Airat Valiakhmetov

Sugar-induced cell death (SICD) remains an intriguing but poorly studied phenomenon in the physiology of Saccharomyces cerevisiae . Recently, it was shown that SICD development largely depends on the redirection of glucose fluxes between glycolysis and the pentose phosphate pathway (PPP). In particular, inhibition of glycolysis by iodoacetamide (IAA) was observed to reduce SICD levels. This study is devoted to further investigation of the relationship between SICD and the functionality of the two PPP branches. It was shown that deletion of the ZWF1 gene does not affect the decrease in SICD levels in IAA-treated cells. This allows us to conclude that the oxidative branch of the PPP is not involved in the suppression of SICD/ROS. Deletion of the GLR1 gene and attenuation of the TRR1 gene also did not restore SICD levels in cells after IAA treatment. The obtained results indicate that the level of reduced glutathione or thioredoxin does not affect SICD genesis. The addition of 5 mM ribose-5-phosphate (R5P) to the incubation medium led to suppression of SICD by 79%. At the same time, the addition of 5 mM ribose + 5 mM Pi suppressed SICD by only 20%. Suppression of SICD by 5 mM R5P in the Δpho3 strain (83%) excludes the mechanism of extracellular dephosphorylation of R5P to ribose, its subsequent transport into the cell, and re-phosphorylation inside the cell. Furthermore, more than 70% suppression of SICD in the Δend3 strain with 5 mM R5P excludes endocytosis as a mechanism of R5P import into the cell. The observed effect of R5P can be explained by the moonlighting function of some unknown protein. Thus, SICD development in S. cerevisiae cells depends on the final product of the non-oxidative PPP—R5P.

Sugar-induced cell death (SICD) remains an intriguing but poorly studied phenomenon in the physiology of Saccharomyces cerevisiae. Recently, it was shown that SICD development largely depends on the redirection of glucose fluxes between glycolysis and the pentose phosphate pathway (PPP). In particular, inhibition of glycolysis by iodoacetamide (IAA) was observed to reduce SICD levels. This study is devoted to further investigation of the relationship between SICD and the functionality of the two PPP branches. It was shown that deletion of the ZWF1 gene does not affect the decrease in SICD levels in IAA-treated cells. This allows us to conclude that the oxidative branch of the PPP is not involved in the suppression of SICD/ROS. Deletion of the GLR1 gene and attenuation of the TRR1 gene also did not restore SICD levels in cells after IAA treatment. The obtained results indicate that the level of reduced glutathione or thioredoxin does not affect SICD genesis. The addition of 5 mM ribose-5-phosphate (R5P) to the incubation medium led to suppression of SICD by 79%. At the same time, the addition of 5 mM ribose + 5 mM Pi suppressed SICD by only 20%. Suppression of SICD by 5 mM R5P in the Δpho3 strain (83%) excludes the mechanism of extracellular dephosphorylation of R5P to ribose, its subsequent transport into the cell, and re-phosphorylation inside the cell. Furthermore, more than 70% suppression of SICD in the Δend3 strain with 5 mM R5P excludes endocytosis as a mechanism of R5P import into the cell. The observed effect of R5P can be explained by the moonlighting function of some unknown protein. Thus, SICD development in S. cerevisiae cells depends on the final product of the non-oxidative PPP–R5P.

Although 30 years have passed since the description of sugar-induced cell death (SICD), the specific molecular mechanism that triggers this process remains unclear. This paper attempts to shed light on the relationship between SICD and glucose catabolism. In yeast cells, glucose is involved not only in energy-producing processes but also in the synthesis of reserve hydrocarbons. It is known that disruption of trehalose synthesis leads to significant changes in the physiology of Saccharomyces cerevisiae. The present study shows that deletion of the TPS1 gene resulted in a 44% suppression of SICD and a 75% reduction in the number of cells with excess ROS (reactive oxygen species). The suppression was comparable to the suppression of SICD (38%) and ROS (71%) with deletion of the HXK2 gene. Since HXK2 is the first enzyme in the glycolytic pathway, the effect of two other key glycolytic enzymes on SICD was tested. Deletion of the TDH3 gene (glyceraldehyde-3-phosphate dehydrogenase) resulted in a 39% suppression of SICD and ROS by 48%. Inhibition of Tdh3p with 1 mM iodoacetamide also suppressed SICD by 67% and ROS by 58%. Deletion of the PFK1 (phosphofructokinase 1) gene resulted in a complete block of SICD (97%) but unexpectedly resulted in a significant increase in the number of cells with excess ROS. The results obtained suggest that such a phenomenon as SICD is the result of an imbalance in the cellular pathways of glucose catabolism.

Inorganic polyphosphates and respective metabolic pathways and enzymes are important factors for yeast active growth in unfavorable conditions. However, particular proteins of polyphosphate metabolism remain poorly explored in this context. Here we report biochemical and transcriptomic characterization of the CRN/PPN2 yeast strain (derived from Ppn1-lacking CRN strain) overexpressing poorly studied Ppn2 polyphosphatase. We showed that Ppn2 overexpression significantly reduced lag phase in the alkaline medium presumably due to the ability of Ppn2 to efficiently hydrolyze inorganic polyphosphates and thus neutralize hydroxide ions in the cell. With RNA-Seq, we compared the molecular phenotypes of CRN/PPN2 and its parent CRN strain grown in YPD or alkaline medium and detected transcriptomic changes induced by Ppn2 overexpression and reflecting the adaptation to alkaline conditions. The core set of upregulated genes included several genes with a previously unknown function. Respective knockout strains (∆ecm8, ∆yol160w, ∆cpp3, ∆ycr099c) exhibited defects of growth or cell morphology in the alkaline medium, proving the functional involvement of the respective proteins in sustaining growth in alkaline conditions.

Although 30 years have passed since the description of sugar-induced cell death (SICD), the specific molecular mechanism that triggers this process remains unclear. This paper attempts to shed light on the relationship between SICD and glucose catabolism. Deletion of the TPS1 gene resulted in a 44% suppression of SICD and a 75% reduction in the number of cells with excess ROS. The suppression was comparable to the suppression of SICD (38%) and ROS (71%) with deletion of the HXK2 gene. Since HXK2 is the first enzyme in the glycolytic pathway, the effect of two other key glycolytic enzymes on SICD was tested. Deletion of the TDH3 gene (glyceraldehyde-3-phosphate dehydrogenase) resulted in a 39% suppression of SICD and ROS by 48%. Inhibition of Tdh3p with 1 mM iodoacetamide also suppressed SICD by 67% and ROS by 58%. Deletion of the PFK1 (phosphofructokinase 1) gene resulted in a complete block of SICD (97%), but unexpectedly resulted in a significant increase in the number of cells with excess ROS. All strains tested (except ΔPFK1) showed increased glucose consumption, suggesting that redistribution of glucose fluxes between glycolysis and the pentose phosphate shunt is a key regulator of SICD development.

Background: incubation of exponentially growing yeast S.cerevisiae with glucose in the absence of other nutrients results in S ugar I nduced C ell D eath (SICD). SICD is accompanied by the accumulation of reactive oxygen species (ROS), has the nature of primary necrosis, affects cells in the S-phase of the cell cycle, and is completely suppressed by dissipation of ΔΨ. The specific mechanism linking the ΔΨ status to the induction of SICD remains unclear. This study aimed to attempt to identify the specific molecular mechanism responsible for ROS overproduction and the development of SICD. Methods: The main method employed was the analysis of SICD development in a set of knockout mutants targeting key participants in metabolic processes. Results: A statistically significant decrease in the number of cells with ROS overproduction was observed in the ΔAFO1, ΔPOX1, ΔYNO1, ΔTRK1, ΔTRK2, ΔVSB1, and ΔYPR003C strains. A significant decrease in the number of cells with SICD was shown in the ΔTRK1, ΔVSB1, and ΔYPR003C strains. The development of SICD is not due to the presence of a nitrogen reactive species (NRS). Deletion of certain genes expressed during the S-phase of the cell cycle did not alter the dynamics of ROS accumulation and the development of SICD. The presence of exogenous or endogenous glutathione significantly suppresses both processes studied, although not as effectively as ΔΨ dissipation. Conclusions: The development of SICD is dependent on the presence of ROS, but is not strictly linked to it, as evidenced by the effects of glutathione and mutations related to its biosynthesis. In all strains tested, SICD was critically dependent on ΔΨ, although the nature of its generator remains unclear.

Incubation of the yeast S. cerevisiae with glucose, in the absence of other nutrients, leads to Sugar-Induced Cell Death (SICD), accompanied by the accumulation of Reactive Oxygen Species (ROS). Yeast acidifies the environment during glucose metabolism not only as a result of the activity of the H⁺-ATPase of the plasma membrane but also due to the release of carboxylic acids. Acetic acid is known to induce apoptosis in growing yeast. We analyzed the composition of the incubation medium and found octanoic acid (OA) but no other carboxylic acids. Its concentration (0.675 µM) was significantly lower than the one at which OA had a toxic effect on the cell. However, the theoretically calculated concentration of OA inside the cell (about 200 μM) was found to be high enough to lead to cell necrosis. To test the hypothesis that OA might cause SICD, we used a ΔACP1 strain incapable of synthesizing OA in the yeast mitochondrial Fatty Acid Synthesis type II system (FAS-II). The deletion of the ACP1 gene did not affect the OA content in the medium. But, on the other hand, OA is a precursor of lipoic acid, which has antioxidant properties. However, strains with deleted genes for lipoic acid biosynthesis from OA (ΔPPT2, ΔLIP2, ΔLIP5, and ΔSGV3) showed no change in ROS and SICD levels. Thus, lipoic acid synthesized in FAS-II does not protect cells from ROS accumulated during SICD. We conclude that OA synthesized in the mitochondrial FAS-II system and its derivative lipoic acid are not involved in SICD in yeast S. cerevisiae.

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test the expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions, such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. The expression level of YDR034W-B was higher than that of YBR056W-A under alkali and cadmium stresses. The Ydr034w-b-GFP and Ybr056w-a-GFP proteins differ in the cellular localization: Ydr034w-b-GFP was mainly observed in the plasma membrane and vacuolar membrane, while Ybr056w-a-GFP was observed in the cytoplasm, probably in intracellular membranes. The null-mutants in both genes demonstrated decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This allows for speculations about the involvement of Mnc1 and Ydr034w-b proteins in manganese stress overcoming.

The CYSTM (cysteine-rich transmembrane module) protein family comprises small molecular cysteine-rich tail-anchored membrane proteins found in many eukaryotes. The Saccharomyces cerevisiae strains carrying the CYSTM genes YDRO34W-B and YBR056W-A (MNC1) fused with GFP were used to test expression of these genes under different stresses. The YBR056W-A (MNC1) and YDR034W-B genes are expressed under stress conditions caused by the toxic concentrations of heavy metal ions such as manganese, cobalt, nickel, zinc, cuprum, and 2.4-dinitrophenol uncoupler. YDR034W-B but not YBR056W-A is expressed under alkali and cadmium stresses. YBR056W-A is localized in intracellular membranes, while YDR034W-B is localized in the cytoplasmic membrane. The null-mutants in both genes demonstrated both decreased cell concentration and lytic phenotype when cultivated in the presence of excess manganese. This indicates the involvement of Mnc1 and YDR034W-B proteins in manganese detoxification, probably by complexation of its ion.

Extracellular pH and concentration of K⁺ as well as their gradient across the plasma membrane have a significant impact on the physiology of the yeast cell, but their role in cell death has not been thoroughly investigated. Here we observed that increasing extracellular pH, as well as supplementing with K⁺ ions had a mitigating effect on cell death in yeast occurring under several conditions. The first is sugar induced cell death (SICD), and the second is death caused by several specific gene deletions, which have been recently identified in a systematic screen. It was shown that in both cases, primary necrosis is suppressed at neutral pH. SICD was also inhibited by the protonophore dinitrophenol (DNP) and 150 mM extracellular K⁺, with the latter condition also benefiting survival of cell dying due to gene mutations. In the case of SICD, these effects could not be mitigated by perturbing known pH-dependent signaling pathways, and thus are likely to be realized via direct effects on the plasma membrane potential. Thus, (a)—we show that stabilization of external pH at a neutral level can suppress different types of primary necrosis, and (b)—we suggest that changes to the cellular membrane potential can play a central role in yeast cell death caused by different factors.