Contribution of the yeast bi-chaperone system in the restoration of the RNA helicase Ded1 and translational activity under severe ethanol stress.

Preexposure to mild stress often improves cellular tolerance to subsequent severe stress. Severe ethanol stress (10% v/v) causes persistent and pronounced translation repression in Saccharomyces cerevisiae. However, it remains unclear whether preexposure to mild stress can mitigate translation repression in yeast cells under severe ethanol stress. We found that the translational activity of yeast cells pretreated with 6% (v/v) ethanol was initially significantly repressed under subsequent 10% ethanol but was then gradually restored even under severe ethanol stress. We also found that 10% ethanol caused the aggregation of Ded1, which plays a key role in translation initiation as a DEAD-box RNA helicase. Pretreatment with 6% ethanol led to the gradual disaggregation of Ded1 under subsequent 10% ethanol treatment in wild-type cells but not in fes1Δhsp104Δ cells, which are deficient in Hsp104 with significantly reduced capacity for Hsp70. Hsp104 and Hsp70 are key components of the bi-chaperone system that play a role in yeast protein quality control. fes1Δhsp104Δ cells did not restore translational activity under 10% ethanol, even after pretreatment with 6% ethanol. These results indicate that the regeneration of Ded1 through the bi-chaperone system leads to the gradual restoration of translational activity under continuous severe stress. This study provides new insights into the acquired tolerance of yeast cells to severe ethanol stress and the resilience of their translational activity.

Yeast Tor complex 1 phosphorylates eIF4E-binding protein, Caf20.

Tor complex 1 (TORC1), a master regulator of cell growth, is an evolutionarily conserved protein kinase within eukaryotic organisms. To control cell growth, TORC1 governs translational processes by phosphorylating its substrate proteins in response to cellular nutritional cues. Mammalian TORC1 (mTORC1) assumes the responsibility of phosphorylating the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) to regulate its interaction with eIF4E. The budding yeast Saccharomyces cerevisiae possesses a pair of 4E-BP genes, CAF20 and EAP1. However, the extent to which the TORC1-4E-BP axis regulates translational initiation in yeast remains uncertain. In this study, we demonstrated the influence of TORC1 on the phosphorylation status of Caf20 in vivo, as well as the direct phosphorylation of Caf20 by TORC1 in vitro. Furthermore, we found the TORC1-dependent recruitment of Caf20 to the 80S ribosome. Consequently, our study proposes a plausible involvement of yeast's 4E-BP in the efficacy of translation initiation, an aspect under the control of TORC1.

Severe ethanol stress inhibits yeast proteasome activity at moderate temperatures but not at low temperatures.

Since yeast research under laboratory conditions is usually conducted at 25-30°C (moderate temperature range), most of the findings on yeast physiology are based on analyses in this temperature range. Due to inefficiencies in cultivation and analysis, insufficient information is available on yeast physiology in the low-temperature range, although alcoholic beverage production is often conducted at relatively low temperatures (around 15°C). Recently, we reported that severe ethanol stress (10% v/v) inhibits proteasomal proteolysis in yeast cells under laboratory conditions at 28°C. In this study, proteasomal proteolysis at a low temperature (15°C) was evaluated using cycloheximide chase analysis of a short-lived protein (Gic2-3HA), an auxin-inducible degron system (Paf1-AID*-6FLAG), and Spe1-3HA, which is degraded ubiquitin-independently by the proteasome. At 15°C, proteasomal proteolysis was not inhibited under severe ethanol stress, and sufficient proteasomal activity was maintained. These results provide novel insights into the effects of low temperature and ethanol on yeast physiology.

Amino acid homeostatic control by TORC1 in Saccharomyces cerevisiae under high hydrostatic pressure.

Mechanical stresses, including high hydrostatic pressure, elicit diverse physiological effects on organisms. Gtr1, Gtr2, Ego1 (also known as Meh1) and Ego3 (also known as Slm4), central regulators of the TOR complex 1 (TORC1) nutrient signaling pathway, are required for the growth of Saccharomyces cerevisiae cells under high pressure. Here, we showed that a pressure of 25 MPa (∼250 kg/cm2) stimulates TORC1 to promote phosphorylation of Sch9, which depends on the EGO complex (EGOC) and Pib2. Incubation of cells at this pressure aberrantly increased glutamine and alanine levels in the ego1Δ, gtr1Δ, tor1Δ and pib2Δ mutants, whereas the polysome profiles were unaffected. Moreover, we found that glutamine levels were reduced by combined deletions of EGO1, GTR1, TOR1 and PIB2 with GLN3 These results suggest that high pressure leads to the intracellular accumulation of amino acids. Subsequently, Pib2 loaded with glutamine stimulates the EGOC-TORC1 complex to inactivate Gln3, downregulating glutamine synthesis. Our findings illustrate the regulatory circuit that maintains intracellular amino acid homeostasis and suggest critical roles for the EGOC-TORC1 and Pib2-TORC1 complexes in the growth of yeast under high hydrostatic pressure.

Adaptability of wine yeast to ethanol-induced protein denaturation.

This year marks the 200th anniversary of the birth of Dr Louis Pasteur (1822-1895), who revealed that alcoholic fermentation is performed by yeast cells. Subsequently, details of the mechanisms of alcoholic fermentation and glycolysis in yeast cells have been elucidated. However, the mechanisms underlying the high tolerance and adaptability of yeast cells to ethanol are not yet fully understood. This review presents the response and adaptability of yeast cells to ethanol-induced protein denaturation. Herein, we describe the adverse effects of severe ethanol stress on intracellular proteins and the responses of yeast cells. Furthermore, recent findings on the acquired resistance of wine yeast cells to severe ethanol stress that causes protein denaturation are discussed, not only under laboratory conditions, but also during the fermentation process at 15°C to mimic the vinification process of white wine.

Acquired Resistance to Severe Ethanol Stress in Saccharomyces cerevisiae Protein Quality Control.

Acute severe ethanol stress (10% [vol/vol]) damages proteins and causes the intracellular accumulation of insoluble proteins in Saccharomyces cerevisiae On the other hand, a pretreatment with mild stress increases tolerance to subsequent severe stress, which is called acquired stress resistance. It currently remains unclear whether the accumulation of insoluble proteins under severe ethanol stress may be mitigated by increasing protein quality control (PQC) activity in cells pretreated with mild stress. In the present study, we examined the induction of resistance to severe ethanol stress in PQC and confirmed that a pretreatment with 6% (vol/vol) ethanol or mild thermal stress at 37°C significantly reduced insoluble protein levels and the aggregation of Lsg1, which is prone to denaturation and aggregation by stress, in yeast cells under 10% (vol/vol) ethanol stress. The induction of this stress resistance required the new synthesis of proteins; the expression of proteins comprising the bichaperone system (Hsp104, Ssa3, and Fes1), Sis1, and Hsp42 was upregulated during the pretreatment and maintained under subsequent severe ethanol stress. Since the pretreated cells of deficient mutants in the bichaperone system (fes1Δ hsp104Δ and ssa2Δ ssa3Δ ssa4Δ) failed to sufficiently reduce insoluble protein levels and Lsg1 aggregation, the enhanced activity of the bichaperone system appears to be important for the induction of adequate stress resistance. In contrast, the importance of proteasomes and aggregases (Btn2 and Hsp42) in the induction of stress resistance has not been confirmed. These results provide further insights into the PQC activity of yeast cells under severe ethanol stress, including the brewing process.IMPORTANCE Although the budding yeast S. cerevisiae, which is used in the production of alcoholic beverages and bioethanol, is highly tolerant of ethanol, high concentrations of ethanol are also stressful to the yeast and cause various adverse effects, including protein denaturation. A pretreatment with mild stress improves the ethanol tolerance of yeast cells; however, it currently remains unclear whether it increases PQC activity and reduces the levels of denatured proteins. In the present study, we found that a pretreatment with mild ethanol upregulated the expression of proteins involved in PQC and mitigated the accumulation of insoluble proteins, even under severe ethanol stress. These results provide novel insights into ethanol tolerance and the adaptive capacity of yeast. They may also contribute to research on the physiology of yeast cells during the brewing process, in which the concentration of ethanol gradually increases.

Persistent actin depolarization caused by ethanol induces the formation of multiple small cortical septin rings in yeast.

Short-term exposure to severe ethanol stress has adverse effects on yeast cells. However, limited information is available on the effects of long-term exposure to severe ethanol stress. In this study, we examined the effects of a long-term treatment with a high ethanol concentration [10% (v/v)] on yeast morphology. We found that long-term severe ethanol stress induced the continuous depolarization of the actin cytoskeleton and hypertrophy in yeast cells, accompanied by the aberrant localization of septins, which formed multiple small cortical rings (MSCRs). The formation of MSCRs was also induced by the continuous depolarization of the actin cytoskeleton caused by a treatment with latrunculin-A, an effective inhibitor of actin polymerization. Unlike the formation of conventional septin rings, the formation of MSCRs did not require Cdc42 and its effectors, Gic1, Gic2 and Cla4. These results provide novel insights into the effects of persistent actin depolarization caused by long-term exposure to severe ethanol stress on yeast cytomorphology.

Nutrient Signaling via the TORC1-Greatwall-PP2AB55δ Pathway Is Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae.

Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 (K7) and its relatives carry a homozygous loss-of-function mutation in the RIM15 gene, which encodes a Greatwall family protein kinase. Disruption of RIM15 in nonsake yeast strains leads to improved alcoholic fermentation, indicating that the defect in Rim15p is associated with the enhanced fermentation performance of sake yeast cells. In order to understand how Rim15p mediates fermentation control, we here focused on target-of-rapamycin protein kinase complex 1 (TORC1) and protein phosphatase 2A with the B55δ regulatory subunit (PP2AB55δ), complexes that are known to act upstream and downstream of Rim15p, respectively. Several lines of evidence, including our previous transcriptomic analysis data, suggested enhanced TORC1 signaling in sake yeast cells during sake fermentation. Fermentation tests of the TORC1-related mutants using a laboratory strain revealed that TORC1 signaling positively regulates the initial fermentation rate in a Rim15p-dependent manner. Deletion of the CDC55 gene, encoding B55δ, abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55δ mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55δ pathway similarly affected the fermentation rate in the fission yeast Schizosaccharomyces pombe, strongly suggesting that the evolutionarily conserved pathway governs alcoholic fermentation in yeasts. It is likely that elevated PP2AB55δ activity accounts for the high fermentation performance of sake yeast cells. Heterozygous loss-of-function mutations in CDC55 found in K7-related sake strains may indicate that the Rim15p-deficient phenotypes are disadvantageous to cell survival.IMPORTANCE The biochemical processes and enzymes responsible for glycolysis and alcoholic fermentation by the yeast S. cerevisiae have long been the subject of scientific research. Nevertheless, the factors determining fermentation performance in vivo are not fully understood. As a result, the industrial breeding of yeast strains has required empirical characterization of fermentation by screening numerous mutants through laborious fermentation tests. To establish a rational and efficient breeding strategy, key regulators of alcoholic fermentation need to be identified. In the present study, we focused on how sake yeast strains of S. cerevisiae have acquired high alcoholic fermentation performance. Our findings provide a rational molecular basis to design yeast strains with optimal fermentation performance for production of alcoholic beverages and bioethanol. In addition, as the evolutionarily conserved TORC1-Greatwall-PP2AB55δ pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.

Btn2 is involved in the clearance of denatured proteins caused by severe ethanol stress in Saccharomyces cerevisiae.

Saccharomyces cerevisiae shows similar responses to heat shock and ethanol stress. Cells treated with severe ethanol stress activate the transcription of HSP genes and cause the aggregation of Hsp104-GFP, implying that severe ethanol stress as well as heat shock causes the accumulation of denatured proteins in yeast cells. However, there is currently no concrete evidence to show that severe ethanol stress causes protein denaturation in living yeast cells. In the present study, we investigated whether severe ethanol stress causes protein denaturation, and confirmed that a treatment with 10% (v/v) ethanol stress resulted in the accumulation of insoluble proteins and ubiquitinated proteins in yeast cells. We also found that increased denatured protein levels were efficiently reduced by the ubiquitin-proteasome system after the elimination of ethanol. Since our previous findings demonstrated that the expression of Btn2 was induced by severe ethanol stress, we herein examined the importance of Btn2 in protein quality control in cells treated with severe ethanol stress. btn2∆ cells showed a significant delay in the clearance of denatured proteins during the recovery process. These results provide further insights into the effects of severe ethanol stress on yeast proteostasis and the contribution of Btn2 to the efficient clearance of denatured proteins.

The VFH1 (YLL056C) promoter is vanillin-inducible and enables mRNA translation despite pronounced translation repression caused by severe vanillin stress in Saccharomyces cerevisiae.

Vanillin, furfural and 5-hydroxymethylfurfural (HMF) are representative fermentation inhibitors generated during the pretreatment process of lignocellulosic biomass in bioethanol production. These biomass conversion inhibitors, particularly vanillin, are known to repress translation activity in Saccharomyces cerevisiae. We have reported that the mRNAs of ADH7 and BDH2 were efficiently translated under severe vanillin stress despite marked repression of overall protein synthesis. In this study, we found that expression of VFH1 (YLL056C) was also significantly induced at the protein level by severe vanillin stress. Additionally, we demonstrated that the VFH1 promoter enabled the protein synthesis of other genes including GFP and ALD6 under severe vanillin stress. It is known that transcriptional activation of VFH1 is induced by furfural and HMF, and we verified that Vfh1 protein synthesis was also induced by furfural and HMF. The null mutant of VFH1 delayed growth in the presence of vanillin, furfural and HMF, indicating the importance of Vfh1 for sufficient tolerance against these inhibitors. The protein levels of Vfh1 induced by the inhibitors tested were markedly higher than those of Adh7 and Bdh2, suggesting the superior utility of the VFH1 promoter over the ADH7 or BDH2 promoter for breeding optimized yeast strains for bioethanol production from lignocellulosic biomass.

Protein synthesis of Btn2 under pronounced translation repression during the process of alcoholic fermentation and wine-making in yeast.

Acute high-concentration ethanol (> 9% v/v) has adverse effects on Saccharomyces cerevisiae, including the remarkable repression of bulk mRNA translation. Therefore, increased mRNA levels do not necessarily lead to an increase in the corresponding protein levels in yeast cells under severe ethanol stress. We previously identified that synthesis of Btn2 protein was efficiently induced even under the pronounced translation repression caused by acute severe ethanol stress under laboratory conditions. However, it remains to be clarified whether the translational activity is also repressed and whether the synthesis of Btn2 protein is induced during the process of alcoholic fermentation, in which the ethanol concentration increases gradually to reach high levels. In this study, we revealed that the pronounced translation repression and the translation of BTN2 are induced by high ethanol concentrations that form gradually during alcoholic fermentation using a wine yeast strain EC1118. Furthermore, we confirmed the induced expression of non-native genes driven by the BTN2 promoter during the later stage of the wine-making process. Our findings provide new information on the translation activity in yeast cells during alcoholic fermentation and suggest the utility of the BTN2 promoter for sustaining the fermentation efficiency and quality modification of alcoholic beverages.

Cold atmospheric pressure plasma causes protein denaturation and endoplasmic reticulum stress in Saccharomyces cerevisiae.

Cold atmospheric pressure plasma (CAP) does not cause thermal damage or generate toxic residues; hence, it is projected as an alternative agent for sterilization in food and pharmaceutical industries. The fungicidal effects of CAP have not yet been investigated as extensively as its bactericidal effects. We herein examined the effects of CAP on yeast proteins using a new CAP system with an improved processing capacity. We demonstrated that protein ubiquitination and the formation of protein aggregates were induced in the cytoplasm of yeast cells by the CAP treatment. GFP-tagged Tsa1 and Ssa1, an H2O2-responsive molecular chaperone and constitutively expressed Hsp70, respectively, formed cytoplasmic foci in CAP-treated cells. Furthermore, Tsa1 was essential for the formation of Ssa1-GFP foci. These results indicate that the denaturation of yeast proteins was caused by CAP, at least partially, in a H2O2-dependent manner. Furthermore, misfolded protein levels in the endoplasmic reticulum (ER) and the oligomerization of Ire1, a key sensor of ER stress, were enhanced by the treatment with CAP, indicating that CAP causes ER stress in yeast cells as a specific phenomenon to eukaryotic cells. The pretreatment of yeast cells at 37 °C significantly alleviated cell death caused by CAP. Our results strongly suggest that the induction of protein denaturation is a primary mechanism of the fungicidal effects of CAP.

The novel heme-dependent inducible protein, SRRD regulates heme biosynthesis and circadian rhythms.

Heme plays a role in the regulation of the expression of genes related to circadian rhythms and heme metabolism. In order to identify new heme-regulated proteins, an RNA sequence analysis using mouse NIH3T3 cells treated without or with 5-aminolevulinic acid (ALA) was performed. Among the changes observed in the levels of various mRNAs including heme oxygenase-1 (HO-1) and ALA synthase-1 (ALAS1), a mouse homologue of the plant circadian-regulating protein SRR1, SRR1 domain containing (SRRD) was induced by the ALA treatment. The expression of SRRD was dependent on heme biosynthesis, and increased the production of heme. SRRD was expressed under circadian rhythms, and influenced the expression of clock genes including PER2, BMAL1, and CLOCK. The knockout of SRRD arrested the growth of cells, indicating that SRRD plays roles in heme-regulated circadian rhythms and cell proliferation.

Potential role of heme metabolism in the inducible expression of heme oxygenase-1.

BACKGROUND: The degradation of heme significantly contributes to cytoprotective effects against oxidative stress and inflammation. The enzyme heme oxygenase-1 (HO-1), involved in the degradation of heme, forms carbon monoxide (CO), ferrous iron, and bilirubin in conjunction with biliverdin reductase, and is induced by various stimuli including oxidative stress and heavy metals. We examined the involvement of heme metabolism in the induction of HO-1 by the inducers sulforaphane and sodium arsenite. METHODS: We examined the expression of HO-1 in sulforaphane-, sodium arsenite- and CORM3-treated HEK293T cells, by measuring the transcriptional activity and levels of mRNA and protein. RESULTS: The blockade of heme biosynthesis by succinylacetone and N-methyl protoporphyrin, which are inhibitors of heme biosynthesis, markedly decreased the induction of HO-1. The knockdown of the first enzyme in the biosynthesis of heme, 5-aminolevulinic acid synthase, also decreased the induction of HO-1. The cessation of HO-1 induction occurred at the transcriptional and translational levels, and was mediated by the activation of the heme-binding transcriptional repressor Bach1 and translational factor HRI. CO appeared to improve the expression of HO-1 at the transcriptional and translational levels. CONCLUSIONS: We demonstrated the importance of heme metabolism in the stress-inducible expression of HO-1, and also that heme and its degradation products are protective factors for self-defense responses. GENERAL SIGNIFICANCE: The key role of heme metabolism in the stress-inducible expression of HO-1 may promote further studies on heme and its degradation products as protective factors of cellular stresses and iron homeostasis in specialized cells, organs, and whole animal systems.

Fluorescence microscopic analysis of antifungal effects of cold atmospheric pressure plasma in Saccharomyces cerevisiae.

Cold atmospheric pressure plasma (CAP) has potential to be utilized as an alternative method for sterilization in food industries without thermal damage or toxic residues. In contrast to the bactericidal effects of CAP, information regarding the efficacy of CAP against eukaryotic microorganisms is very limited. Therefore, herein we investigated the effects of CAP on the budding yeast Saccharomyces cerevisiae, with a focus on the cellular response to CAP. The CAP treatment caused oxidative stress responses including the nuclear accumulation of the oxidative stress responsive transcription factor Yap1, mitochondrial fragmentation, and enhanced intracellular oxidation. Yeast cells also induced the expression of heat shock protein (HSP) genes and formation of Hsp104 aggregates when treated with CAP, suggesting that CAP denatures proteins. As phenomena unique to eukaryotic cells, the formation of cytoplasmic mRNP granules such as processing bodies and stress granules and changes in the intracellular localization of Ire1 were caused by the treatment with CAP, indicating that translational repression and endoplasmic reticulum (ER) stress were induced by the CAP treatment. These results suggest that the fungicidal effects of CAP are attributed to the multiple severe stresses.

Plasma membrane proteins Yro2 and Mrh1 are required for acetic acid tolerance in Saccharomyces cerevisiae.

Yro2 and its paralogous protein Mrh1 of Saccharomyces cerevisiae have seven predicted transmembrane domains and predominantly localize to the plasma membrane. Their physiological functions and regulation of gene expression have not yet been elucidated in detail. We herein demonstrated that MRH1 was constitutively expressed, whereas the expression of YRO2 was induced by acetic acid stress and entering the stationary phase. Fluorescence microscopic analysis revealed that Mrh1 and Yro2 were distributed as small foci in the plasma membrane under acetic acid stress conditions. The null mutants of these genes (mrh1∆, yro2∆, and mrh1∆yro2∆) showed delayed growth and a decrease in the productivity of ethanol in the presence of acetic acid, indicating that Yro2 and Mrh1 are involved in tolerance to acetic acid stress.

Adaptive response in stress granule formation and bulk translational repression upon a combined stress of mild heat shock and mild ethanol stress in yeast.

The formation of cytoplasmic mRNA-protein complex granules termed 'processing bodies and stress granules' is often induced in the stress responses of eukaryotic cells. Most previous studies on stress granules have focused on the response to a single type of stress, and little information is available about the response to combined stress. Additionally, the effects of adaptation on stress granule formation and bulk translation activity are poorly understood. We investigated the formation of stress granules upon combined exposure to mild heat shock (37 °C) and mild ethanol stress (5% v/v) in Saccharomyces cerevisiae. Although neither stress alone induced stress granule formation, their combination caused a pronounced repression of translation activity and the formation of stress granules. Pretreatment with each mild stress significantly attenuated the formation of stress granules and caused changes in the composition of stress granules upon the subsequent combined stress and facilitated stress granule disassembly accompanied by smooth translational resurrection during the recovery process, indicating that yeast cells can induce adaptations in stress granule formation. However, the pretreated cells still exhibited a severe repression of translation activity. These findings provide novel and fundamental insight into the regulation of yeast stress granules.

Reduction of glucose uptake through inhibition of hexose transporters and enhancement of their endocytosis by methylglyoxal in Saccharomyces cerevisiae.

Diabetes mellitus is characterized by an impairment of glucose uptake even though blood glucose levels are increased. Methylglyoxal is derived from glycolysis and has been implicated in the development of diabetes mellitus, because methylglyoxal levels in blood and tissues are higher in diabetic patients than in healthy individuals. However, it remains to be elucidated whether such factors are a cause, or consequence, of diabetes. Here, we show that methylglyoxal inhibits the activity of mammalian glucose transporters using recombinant Saccharomyces cerevisiae cells genetically lacking all hexose transporters but carrying cDNA for human GLUT1 or rat GLUT4. We found that methylglyoxal inhibits yeast hexose transporters also. Glucose uptake was reduced in a stepwise manner following treatment with methylglyoxal, i.e. a rapid reduction within 5 min, followed by a slow and gradual reduction. The rapid reduction was due to the inhibitory effect of methylglyoxal on hexose transporters, whereas the slow and gradual reduction seemed due to endocytosis, which leads to a decrease in the amount of hexose transporters on the plasma membrane. We found that Rsp5, a HECT-type ubiquitin ligase, is responsible for the ubiquitination of hexose transporters. Intriguingly, Plc1 (phospholipase C) negatively regulated the endocytosis of hexose transporters in an Rsp5-dependent manner, although the methylglyoxal-induced endocytosis of hexose transporters occurred irrespective of Plc1. Meanwhile, the internalization of hexose transporters following treatment with methylglyoxal was delayed in a mutant defective in protein kinase C.

Biomass conversion inhibitors furfural and 5-hydroxymethylfurfural induce formation of messenger RNP granules and attenuate translation activity in Saccharomyces cerevisiae.

Various forms of stress can cause an attenuation of bulk translation activity and the accumulation of nontranslating mRNAs into cytoplasmic messenger RNP (mRNP) granules termed processing bodies (P-bodies) and stress granules (SGs) in eukaryotic cells. Furfural and 5-hydroxymethylfurfural (HMF), derived from lignocellulosic biomass, inhibit yeast growth and fermentation as stressors. Since there is no report regarding their effects on the formation of cytoplasmic mRNP granules, here we investigated whether furfural and HMF cause the assembly of yeast P-bodies and SGs accompanied by translational repression. We found that furfural and HMF cause the attenuation of bulk translation activity and the assembly of cytoplasmic mRNP granules in Saccharomyces cerevisiae. Notably, a combination of furfural and HMF induced the remarkable repression of translation initiation and SG formation. These findings provide new information about the physiological effects of furfural and HMF on yeast cells, and also suggest the potential usefulness of cytoplasmic mRNP granules as a warning sign or index of the deterioration of cellular physiological status in the fermentation of lignocellulosic hydrolysates.

Acidic stress induces the formation of P-bodies, but not stress granules, with mild attenuation of bulk translation in Saccharomyces cerevisiae.

The stress response of eukaryotic cells often causes an attenuation of bulk translation activity and the accumulation of non-translating mRNAs into cytoplasmic mRNP (messenger ribonucleoprotein) granules termed cytoplasmic P-bodies (processing bodies) and SGs (stress granules). We examined effects of acidic stress on the formation of mRNP granules compared with other forms of stress such as glucose deprivation and a high Ca²âº level in Saccharomyces cerevisiae. Treatment with lactic acid clearly caused the formation of P-bodies, but not SGs, and also caused an attenuation of translation initiation, albeit to a lesser extent than glucose depletion. P-body formation was also induced by hydrochloric acid and sulfuric acid. However, lactic acid in SD (synthetic dextrose) medium with a pH greater than 3.0, propionic acid and acetic acid did not induce P-body formation. The results of the present study suggest that the assembly of yeast P-bodies can be induced by external conditions with a low pH and the threshold was around pH 2.5. The P-body formation upon acidic stress required Scd6 (suppressor of clathrin deficiency 6), a component of P-bodies, indicating that P-bodies induced by acidic stress have rules of assembly different from those induced by glucose deprivation or high Ca²âº levels.