionic radius but also in its affinity to oxygen-containing ligands, a trait which sometimes makes Mn²⁺ a good substitute of Ca²⁺ [52]; this would explain why other essential cations tested (Cu²⁺, Fe³⁺, Zn²⁺) failed to alleviate the haploinsufficiency showed by trpy1Δ/TRPY1 strain. That TRPY1 haploinsufficiency in LMeMM is rescued by Mn²⁺ can be explained in three ways: (1) the supplemental Mn²⁺ simply counteracts the deficiency of essential metals of the LMeMM, providing the necessary amount of cations (albeit surrogate in certain cases) that support cell fitness; (2) Mn²⁺ stimulates TRPY1 activity by increasing the Ca²⁺ release to the cytososl, and consequently by stimulating other components involved in maintaining the cell fitness; (3) Mn²⁺ generates reactive oxygen species (ROS) by a Fenton-like reaction augmenting the oxidative stress and indirectly stimulating TRPY1. The fact that surplus Mn²⁺ augments the TRPY1-related increase in [Ca²⁺]_cyt under oxidative stress clearly correlates with the Mn²⁺ cytosolic level, as the strain lacking the high-affinity plasma membrane Mn²⁺ transporter Smf1 exhibited only a modest increase in [Ca²⁺]_cyt under H₂O₂, when compared with WT (Figure 4c). In this line of evidence, it was shown that a haploid smf1Δ was sensitive to H₂O₂ [20] probably by not attaining the optimum TRPY1 activation for adaptation to oxidative stress. On the other hand, it had been shown that deletion of PMR1—which leads to increased cytosolic Mn²⁺—suppresses the sensitivity of superoxide dismutase (SOD) mutants to superoxide-generating drugs due to the Mn²⁺ capacity to scavenge superoxide ions [50]. In the light of our findings, it is also possible that the high cytosolic Mn²⁺ in cells devoid of Pmr1 rescue the SOD mutants from ROS attack not due to the scavenger traits of Mn²⁺, but through TRPY1 activation. Whether Mn²⁺ rescues the haploinsufficient trpy1Δ/TRPY1 by neutralizing ROS or by directly stimulating TRPY1 are issues to be addressed in the future; nevertheless, the observation that well-known antioxidants such as ascorbate or glutathione did not rescue the trpy1Δ/TRPY1 haploinsufficiency rather supports the latter hypothesis. An open question remains: why is the homozygous trpy1Δ/trpy1Δ apparently more fit than the heterozygous trpy1Δ/TRPY1. The calcium-mediated responses to environmental insults are diverse: depending on the intensity or duration of the [Ca²⁺]_cyt waves, the cell can be led towards adaptation, survival (sometimes with growth arrest or slow growth) or death [19,20,21,23]. The trpy1Δ/trpy1Δ cells are probably more fit because they are never “bothered” by periodic nuisance caused by Ca²⁺ release from the vacuole; on the other hand, trpy1Δ/TRPY1 cells need to find the right balance in Ca²⁺ gating while depending on only one gene copy, and sometimes extra help—external Ca²⁺ carried by Cch1/Mid1, mechanical force [34,51] or even surplus Mn²⁺—may contribute to find the most suitable path to be followed.