Glucose and osmotic stress-dependent calcium signalling in saccharomyces cerevisiae: evidences for novel transporter systems and calcineurin involvement

The major goal of this work is to understand function and regulation mechanisms of calcium transport systems in Saccharomyces cerevisiae, in response to nutrients and hypotonic shock. Calcium represents, in fact, one of the most important second messengers in all eukaryotic cells, and particularly in budding yeast, where it plays essential roles in regulating many fundamental cellular processes, such as cell cycle, mating, sensing of glucose and glucose starvation, resistance to salt stress and cell survival. Yeast cells actively maintain cytosolic free Ca2+ concentration at extremely low levels, in a range of 50-200 nM, through a finely regulated homeostasis maintaining mechanism. Glucose addition to nutrient-deprived cells triggers a rapid and transient increase in cytosolic Ca2+ level, mainly due to an influx of calcium from the extracellular environment (Eilam et al., 1990). Conversely, hypotonic shock induces an increase in cytosolic Ca2+ level, mainly mediated by calcium release from intracellular stores (Batiza et al., 1996), though the intracellular transporters involved in this signalling are not yet identified. Glucose-triggered calcium influx from extracellular environment is mediated by a high affinity calcium transporter (HACS), composed by Mid1p/Cch1p subunits, during growth in minimal medium, whereas in rich media cultured cells it seems to be mediated by a still unidentified transporter, named GIC (for Glucose Induced Calcium Channel). By taking advantage of a bioluminescent assay in vivo allowing us to monitor cytosolic Ca2+ level changes, based on aequorin bioluminescent protein, the role of the known calcium channels and of the still unidentified putative transporters in glucose and hypotonic shock-dependent Ca2+ signalling, was here investigated. In order to better characterize GIC and the unknown hypotonic shock-responsive intracellular transporters, a pharmacological approach was applied, testing their sensitivity to common blockers of mammalian voltage-gated calcium channels. In addition, the effects of glucose-induced Ca2+ signalling on calcineurin, the major effector for intracellular calcium, were here investigated, demonstrating for the first time that calcineurin activation, normally recognized as being essential for survival under diverse stress conditions, can be also responsive to nutrients. The emergent role of calcineurin in regulating functionality of calcium transporters depending on nutrient availability and the crosstalk between calcineurin pathway and nutrient sensing were also investigated in this work.