Coupling/Uncoupling Reversibility in Isolated Mitochondria from Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae uses fermentation as the preferred pathway to obtain ATP and requires the respiratory chain to re-oxidize the NADH needed for activity of Glyceraldehyde-3-phosphate. This process is favored by uncoupling of oxidative phosphorylation (OxPhos), which is at least partially controlled by the mitochondrial unspecific pore (ScMUC). When mitochondrial ATP synthesis is needed as in the diauxic phase or during mating, a large rise in Ca2+ concentration ([Ca2+]) closes ScMUC, coupling OxPhos. In addition, ScMUC opening/closing is mediated by the ATP/ADP ratio, which indicates cellular energy needs. Here, opening and closing of ScMUC was evaluated in isolated mitochondria from S. cerevisiae at different incubation times and in the presence of different ATP/ADP ratios or varying [Ca2+]. Measurements of the rate of O2 consumption, mitochondrial swelling, transmembrane potential and ROS generation were conducted. It was observed that ScMUC opening was reversible, a high ATP/ADP ratio promoted opening and [Ca2+] closed ScMUC even after several minutes of incubation in the open state. In the absence of ATP synthesis, closure of ScMUC resulted in an increase in ROS.


Introduction
Fermentative metabolism in Saccharomyces cerevisiae requires an active mitochondrial respiratory chain to re-oxidize the NADH produced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [1]. NAD + is needed in the glycolytic pathway by glyceraldehyde-3-Phosphate dehydrogenase (GAPDH), which may bind to mitochondrial porin and exchange produced NADH for required NAD + [2]. A basal high rate of oxygen consumption is possible in S. cerevisiae mitochondria due to the substitution of Complex I by three alternative NADH dehydrogenases (NDH2), which do not pump protons and thus decrease the ATP/O [3]. In addition, as Complex-I contains the two most important sites for ROS production in the respiratory chain [4] this substitution probably helps to avoid ROS overproduction in S. cerevisiae. In addition, the S. cerevisiae mitochondrial unspecific channel ( Sc MUC) may open, depleting the transmembrane potential (∆Ψ), uncoupling oxidative phosphorylation and increasing the rate of oxygen consumption, thus decreasing ROS production further [5][6][7].
When fermentative substrates are exhausted and O 2 is available, S. cerevisiae cells shift their metabolism to become aerobic using accumulated ethanol as respiratory substrate to begin the diauxic phase of growth [8,9]. Physiologically important effectors that alternatively open and close Sc MUC are adenine nucleotides, where ATP opens Sc MUC [7] while hydrolysis products ADP and Pi close it, i.e., a high energy charge opens Sc MUC Life 2021, 11, 1307 2 of 13 while a low energy charge closes it. The second effector of physiological interest is Ca 2+ , which closes Sc MUC at high concentrations [10]. In resting yeast, cytoplasmic [Ca 2+ ] is near 0.1 µM, while in most extracellular media [Ca 2+ ] is 1 to 2 mM [11]. Upon stimulation, specialized channels allow large amounts of Ca 2+ to enter the cell, closing Sc MUC and enhancing energy production [12]. Thus, Ca 2+ is an ideal second messenger [12,13]. Activation by Ca 2+ influx is observed during mating: when an a-type haploid cell is exposed to α-pheromone, this pheromone binds to a specific receptor [14]. When mating, cytoplasmic [Ca 2+ ] may remain high for up to 20 min signaling the need large morphological changes in the cell due to large modifications in the cellular cytoskeleton [14]. Eventually, an a-cell and an α-cell undergo fusion to generate a diploid [15]. As these processes require a large amount of energy, Ca 2+ is also needed to close Sc MUC, promote OxPhos coupling and increase ATP availability. When the mating process is over, Ca 2+ is expelled and a basal metabolism is reinstated [13,16].
Here, we tested in isolated mitochondria the response to either the ratio ATP/ADP + Pi or to [Ca 2+ ] variations. The role of each effector to control alternative opening and closing was followed using measurements of the rate of O 2 consumption, mitochondrial transmembrane potential (∆Ψ), mitochondrial swelling and ROS production. The open/close state of Sc MUC was dependent on the addition of different effectors. Opening and closing were reversible. In addition it was observed that opening was gradual as effector mixtures elicited partial effects. These effects suggest that in fermenting yeast, mitochondria are uncoupled, becoming coupled when a large amount of ATP is required.
Isolation of yeast mitochondria. After incubation, yeast was centrifuged (5000× g for 5 min and washed twice) and resuspended in 0.6 M mannitol, 5 mM MES, 0.1% bovine serum albumin, pH 6.8 (TEA). The cell suspension was mixed with 0.5 mm diameter glass beads 50% (v/v) and disrupted in a Bead Beater, Biospec Products Inc, OK. Mitochondria were isolated from the homogenate by differential centrifugation as previously described [17]. The concentration of mitochondrial protein was determined by Biuret [19]. In all assays we used mitochondria at a final concentration of 0.5 mg prot/mL. Oxygen consumption. Experiments were conducted using a Clark electrode (Oximeter model 782, Warner/Strathkelvin Instruments, North Lanarkshire, Scotland) in a waterjacketed chamber. Temperature was kept at 30 • C using a water bath (PolyScience 7 L, IL). Total volume 1.0 mL. The reaction mixture was 0.6 M mannitol, 5 mM MES (TEA), pH 6.8 plus 0.1 M KCl, 0.5 mM MgCl 2 and 2 µL/mL ethanol. In all experiments, samples were preincubated for 5 min with oligomycin 4 µg/mg prot.
Transmembrane potential. ∆Ψ was determined as described by Åkerman and Wikström [20], following the changes in absorbance of safranine-O at 511-533 nm using a double beam Aminco-Olis spectrophotometer (GA) in dual mode. The concentrations of ATP, ADP, Ca 2+ and EGTA are indicated under each figure. At the end of each trace, Life 2021, 11, 1307 3 of 13 the collapse of ∆Ψ was induced adding 6 µM CCCP. In all experiments, samples were preincubated for 5 min with oligomycin 4 µg/mg prot.
Mitochondrial swelling. The decrease in absorbance of a mitochondrial suspension at 540 nm was followed as described in the literature [6]. We used a DW2000 Olis/Aminco spectrophotometer in split mode. In all experiments, samples were preincubated for 5 min with oligomycin 4 µg/mg prot.
Reactive oxygen species were measured in freshly mitochondria using the Amplex Red (Invitrogen, Molecular Probes, Carlsbad, CA, USA) as in [21]. Samples were incubated for 1 min, then 50 µg were placed into a 96-well micro plate with working solution 20 µL (10 µM Amplex red, 0.2 units/mL horseradish peroxidase and 0.2 units superoxide dismutase/mL in 250 mM sodium phosphate pH 7.4), final volume 100 µL. Fluorescence was measured after 30 mins in a POLARstar Omega detector (BGM LABTECH, Offenburg, Germany) set at 571 and 585 nm and results were interpolated against a calibration curve.

Results
During fermentation, Saccharomyces cerevisiae uncouples OxPhos to oxidize NADH optimizing the rate of glycolysis while in contrast, after glucose depletion a coupled OxPhos is needed and thus Sc MUC is closed [8]. Among Sc MUC effectors, the molecules that vary in concentration in different metabolic states such as adenine nucleotides and Pi seem important. Indeed, ATP opens Sc MUC uncoupling OxPhos, while ADP closes it, coupling OxPhos [22]. Another effector is [Ca 2+ ], which in eukaryotes increases several orders of magnitude when plasma membrane Ca 2+ channels open [12][13][14].
To analyze the possible physiological role of Sc MUC, mitochondria were isolated and used to measure Oxygen consumption, mitochondrial swelling, the transmembrane potential (∆Ψ) and reactive oxygen species (ROS) production under conditions where Sc MUC was open or closed ( Figure 1). ATP, ADP, Ca 2+ and EGTA are indicated under each figure. At the end of each trace, the collapse of ΔΨ was induced adding 6 μM CCCP. In all experiments, samples were preincubated for 5 min with oligomycin 4 μg/mg prot. Mitochondrial swelling. The decrease in absorbance of a mitochondrial suspension at 540 nm was followed as described in the literature [6]. We used a DW2000 Olis/Aminco spectrophotometer in split mode. In all experiments, samples were preincubated for 5 min with oligomycin 4 μg/mg prot.
Reactive oxygen species were measured in freshly mitochondria using the Amplex Red (Invitrogen, Molecular Probes, Carlsbad, CA, USA) as in [21]. Samples were incubated for 1 min, then 50 μg were placed into a 96-well micro plate with working solution 20 μL (10 μM Amplex red, 0.2 units/mL horseradish peroxidase and 0.2 units superoxide dismutase/mL in 250 mM sodium phosphate pH 7.4), final volume 100 μL. Fluorescence was measured after 30 mins in a POLARstar Omega detector (BGM LABTECH, Offenburg, Germany) set at 571 and 585 nm and results were interpolated against a calibration curve.

Results
During fermentation, Saccharomyces cerevisiae uncouples OxPhos to oxidize NADH optimizing the rate of glycolysis while in contrast, after glucose depletion a coupled OxPhos is needed and thus ScMUC is closed [8]. Among ScMUC effectors, the molecules that vary in concentration in different metabolic states such as adenine nucleotides and Pi seem important. Indeed, ATP opens ScMUC uncoupling OxPhos, while ADP closes it, coupling OxPhos [22]. Another effector is [Ca 2+ ], which in eukaryotes increases several orders of magnitude when plasma membrane Ca 2+ channels open [12][13][14].
To analyze the possible physiological role of ScMUC, mitochondria were isolated and used to measure Oxygen consumption, mitochondrial swelling, the transmembrane potential (ΔΨ) and reactive oxygen species (ROS) production under conditions where ScMUC was open or closed ( Figure 1). Oximetry. The rate of O2 consumption was measured in the presence of different, fixed Pi concentrations. Then the reversibility of ScMUC opening in response to the addition of ATP and/or ADP was evaluated. As described in the literature [5][6][7], at 2.0 mM Pi

Effects of ADP/ATP on Sc MUC
Oximetry. The rate of O 2 consumption was measured in the presence of different, fixed Pi concentrations. Then the reversibility of Sc MUC opening in response to the addition of ATP and/or ADP was evaluated. As described in the literature [5][6][7], at 2.0 mM Pi we observed a slow rate of oxygen consumption, indicating that OxPhos was coupled ( Figure 2A, trace a). Under these conditions, the addition of different concentrations of ATP proportionally increased the rate of oxygen consumption, suggesting that different concentrations of ATP gradually uncoupled OxPhos ( Figure 2A, traces b to e). In contrast, as reported by others [5][6][7], at 0.1 mM Pi the rate of oxygen consumption was high, indicating that OxPhos was uncoupled due to opening of Sc MUC ( Figure 2B trace a). Then, the rate of oxygen consumption gradually decreased upon addition of increasing concentrations of ADP, suggesting different ADP concentrations promoted closure of Sc MUC, promoting coupling ( Figure 2B traces b to e). Oxygen consumption results confirmed results by others that increasing concentrations of Pi, and ADP close, while ATP opens Sc MUC [5][6][7]. we observed a slow rate of oxygen consumption, indicating that OxPhos was coupled ( Figure 2A, trace a). Under these conditions, the addition of different concentrations of ATP proportionally increased the rate of oxygen consumption, suggesting that different concentrations of ATP gradually uncoupled OxPhos ( Figure 2A, traces b to e). In contrast, as reported by others [5][6][7], at 0.1 mM Pi the rate of oxygen consumption was high, indicating that OxPhos was uncoupled due to opening of ScMUC ( Figure 2B trace a). Then, the rate of oxygen consumption gradually decreased upon addition of increasing concentrations of ADP, suggesting different ADP concentrations promoted closure of ScMUC, promoting coupling ( Figure 2B traces b to e). Oxygen consumption results confirmed results by others that increasing concentrations of Pi, and ADP close, while ATP opens ScMUC [5][6][7]. Transmembrane potential. The reversibility of ScMUC opening and closing by ATP and ADP, respectively was evaluated measuring the ΔΨ ( Figure 3). In these experiments, one adenine nucleotide was added first and the second was added 1.5 min later. At 0.1 mM Pi, ΔΨ was almost depleted ( Figure 3A trace a) and addition of 2 mM ADP led to ΔΨ recovery ( Figure 3A trace b). Then, the addition of increasing ATP led to a second fall in ΔΨ ( Figure 3A, traces c, d and e). The opposite experiment was also performed: when in the presence of 2 mM Pi, ΔΨ was high ( Figure 3B) and addition of ATP decreased ΔΨ ( Figure 3B, traces b to e). Then, a later addition of different ADP concentrations resulted in increasing recovery of ΔΨ, suggesting that ScMUC closed gradually ( Figure 3B traces c, d and e). The reversibility in ΔΨ rise/decrease suggests that the alternative addition of ADP and ATP led to alternative, gradual opening/closing of ScMUC. Transmembrane potential. The reversibility of Sc MUC opening and closing by ATP and ADP, respectively was evaluated measuring the ∆Ψ (Figure 3). In these experiments, one adenine nucleotide was added first and the second was added 1.5 min later. At 0.1 mM Pi, ∆Ψ was almost depleted ( Figure 3A trace a) and addition of 2 mM ADP led to ∆Ψ recovery ( Figure 3A trace b). Then, the addition of increasing ATP led to a second fall in ∆Ψ ( Figure 3A, traces c, d and e). The opposite experiment was also performed: when in the presence of 2 mM Pi, ∆Ψ was high ( Figure 3B) and addition of ATP decreased ∆Ψ ( Figure 3B, traces b to e). Then, a later addition of different ADP concentrations resulted in increasing recovery of ∆Ψ, suggesting that Sc MUC closed gradually ( Figure 3B traces c, d and e). The reversibility in ∆Ψ rise/decrease suggests that the alternative addition of ADP and ATP led to alternative, gradual opening/closing of Sc MUC.
Mitochondrial swelling. Another parameter commonly used to follow the open/close state of Sc MUC is mitochondrial swelling [6,23]. At low Pi plus ADP, Sc MUC was closed, so the rate of K + -mediated swelling was slow ( Figure 4A). Under these conditions, addition of different concentrations of ATP promoted increasing rates in swelling. These data suggested that ATP reverted the ADP-mediated closing of Sc MUC. The opposite experiment, where mitochondria were incubated in the presence of 2 mM Pi plus 2 mM ATP, a rapid rate of K + -mediated swelling was observed which indicated Sc MUC was open ( Figure 4B, trace b). Then, addition of different concentrations of ADP ( Figure 4B, traces c, d, e) inhibited the rate of swelling ( Figure 4B traces c, d, and e) suggesting that ADP closed Sc MUC. Therefore, the effect of Pi, ADP or ATP at different times led to changes of swelling rates suggesting that Sc MUC open and close states were reversible [24][25][26]. Mitochondrial swelling. Another parameter commonly used to follow the open/close state of ScMUC is mitochondrial swelling [6,23]. At low Pi plus ADP, ScMUC was closed, so the rate of K + -mediated swelling was slow ( Figure 4A). Under these conditions, addition of different concentrations of ATP promoted increasing rates in swelling. These data suggested that ATP reverted the ADP-mediated closing of ScMUC. The opposite experiment, where mitochondria were incubated in the presence of 2 mM Pi plus 2 mM ATP, a rapid rate of K + -mediated swelling was observed which indicated ScMUC was open (Figure 4B, trace b). Then, addition of different concentrations of ADP ( Figure 4B, traces c, d, e) inhibited the rate of swelling ( Figure 4B traces c, d, and e) suggesting that ADP closed ScMUC. Therefore, the effect of Pi, ADP or ATP at different times led to changes of swelling rates suggesting that ScMUC open and close states were reversible [24][25][26].  Mitochondrial swelling. Another parameter commonly used to follow the open/close state of ScMUC is mitochondrial swelling [6,23]. At low Pi plus ADP, ScMUC was closed, so the rate of K + -mediated swelling was slow ( Figure 4A). Under these conditions, addition of different concentrations of ATP promoted increasing rates in swelling. These data suggested that ATP reverted the ADP-mediated closing of ScMUC. The opposite experiment, where mitochondria were incubated in the presence of 2 mM Pi plus 2 mM ATP, a rapid rate of K + -mediated swelling was observed which   Effects of incubation time on the reversibility ATP-mediated opening of Sc MUC. Preservation of Sc MUC opening reversibility after different incubation times is critical for survival of the cell. Reversibility of Sc MUC opening was explored measuring both ∆Ψ ( Figure 5A) and the rate of swelling ( Figure 5B). Yeast mitochondria were incubated for different times, from 30 s to 4 min under open Sc MUC conditions, and then ADP was added ( Figure 5). In all cases, ADP promoted partial recovery of ∆Ψ ( Figure 5A) and slight reversal of swelling ( Figure 5B). Thus the ATP-mediated opening of Sc MUC was responsive to ADP for at least 4 min. (Figure 5). tion of ScMUC opening reversibility after different incubation times is critical for survival of the cell. Reversibility of ScMUC opening was explored measuring both ΔΨ ( Figure 5A) and the rate of swelling ( Figure 5B). Yeast mitochondria were incubated for different times, from 30 s to 4 min under open ScMUC conditions, and then ADP was added ( Figure  5). In all cases, ADP promoted partial recovery of ΔΨ ( Figure 5A) and slight reversal of swelling ( Figure 5B). Thus the ATP-mediated opening of ScMUC was responsive to ADP for at least 4 min. (Figure 5).

Ca 2+ /EGTA Alternating Effects on ScMUC
The above results indicate that the opening response of ScMUC to the sequential additions of ATP and ADP is reversible even after several minutes of incubation. However, neither mitochondrial ΔΨ nor swelling returned to the values observed when ScMUC was closed from the beginning. Thus, it was decided to test whether full reversal of ScMUC opening could be observed upon depletion of a positive effector after different times of incubation. From previous data, it was reasoned that this could be achieved with Ca 2+ , an effector closing ScMUC, and its full chelation with EGTA to open ScMUC [10,27]. To test whether the EGTA-mediated opening of ScMUC was reverted adding Ca 2+ at different incubation times, we conducted measurements of ΔΨ ( Figure 6A) and mitochondrial swelling ( Figure 6B). Mitochondria were incubated in the presence of EGTA for 0.5, 1, 2 and 4 min and then Ca 2+ was added. Up to 2 min, the addition of Ca 2+ promoted recovery of a large proportion of ΔΨ while a partial effect was obtained at 4 min. (Figure 6A) Mitochondrial swelling was reverted by Ca 2+ , indicating ScMUC was closed ( Figure 6B). The above data suggest that in isolated mitochondria from S. cerevisiae, Ca 2+ chelation by EGTA evoked the reversible opening of ScMUC. In contrast to the partial effects of ADP ( Figure  5), Ca 2+ addition resulted in better recovery of ΔΨ ( Figure 6A) and also at variance with ADP, actual reversal of swelling ( Figure 6B).

Ca 2+ /EGTA Alternating Effects on Sc MUC
The above results indicate that the opening response of Sc MUC to the sequential additions of ATP and ADP is reversible even after several minutes of incubation. However, neither mitochondrial ∆Ψ nor swelling returned to the values observed when Sc MUC was closed from the beginning. Thus, it was decided to test whether full reversal of Sc MUC opening could be observed upon depletion of a positive effector after different times of incubation. From previous data, it was reasoned that this could be achieved with Ca 2+ , an effector closing Sc MUC, and its full chelation with EGTA to open Sc MUC [10,27]. To test whether the EGTA-mediated opening of Sc MUC was reverted adding Ca 2+ at different incubation times, we conducted measurements of ∆Ψ ( Figure 6A) and mitochondrial swelling ( Figure 6B). Mitochondria were incubated in the presence of EGTA for 0.5, 1, 2 and 4 min and then Ca 2+ was added. Up to 2 min, the addition of Ca 2+ promoted recovery of a large proportion of ∆Ψ while a partial effect was obtained at 4 min. (Figure 6A) Mitochondrial swelling was reverted by Ca 2+ , indicating Sc MUC was closed ( Figure 6B). The above data suggest that in isolated mitochondria from S. cerevisiae, Ca 2+ chelation by EGTA evoked the reversible opening of Sc MUC. In contrast to the partial effects of ADP ( Figure 5), Ca 2+ addition resulted in better recovery of ∆Ψ ( Figure 6A) and also at variance with ADP, actual reversal of swelling ( Figure 6B).

Reactive Oxygen Species (ROS) Production under Conditions That Open or Close
ScMUC.
Physiological uncoupling increases electron flow in the respiratory chain, preventing the over-production of ROS. It has been proposed that permeability transition pores work

Reactive Oxygen Species (ROS) Production under Conditions That Open or Close Sc MUC
Physiological uncoupling increases electron flow in the respiratory chain, preventing the over-production of ROS. It has been proposed that permeability transition pores work as unspecific proton sinks thus promoting physiological uncoupling and therefore decrease ROS production [5][6][7]25]. With this in mind, it was decided to determine whether a correlation exists between the opening/closing of Sc MUC reported above and the control of ROS production (Figure 7). Figure 6. Effect of the addition of Ca 2+ on mitochondrial transmembrane potential and mitochondrial swelling. (A) Reaction mixture as in Figure 4A. Ca 2+ 600 μM (upward arrows) was added at: 30 s, 1, 2 and 4 min. (B) Reaction mixture as in Figure 4B. Where indicated 0.1 M KCl. (upward arrows).

Reactive Oxygen Species (ROS) Production under Conditions That Open or Close
ScMUC.
Physiological uncoupling increases electron flow in the respiratory chain, preventing the over-production of ROS. It has been proposed that permeability transition pores work as unspecific proton sinks thus promoting physiological uncoupling and therefore decrease ROS production [5][6][7]25]. With this in mind, it was decided to determine whether a correlation exists between the opening/closing of ScMUC reported above and the control of ROS production (Figure 7).

Reactive Oxygen Species (ROS) Production under Conditions That Open or Clo
ScMUC.
Physiological uncoupling increases electron flow in the respiratory chain, prev the over-production of ROS. It has been proposed that permeability transition pores as unspecific proton sinks thus promoting physiological uncoupling and the decrease ROS production [5,6,7,25]. With this in mind, it was decided to dete whether a correlation exists between the opening/closing of ScMUC reported abov the control of ROS production (Figure 7).  Supplementary Table S1.
At 0.4 m Pi, ROS production was lower than at 4.0 mM Pi, suggesting as expected, that a closed Sc MUC enhanced ROS production ( Figure 7A, Table S1). Then, to test other effectors used here, at 0.4 mM Pi, ADP was added at 1 or 2 mM, which closed Sc MUC (Figures 2-5), observing that ROS production increased ( Figure 7B, Table S1). In contrast, in the presence of 4 mM Pi where Sc MUC was closed, addition of ATP, which opens Sc MUC (Figures 2-5), inhibited ROS production ( Figure 7C, Table S1). Under the same low or high Pi concentrations, the effects of Ca 2+ chelation or addition were tested. As expected from the results shown in Figure 6, at 0.4 mM Pi, EGTA decreased ROS production, while Ca 2+ addition mildly increased ROS production ( Figure 7D, Table S1). Then, at 4.0 mM Pi, EGTA inhibited ROS production, while Ca 2+ addition resulted in more that twice as  Table S1). Thus, when Sc MUC is closed ROS production is higher that when Sc MUC is open. These results are in agreement with the idea that an open Sc MUC is a physiological uncoupler of oxidative phosphorylation that inhibits production of ROS.
Together, our results add to the idea that the permeability transition protects mitochondria and thus the cell against stress. Effectors such as ATP and ADP probably control OxPhos in response with the energy charge, where high ATP signals for uncoupling. In addition, Ca 2+ promotes a stronger response and prepares the cell in advance for high energy-requiring processes such as duplication of gene exchange with other cells [11][12][13] ( Table 1).

Discussion
During fermentation, S. cerevisiae extracts all the energy it needs from glucose, while producing ethanol [28]. Under these conditions, ATP is high, so the F 1 F 0 ATP synthase is not required and thus it does not dissipate ∆Ψ. A high ∆Ψ would inhibit the rate of oxygen consumption by the respiratory chain (RC) [1], however RC does work at a high rate in order to oxidize NADH and produce the NAD + needed for glyceraldehyde-3-phosphate dehydrogenase activity [29]. Therefore, during fermentation, mitochondrial OxPhos has to be uncoupled. Uncoupling is due to ATP itself, as it promotes opening of Sc MUC [5][6][7] (Figures 2-4). An open Sc MUC dissipates the mitochondrial ∆Ψ, accelerating the rate of electron transfer in the respiratory chain [1]. In contrast, when fermentation substrates are depleted, ATP is hydrolyzed to ADP, which is a negative Sc MUC effector and thus promotes the recovery of a high ∆Ψ [7,8]. Different reports [30][31][32] and data presented here (Figures 2-4), suggest that opening/closing of Sc MUC is not an all-or-nothing event, but instead partial opening states modulate the degree of uncoupling. Such modulation would be a mechanism to coordinate OxPhos and fermentation as needed [32]. Indeed, our results suggest that the degree of Sc MUC opening is gradual and depends on ATP/(ADP + Pi) concentrations (Figures 2-4). The physiological role of permeability transition may profit from the eventual elucidation of the structure of the pore, which seems to be approaching fast [33].
In addition to adenine nucleotides, Ca 2+ is an important negative effector of Sc MUC. This contrasts with mammalian cells, where Ca 2+ works in the opposite sense, i.e., in the mammalian mitochondrial permeability transition pore (mPTP) opening is triggered by Ca 2+ [34,35]. In the resting state cytoplasmic Ca 2+ is below 100 nM both in S. cerevisiae, and in mammalian cells, while external Ca 2+ is in the mM range [11]. Several transport systems use this massive gradient to allow Ca 2+ entry through specialized channels in the plasma membrane to the cytoplasm where it acts as a secondary messenger [36,37]. Again, at variance with most cells, where these transients last less than a minute, in S. cerevisiae cytoplasmic Ca 2+ may remain high for as long as 20 min [12,13]. Thus, Ca 2+ closes Sc MUC, enhancing OxPhos, while Sc MUC may remain open for several minutes and still be reversible. In contrast, in mammalians, mPTP opening is dangerous as it uncouples OxPhos and may eventually deplete ATP and trigger cell death through mitochondrial signaling [38].
In yeast, when an event requiring high amounts of energy occurs, Ca 2+ enters the cell increasing ATP production. One such event may be the mating response, where massive morphologic changes occur that seem to consume a large amount of ATP [39]. Another event where Ca 2+ enters the cell is phase S1 of the S. cerevisiae cell cycle when again it is likely that large amounts of ATP are needed in order to synthesize macromolecules [40]. Both mating and the S1 phase of the cell cycle are events where high mitochondrial ATP production is needed [16]. In these circumstances Ca 2+ would enable OxPhos coupling by closing Sc MUC and increasing ∆Ψ.
The mammalian mitochondrial permeability transition pore (mPTP) is widely considered as an equivalent entity to Sc MUC. Indeed, both mPTP and Sc MUC possess similar cutoff diameters [41] and react in similar fashion to some effectors such as adenine nucleotides [6,22] or to pharmacological agents such as bongkrekic acid or atractyloside [40]. However, the response to Ca 2+ is the opposite as Ca 2+ opens mPTP [42] while it closes Sc MUC [22,27]. In order to obtain coupled mammalian mitochondria, Mg 2+ or Ca 2+ chelators have to be added to the isolation medium, while this is not needed in yeast mitochondria [5][6][7]18]. Additionally, it must be considered that the structure of the channel is still under debate and some components that seem to participate in one channel are not present in the other. One such component may be PiC, which in yeast controls opening and confers sensitivity to Pi [24], but when manipulated in heart mice, it does not modify the properties of mPTP [43]. These results suggest that the still undefined components of mitochondrial permeability transition pores may be different for each species [44]. Furthermore, in mPTP, components of the Ca 2+ uniporter machinery modify mPTP activity [45,46], while this is not possible in S. cerevisiae where there is no Ca 2+ uniporter [47][48][49]. Thus, it is probably not safe to assume that mPTP and Sc MUC are identical entities.
In the mammalian heart, reversal of pore opening has also been reported [24][25][26][27], although opening of mPTP seems to react to different events than in S. cerevisiae. It is observed during cardiac stress, intermittent episodes of reperfusion allow the cell to empty Ca 2+ while replenishing ATP pools. Thus, in mammals, mPTP seems important to prevent ROS overproduction through mitochondrial uncoupling, greatly improving survival probabilities [50]. This procedure is termed conditioning. Thus, even though both Sc MUc and mPTP constitute a mitochondrial uncoupling device, the role of Sc MUC opening is to oxidize NADH in a species that does not suffer from hypoxia [51].
Other yeast species also present some peculiarities that contribute to the idea that perhaps permeability transition pores are different. It is not clear whether Yarrowia lipolytica and perhaps Endomyces magnusii undergo mitochondrial permeability transition [52], as this event does not occur except under extreme circumstances, when a calcium ionophore plus massive amounts of calcium are added [53]. Another yeast, Debaryomyces hansenii, does undergo PT, however, its channel exhibits a unique sensitivity to Na + and K + , which are negative effectors and such sensitivity is probably quite important as this yeast resides in marine environments and is used in salted cheese maturation [54].
In many species including S. cerevisiae, aging damages mtDNA, possibly through an excess in ROS that in turn increases activity of proteins that promote mtDNA recombination. Then mutations accumulate damaging mitochondria irreversibly in a "vicious cycle" [55,56].
Opening of Sc MUC increases the rate of O 2 flow in the respiratory chain, preventing overproduction of ROS. This is not the only mechanism accelerating electron flow, as other proton sinks such as uncoupling proteins are differentially expressed upon exposure to stress [31]. A second mechanism promoting physiological uncoupling, where branched Life 2021, 11, 1307 10 of 13 respiratory chains differentially express their alternative redox enzymes such as alternative oxidase (AOX) or NADH dehydrogenases type 2 (NDH2), in order to increase electron flux in the respiratory chain either at the beginning of the stationary phase or when the cell is subjected to stress [56,57].
The Index of Hydrogen Defficiency (IHF) is another exceedingly interesting mechanism mitochondria use to prevent damage by ROS. In aging Endomyces magnusii (grown for up to 168 h), unsaturated fatty acids are replaced by their saturated counterparts. So, as palmitic and oleic acid are less susceptible to damage by ROS than unsaturated fatty acids, mitochondrial membranes are protected. In addition, in this species, an alternative AOX is expressed from the beginning of the stationary phase, so physiological uncoupling is also used to protect mitochondria [58].
The "Co-localization for Redox Regulation (CoRR) Hypothesis" must also be considered when explaining how damaged mitochondria are negatively selected to allow cell survival [57]. When protection fails, mitochondria containing damaged mtDNA are negatively selected. This is possible due to the coexistence in the same compartment of mitochondrial DNA with its encoded proteins. These proteins are the most hydrophobic and difficult to move from the nucleus to the mitochondrial matrix. Additionally the CoRR hypothesis proposes that these mtDNA-encoded proteins are vital for the structure of OxPhos complexes I, III, IV and V. Therefore, deleterious mutations in mtDNA proteins would result in OxPhos malfunction and elimination of mitochondria [58].
Together, physiological uncoupling, IHF and mtDNA-encoded proteins (as proposed by the CoRR hypothesis), provide mitochondria with mechanisms to respond to exposure to stress, increased ROS and improve cell survival. Furthermore, regulation of Sc MUC by the energy charge and by Ca 2+ signaling point to a role of Sc MUC in metabolic control, controlling fermentation, OxPhos and ROS production. In addition, a system to deplete O 2 would confer yeast with an advantage over other organisms as few other cells thrive under hypoxic/anoxic conditions. Probably the equilibrium between ATP and ADP works to graduate Sc MUC responses, while Ca 2+ signals an event where OxPhos has to be recruited.

Supplementary Materials:
The following is available online at https://www.mdpi.com/article/ 10.3390/life11121307/s1. Table S1: Raw data used to generate Institutional Review Board Statement: Not applicable as no humans nor animals were involved in these studies.

Informed Consent Statement:
Not applicable as no humans nor animals were involved in these studies.