Factores anatómicos

As already mentioned, Ca2+ ions are fundamental key regulators of a wide variety of processes in eukaryotic cells. Its signaling pathway is triggered by activation of either ion channels or G protein-coupled receptors that are upstream to kinase cascades. The physiological role of Ca2+ signaling ranges from the modulation of enzymes and components of cytoskeleton to that of ion channel permeability, making it particularly relevant in cellular processes e.g. muscle contraction, neuronal transmission and cell-cycle progression. In yeast, in addition to cell growth, Ca2+ controls mating between MATa and MATα cells that secrete specific pheromones able to increase its cytosolic concentration, leading to cellular changes required for agglutination184. Moreover, a key role of Ca2+ signaling has been attributed in the response of yeast cells to an alkaline environment185 as well as to the hypotonic shock through the activation of MAP kinases186. Thus, cytosolic free Ca2+ concentration in yeast cells should be finely regulated and maintained at low levels (50-200 nM)187, through its storage in several compartments e.g. vacuole, endoplasmic reticulum (ER), Golgi apparatus and likely by mitochondria as we will discuss later (Figure 15). Extracellular Ca2+ influx is mainly due to the plasma membrane voltage-gated Ca2+ channel, also referred as Cch1/Mid1 complex, that activates upon several stimuli e.g. depolarization, hypotonic shock and pheromone stimulation184,188. Once entered inside the cytosol, Ca2+ ions can bind the sensor calmodulin, creating a complex able to induce phosphatase calcineurin, promoting the activation of specific set of genes required for cell proliferation and for the response to pheromone189. On the other hand, intracellular Ca2+ is rapidly sequestered by vacuole, which is the major store, through the Ca2+ ATPase Pmc1189 and the high capacity, low-affinity Ca2+/H+ exchanger Vcx1190that might link Ca2+ homeostasis with the regulation of intracellular pH. Furthermore, the contribution of ER/Golgi as alternative Ca2+ storage have been recently described by D’hooge et al., that analyzed the importance of this transport system following specific stimuli191.

Introduction Saccharomyces cerevisiae

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Figure 15. Ca2+ homeostasis in S. cerevisiae.

Upper scheme: In wt yeast extracellular Ca2+ enters the cytosol through Cch1/Mid1 channel complex and an unknown transporter. Cytosolic [Ca2+] is sequestered by vacuole through Vcx1 and Pmc1 and by ER/Golgi via Pmr1 and Cod1. To account for increased Ca2+ influx in Pmr1 or Cod1 cells, two compensatory mechanisms have been proposed. Right lower scheme: Plasma membrane Cch1/Mid1 channels become activated resulting in increased levels of cytosolic Ca2+, activation of calcineurin (CN), a CN-induced compensatory increase in expression of Pmc1 and hence increased vacuolar Ca2+ uptake. Left lower scheme: yeast cells activate a mechanism that enhances Ca2+ influx through not-yet identified plasma membrane Ca2+ transporter, which in turn stimulates vacuolar Ca2+ uptake and induces vacuolar fragmentation or vice versa; from192.

1.6.4.1 Mitochondrial Ca2+ in S. cerevisiae

In mammals, mitochondria play a crucial role in cytosolic Ca2+ homeostasis through an array of transport systems. Yeast mitochondria do not possess an MCU complex193 and therefore their potential role in Ca2+ _{homeostasis is usually not given much consideration. However, also in yeast the}

Ca2+ electrochemical gradient favors Ca2+ accumulation with the same predicted equilibrium distribution as that of mammalian mitochondria. To quote the original conclusions of Carafoli and Lehninger “We consider it likely that all mitochondria, whatever the cell type, possess the electrochemical capacity for moving Ca2+ across the membrane. This capacity cannot be expressed, however, unless a pathway for trans-membrane movement of Ca2+ is available, either through the occurrence of a specific Ca2+ carrier system or through simple physical permeability of the mitochondrial membrane to Ca2+” 194. The emergent hypothesis is that the driving force is so large that Ca2+ uptake could be relevant even if it occurred through a leak pathway rather than through a specific transport system. Consistently (i) S. cerevisae mitochondria have a Ca2+ content of 8-9 ng atoms/mg protein, which is close to that of rat liver mitochondria194, and (ii) electrophoretic Ca2+ uptake coupled to H+ ejection can be easily measured in isolated S. cerevisiae and C. utilis mitochondria when the cation is added at concentrations of 1-10 mM195. It is of note that respiration- driven uptake is observed with Ca2+, Sr2+ and Mn2+ but not with Mg2+, suggesting that cation

Introduction Saccharomyces cerevisiae

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accumulation could be taking place through a low-affinity system whose discrimination for the transported species is strikingly similar to that of the MCU87. It is also interesting to recall that yeast mitochondria are endowed with a very effective 2H+/Ca2+ antiporter activated by fatty acids that mediates mitochondrial Ca2+ release196. Like in mammals, the antiporter could prevent excessive mitochondrial Ca2+ accumulation but also allows rapid mobilization of the matrix Ca2+ pool following activation of phospholipases and perhaps other relevant pathophysiological stimuli.

In contrast to the general assumption that yeast mitochondria lack a specific Ca2+ transport system machinery, a high-capacity Ca2+ uptake system driven by the membrane potential and stimulated by polyamines and ADP has been described in the yeast E. magnusii197,198. Rather than acting as an inhibitor, and at variance from the mammalian MCU, ruthenium red affected Ca2+ transport only marginally or even stimulated it under specific conditions199. Taken together, these findings suggest that a specific Ca2+ transport system may exist also in yeast mitochondria, and that this putative system could be expressed at varying levels in different yeast strains. This might explained more recent studies on the role of Ca2+ in regulating mitochondrial functionality as well as initiation of mitochondrial-dependent cell death. For instance, Gordon Lindsay and Coworkers observed a clear effect of Ca2+ ions on the activity of the pyruvate dehydrogenase complex (PDC)200, responsible for the conversion of pyruvate to acetyl-CoA. From data obtained with isolated mitochondria, it emerged that the activity of PDC, previously inhibited with ATP, could be restored in a Ca2+-dependent manner, suggesting that mitochondrial Ca2+ fraction plays a key role in controlling cellular metabolism. Moreover, occurrence of Ca2+ uptake in S. cerevisiae mitochondria (if over-threshold) might be linked with apoptosis initiation via the activation of the PT pathway, as occurs in mammals.