In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

Lithium is highly toxic to yeast when grown in galactose medium mainly because phosphoglucomutase, a key enzyme of galactose metabolism, is inhibited. We studied the global protein and gene expression profiles of Saccharomyces cerevisiae grown in galactose in different time intervals after addition of lithium. These results were related to physiological studies where both secreted and intracellular metabolites were determined. Microarray analysis showed that 664 open reading frames were down-regulated and 725 up-regulated in response to addition of lithium. Genes involved in transcription, translation, and nucleotide metabolism were down-regulated at the transcriptional level, whereas genes responsive to different stresses as well as genes from energy reserve metabolism and monosaccharide metabolism were up-regulated. Compared with the proteomic data, 26% of the down-regulated and 48% of the up-regulated proteins were also identified as being changed on the mRNA level. Functional clusters obtained from proteome data were coincident with transcriptional clusters. Physiological studies showed that acetate, glycerol, and glycogen accumulate in response to lithium, as reflected in expression data, whereas a change from respiro-fermentative to respiratory growth could not be predicted from the expression analyses.

A gene, TIF2, was identified as corresponding to the translation initiation factor eIF4A and when overexpressed it confers lithium tolerance in galactose medium to Saccharomyces cerevisiae. Incubation of yeast with 6 mM LiCl in galactose medium leads to inhibition of [ 35 S]methionine incorporation. By polysome analysis we show that translation is inhibited by lithium at the initiation step, accumulating 80 S monosomes. We further show by immunoblot analysis that when cells are incubated with lithium eIF4A does not sediment with ribosomal subunits. Overexpression of TIF2 overcomes inhibition of protein synthesis and restores its sedimentation with the initiation complex. In vivo, eIF4A is induced by lithium stress. We have shown previously that lithium is highly toxic to yeast when grown in galactose medium mainly due to inhibition of phosphoglucomutase, an enzyme responsible for the entry of galactose into glycolysis. We show that conditions that revert inhibition of phosphoglucomutase also revert inhibition of protein synthesis. Interestingly, glucose starvation leads to loss of polysomes but not to dissociation of eIF4A from the preinitiation complexes. Overexpression of SIT4, a protein phosphatase related to the TOR kinase pathway, reverts inhibition of protein synthesis by lithium and association of eIF4A with the initiation complex.

The probiotic yeast Saccharomyces cerevisiae var boulardii is widely used as a low cost and efficient adjuvant against gastrointestinal tract disorders such as inflammatory bowel disease and treatment of several types of diarrhea, both in humans and animals. S. boulardii exerts its protective mechanisms by binding and neutralizing enteric pathogens or their toxins, by reducing inflammation and by inducing the secretion of sIgA. Although several S. cerevisiae strains have proven probiotic potential in both humans and animals, only S. boulardii is currently licensed for use in humans. Recently, some researchers started using S. boulardii as heterologous protein expression systems. Combined with their probiotic activity, the use of these strains as prophylactic and therapeutic proteins carriers might result in a positive combined effort to fight specific diseases. Here, we provide an overview of the current use of S. cerevisiae strains as probiotics and their mechanisms of action. We also discuss their potential to produce molecules with biotherapeutic application and the advantages and hurdles of this approach. Finally, we suggest future directions and alternatives for which the combined effort of specific immunomodulatory effects of probiotic S. cerevisiae strains and ability to express desired foreign genes would find a practical application.

bLipid droplets (LDs) are intracellular structures that regulate neutral lipid homeostasis. In mammals, LD synthesis is inhibited by rapamycin, a known inhibitor of the mTORC1 pathway. In Saccharomyces cerevisiae, LD dynamics are modulated by the growth phase; however, the regulatory pathways involved are unknown. Therefore, we decided to study the role of the TORC1 pathway on LD metabolism in S. cerevisiae. Interestingly, rapamycin treatment resulted in a fast LD replenishment and growth inhibition. The discovery that osmotic stress (1 M sorbitol) also induced LD synthesis but not growth inhibition suggested that the induction of LDs in yeast is not a secondary response to reduced growth. The induction of LDs by rapamycin was due to increased triacylglycerol but not sterol ester synthesis. Induction was dependent on the TOR downstream effectors, the PP2A-related phosphatase Sit4p and the regulatory protein Tap42p. The TORC1-controlled transcriptional activators Gln3p, Gat1p, Rtg1p, and Rtg3p, but not Msn2p and Msn4p, were required for full induction of LDs by rapamycin. Furthermore, we show that the deletion of Gln3p and Gat1p transcription factors, which are activated in response to nitrogen availability, led to abnormal LD dynamics. These results reveal that the TORC1 pathway is involved in neutral lipid homeostasis in yeast. Lipid droplets (LDs) are intracellular structures formed by a core of neutral lipids, mainly triacylglycerols (TAG) and sterol esters (SE), which are delimited by a phospholipid monolayer embedded with proteins primarily related to lipid metabolism (1-3). At first, LDs were believed to be mere neutral lipid deposits, but later it became clear that LDs play other important roles in cellular physiology. The main function of LDs is to maintain cellular lipid homeostasis (4, 5), and it was shown that defects in the mobilization of neutral lipids from LDs are related to type 2 diabetes, inflammation, neurodegenerative disorders, and cancer (6-8). In addition to their importance in health, LDs also are studied in oleaginous yeast for their exploitation as cellular oil factories for biofuel production (9).Although the metabolic steps of LD biogenesis are well known, the signals that govern LD dynamics are not clear yet. Because the signaling pathways are well conserved between yeast and mammals, Saccharomyces cerevisiae has been a model for studying LD dynamics. In S. cerevisiae, LDs follow a particular dynamic tightly linked to the growth phase and to the nutritional status of the cell (10, 11). When quiescent yeast cells encounter a rich medium containing glucose, cells must exit G 0 in order to start duplication and cell growth (12). This start demands a large amount of sterols and fatty acids, leading to the strong mobilization of the neutral lipids stored in LDs. As a result, LDs are diminished in number and in size (10). After this strong lipolytic phase, yeast cells shift to a lipogenic phase to replenish the levels of LDs, reaching its maximum at early stationary phase (10, 11).The regu...