The role of the SNF1 signaling pathway in the growth of Saccharomyces cerevisiae in different carbon and nitrogen sources.

Cancer is a leading cause of death worldwide, reporting nearly 10 million deaths in 2020. One of the hallmarks of cancer cells is their capability to evade growth suppressors and sustain proliferative signaling resulting in uncontrolled growth. The AMPK pathway, a catabolic via to economize ATP, has been associated with cancer. AMPK activation is related to cancer progression in advanced stages, while its activation by metformin or phenformin is associated with cancer chemoprevention. Thus, the role of the AMPK pathway in cancer growth modulation is not clear. Saccharomyces cerevisiae might be a useful model to elucidate AMPK participation in growth regulation since it shares a highly conserved AMPK pathway. Therefore, this work is aimed at evaluating the role of the AMPK pathway on S. cerevisiae growth under different nutritional conditions. Herein, we provide evidence that the SNF1 gene is necessary to maintain S. cerevisiae growth with glucose as a sole carbon source at every concentration tested. Resveratrol supplementation inhibited the exponential growth of snf1∆ strain at low glucose levels and decreased it at high glucose levels. SNF1 gene deletion impaired exponential growth in a carbohydrate concentration-dependent manner independently of nitrogen source or concentration. Interestingly, deletion of genes encoding for upstream kinases (SAK1, ELM1, and TOS3) also had a glucose dose-dependent effect upon exponential growth. Furthermore, gene deletion of regulatory subunits of the AMPK complex impacted exponential growth in a glucose-dependent manner. Altogether, these results suggest that the SNF1 pathway affects the exponential growth of S. cerevisiae in a glucose-dependent manner.

SNF1 controls the glycolytic flux and mitochondrial respiration.

The switch between mitochondrial respiration and fermentation as the main ATP production pathway through an increase glycolytic flux is known as the Crabtree effect. The elucidation of the molecular mechanism of the Crabtree effect may have important applications in ethanol production and lay the groundwork for the Warburg effect, which is essential in the molecular etiology of cancer. A key piece in this mechanism could be Snf1p, which is a protein that participates in the nutritional response including glucose metabolism. Thus, this work aimed to recognize the role of the SNF1 gene on the glycolytic flux and mitochondrial respiration through the glucose concentration variation to gain insights about its relationship with the Crabtree effect. Herein, we found that SNF1 deletion in Saccharomyces cerevisiae cells grown at 1% glucose, decreased glycolytic flux, increased NAD(P)H concentration, enhanced HXK2 gene transcription, and decreased mitochondrial respiration. Meanwhile, the same deletion increased the mitochondrial respiration of cells grown at 10% glucose. Altogether, these findings indicate that SNF1 is important to respond to glucose concentration variation and is involved in the switch between mitochondrial respiration and fermentation.

Saccharomyces cerevisiae Exponential Growth Kinetics in Batch Culture to Analyze Respiratory and Fermentative Metabolism.

Saccharomyces cerevisiae cells in the exponential phase sustain their growth by producing ATP through fermentation and/or mitochondrial respiration. The fermentable carbon concentration mainly governs how the yeast cells generate ATP; thus, the variation in fermentable carbohydrate levels drives the energetic metabolism of S. cerevisiae. This paper describes a high-throughput method based on exponential yeast growth to estimate the effects of concentration changes and nature of the carbon source on respiratory and fermentative metabolism. The growth of S. cerevisiae is measured in a microplate or shaken conical flask by determining the optical density (OD) at 600 nm. Then, a growth curve is built by plotting OD versus time, which allows identification and selection of the exponential phase, and is fitted with the exponential growth equation to obtain kinetic parameters. Low specific growth rates with higher doubling times generally represent a respiratory growth. Conversely, higher specific growth rates with lower doubling times indicate fermentative growth. Threshold values of doubling time and specific growth rate are estimated using well-known respiratory or fermentative conditions, such as non-fermentable carbon sources or higher concentrations of fermentable sugars. This is obtained for each specific strain. Finally, the calculated kinetic parameters are compared with the threshold values to establish whether the yeast shows fermentative and/or respiratory growth. The advantage of this method is its relative simplicity for understanding the effects of a substance/compound on fermentative or respiratory metabolism. It is important to highlight that growth is an intricate and complex biological process; therefore, preliminary data from this method must be corroborated by the quantification of oxygen consumption and accumulation of fermentation byproducts. Thereby, this technique can be used as a preliminary screening of compounds/substances that may disturb or enhance fermentative or respiratory metabolism.

Interactions between carbon and nitrogen sources depend on RIM15 and determine fermentative or respiratory growth in Saccharomyces cerevisiae.

Nutritional homeostasis is fundamental for alcoholic fermentation in Saccharomyces cerevisiae. Carbon and nitrogen have been related to this metabolic process; nevertheless, little is known about their interactions with the media and the energetic metabolism. Rim15p kinase is a point of convergence among different nutrient-activated signaling pathways; this makes it a target to investigate the relationship between nutritional status and energetic metabolism. To improve the current knowledge of nutrient interactions and their association with RIM15, we validated the doubling time as an indicator of growth phenotype, confirming that this kinetic parameter can be related to the cellular bioenergetic status. This endorses the usefulness of a threshold in doubling time values as an indicator of fermentative (≤ 6.5 h) and respiratory growth (≥ 13.2 h). Using the doubling time as response variable, we find that (i) two second-order interactions between type and concentration of carbon and nitrogen sources significantly affected the growth phenotype of S. cerevisiae; (ii) these metabolic interactions changed when RIM15 was deleted, suggesting a dependence on this gene; (iii) high concentration of ammonium (5% w/v) is toxic for S. cerevisiae cells; (iv) proline prompted fermentative growth phenotype regardless presence or absence of RIM15; (v) RIM15 deletion reverted ammonium toxicity when cells were grown in glucose (10% w/v); and (vi) RIM15 deletion improves fermentative metabolism probably by a partial inhibition of the respiration capacity. This study reveals the existence of synergic and diverse roles of carbon and nitrogen sources that are affected by RIM15, influencing the fermentative and respiratory growth of S. cerevisiae.

Glutathione levels influence chronological life span of Saccharomyces cerevisiae in a glucose-dependent manner.

Diet plays a key role in determining the longevity of the organisms since it has been demonstrated that glucose restriction increases life span whereas a high-glucose diet decreases it. However, the molecular basis of how diet leads to the aging process is currently unknown. We propose that the quantity of glucose that fuels respiration influences reactive oxygen species generation and glutathione levels, and both chemical species impact in the aging process. Herein, we provide evidence that mutation of the gene GSH1 in Saccharomyces cerevisiae diminishes glutathione levels. Moreover, glutathione levels were higher with 0.5% than in 10% glucose in the gsh1Δ and wild-type strains. Interestingly, the chronological life span was lowered in the gsh1Δ strain cultured with 10% glucose but not under dietary restriction. The gsh1Δ strain also showed inhibition of the mitochondrial respiration in 0.5 and 10% glucose but only increased the H2 O2 levels under dietary restriction. These results correlate well with the GSH/GSSG ratio, which showed a decrease in gsh1Δ strain cultured with 0.5% glucose. Together, these data indicate that glutathione exhaustion impact negatively both the electron transport chain function and the chronological life span of yeast, the latter occurring when a low threshold level of this antioxidant is reached, independently of the H2 O2 levels.

Resveratrol induces mitochondrial dysfunction and decreases chronological life span of Saccharomyces cerevisiae in a glucose-dependent manner.

A broad range of health benefits have been attributed to resveratrol (RSV) supplementation in mammalian systems, including the increases in longevity. Nonetheless, despite the growing number of studies performed with RSV, the molecular mechanism by which it acts still remains unknown. Recently, it has been proposed that inhibition of the oxidative phosphorylation activity is the principal mechanism of RSV action. This mechanism suggests that RSV might induce mitochondrial dysfunction resulting in oxidative damage to cells with a concomitant decrease of cell viability and cellular life span. To prove this hypothesis, the chronological life span (CLS) of Saccharomyces cerevisiae was studied as it is accepted as an important model of oxidative damage and aging. In addition, oxygen consumption, mitochondrial membrane potential, and hydrogen peroxide (H2O2) release were measured in order to determine the extent of mitochondrial dysfunction. The results demonstrated that the supplementation of S. cerevisiae cultures with 100 µM RSV decreased CLS in a glucose-dependent manner. At high-level glucose, RSV supplementation increased oxygen consumption during the exponential phase yeast cultures, but inhibited it in chronologically aged yeast cultures. However, at low-level glucose, oxygen consumption was inhibited in yeast cultures in the exponential phase as well as in chronologically aged cultures. Furthermore, RSV supplementation promoted the polarization of the mitochondrial membrane in both cultures. Finally, RSV decreased the release of H2O2 with high-level glucose and increased it at low-level glucose. Altogether, this data supports the hypothesis that RSV supplementation decreases CLS as a result of mitochondrial dysfunction and this phenotype occurs in a glucose-dependent manner.