BackgroundAcetic acid is mostly known as a toxic by-product of alcoholic fermentation carried out by Saccharomyces cerevisiae, which it frequently impairs. The more recent finding that acetic acid triggers apoptotic programmed cell death (PCD) in yeast sparked an interest to develop strategies to modulate this process, to improve several biotechnological applications, but also for biomedical research. Indeed, acetate can trigger apoptosis in cancer cells, suggesting its exploitation as an anticancer compound. Therefore, we aimed to identify genes involved in the positive and negative regulation of acetic acid-induced PCD by optimizing a functional analysis of a yeast Euroscarf knock-out mutant collection.ResultsThe screen consisted of exposing the mutant strains to acetic acid in YPD medium, pH 3.0, in 96-well plates, and subsequently evaluating the presence of culturable cells at different time points. Several functional categories emerged as greatly relevant for modulation of acetic acid-induced PCD (e.g.: mitochondrial function, transcription of glucose-repressed genes, protein synthesis and modifications, and vesicular traffic for protection, or amino acid transport and biosynthesis, oxidative stress response, cell growth and differentiation, protein phosphorylation and histone deacetylation for its execution). Known pro-apoptotic and anti-apoptotic genes were found, validating the approach developed. Metabolism stood out as a main regulator of this process, since impairment of major carbohydrate metabolic pathways conferred resistance to acetic acid-induced PCD. Among these, lipid catabolism arose as one of the most significant new functions identified. The results also showed that many of the cellular and metabolic features that constitute hallmarks of tumour cells (such as higher glycolytic energetic dependence, lower mitochondrial functionality, increased cell division and metabolite synthesis) confer sensitivity to acetic acid-induced PCD, potentially explaining why tumour cells are more susceptible to acetate than untransformed cells and reinforcing the interest in exploiting this acid in cancer therapy. Furthermore, our results clearly establish a connection between cell proliferation and cell death regulation, evidencing a conserved developmental role of programmed cell death in unicellular eukaryotes.ConclusionsThis work advanced the characterization of acetic acid-induced PCD, providing a wealth of new information on putative molecular targets for its control with impact both in biotechnology and biomedicine.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-838) contains supplementary material, which is available to authorized users.

The highly osmo-and cryotolerant yeast species Torulaspora delbrueckii is an important case study among the non-Saccharomyces yeast species. The strain T. delbrueckii PYCC 5321, isolated from traditional corn and rye bread dough in northern Portugal, is considered particularly interesting for the baking industry. This paper reports the sugar utilization patterns of this strain, using media with glucose, maltose and sucrose, alone or in mixtures. Kinetics of growth, biomass and ethanol yields, fermentation and respiration rates, hydrolase activities and sugar uptake rates were used to infer the potential applied relevance of this yeast in comparison to a conventional baker's strain of Saccharomyces cerevisiae. The results showed that both maltase and maltose transport in T. delbrueckii were subject to glucose repression and maltose induction, whereas invertase was subject to glucose control but not dependent on sucrose induction. A comparative analysis of specific sugar consumption rates and transport capacities suggests that the transport step limits both glucose and maltose metabolism. Specific rates of CO 2 production and O 2 consumption showed a significantly higher contribution of respiration to the overall metabolism in T. delbrueckii than in S. cerevisiae. This was reflected in the biomass yields from batch cultures and could represent an asset for the large-scale production of the former species. This work contributes to a better understanding of the physiology of a non-conventional yeast species, with a view to the full exploitation of T. delbrueckii by the baking industry.

The HSP12 gene encodes one of the two major small heat-shock proteins of Saccharomyces cerevisiae and is induced under different conditions, such as low and high temperatures, osmotic or oxidative stress and high sugar or ethanol concentrations. However, few studies could demonstrate any correlation between HSP12 deletion or overexpression and a phenotype of sensitivity/resistance, making it difficult to attribute a role for Hsp12p under several of these stress conditions. We investigated the possible role of Hsp12p in yeast freezing tolerance. Contrary to what would be expected, the hsp12 null mutant when subjected to prolonged storage at "20 6C showed an increased resistance to freezing when compared with the isogenic wild-type strain. Because the mutant strain displayed a higher intracellular trehalose concentration than the wildtype, which could mask the effect of manipulating HSP12, we overexpressed the HSP12 gene in a trehalose-6-phosphate synthase (TPS1) null mutant. The tps1D strain overexpressing HSP12 showed an increase in resistance to freezing storage, indicating that Hsp12p plays a role in freezing tolerance in a way that seems to be interchangeable with trehalose. In addition, we show that overexpression of HSP12 in this tps1D strain also increased resistance to heat shock and that absence of HSP12 compromises the ability of yeast cells to accumulate high levels of trehalose in response to a mild heat stress. INTRODUCTIONBecause freezing is one of the major abiotic stresses, the adaptation mechanisms that preserve cells at subzero temperatures are extremely important in the development of technology for the cryopreservation of life. Preservation of cell activity is also a very important issue in frozendough technology. This process is well established in the modern baking industry, as it can more easily supply ovenfresh bakery products to consumers while improving labour conditions. Nevertheless, storage of frozen bread dough may lead to the loss of cell viability of baker's yeast as well as of its baking capacity, and consequently to economic losses (Alves-Araú jo et al., 2004;Randez-Gil et al., 1999). In spite of their importance, limited information is available about the mechanisms and determinants of freezing resistance and cold responses in yeast (Kandror & Goldberg, 1997;Kandror et al., 2004;Odani et al., 2003;Zarka et al., 2003). When yeast cells are cultured at 4 u C for a long period, several heat-shock proteins (HSPs) are induced (Homma et al., 2003), suggesting that the induction of these genes might be necessary for adjustment to cold resistance. Considerable evidence indicates that the intracellular level of trehalose may determine the survival response of yeasts under extreme environmental conditions (Diniz-Mendes et al., 1999;Hottiger et al., 1987;Singer & Lindquist, 1998;Van Dijck et al., 1995;Wiemken, 1990). In general, there is wide consensus that trehalose can serve as a stress protectant when yeast cells are confronted with high or low temperatures (Attfield, 1987;Hottiger et ...

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Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides’ cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.