Response Of Yeast Cells Research Articles

Insights into yeast response to chemotherapeutic agent through time series genome-scale metabolic models.

Organism-specific genome-scale metabolic models (GSMMs) can unveil molecular mechanisms within cells and are commonly used in diverse applications, from synthetic biology, biotechnology, and systems biology to metabolic engineering. There are limited studies incorporating time-series transcriptomics in GSMM simulations. Yeast is an easy-to-manipulate model organism for tumor research.Here, a novel approach (TS-GSMM) was proposed to integrate time-series transcriptomics with GSMMs to narrow down the feasible solution space of all possible flux distributions and attain time-series flux samples. The flux samples were clustered using machine learning techniques, and the clusters' functional analysis was performed using reaction set enrichment analysis.A time series transcriptomics response of Yeast cells to a chemotherapeutic reagent-doxorubicin-was mapped onto a Yeast GSMM. Eleven flux clusters were obtained with our approach, and pathway dynamics were displayed. Induction of fluxes related to bicarbonate formation and transport, ergosterol and spermidine transport, and ATP production were captured.Integrating time-series transcriptomics data with GSMMs is a promising approach to reveal pathway dynamics without any kinetic modeling and detects pathways that cannot be identified through transcriptomics-only analysis. The codes are available at https://github.com/karabekmez/TS-GSMM.

Comparative Transcriptome Analysis of Industrial and Laboratory Saccharomyces cerevisiae Strains after Sequential Stresses

While the transcriptional responses of yeast cells to a variety of individual stress conditions have been extensively studied, their responses to sequential stress conditions are less well understood. In this study, we present a comparative analysis of the transcriptome of an industrial strain and a laboratory strain exposed to different sequential stresses to establish a common response profile and also to identify genes whose expression is strain-dependent. Both strains induce pathways related to oxidative stress and osmotic stress response including those involved in glycerol synthesis, glutathione metabolism, and NADPH regeneration. Other genes that may also play an important role in this response include the transcription factor ADR1, SYM1, and most of the heat shock proteins. Induction of genes related to autophagy of mitochondria occurred only in the laboratory strain while possible stress tolerance factors, such as additional genes involved in glutathione production and detoxification, were uniformly enhanced only in the industrial strain. The analysis of the stress response to sequential stresses of two different strains allowed more precise identification of the response of yeast to complex environments. Identification of genes uniquely induced in the industrial strain can also be used to develop strategies to optimize various fermentation processes.

Saccharomyces Cerevisiae as a Model Organism for Retrospective Impedance Biodosimetry.

Previous studies have shown that measuring changes in electrical impedance that follow radiation-induced suppression of metabolic activity in irradiated yeast cells can be used to determine radiation dose. The current work investigates the radiation response of Saccharomyces cerevisiae cells by using metabolic activity of cells as a damage indicator. Impedance biodosimetry was examined as a method to evaluate the radiation response of yeast cells. Active lab-grade dry yeast cells were used as the biological material as these samples are simple to handle and have a long shelf-life. A novel dosimeter design has been developed with a strict fabrication method and measurement procedure to ensure reproducible measurements are possible. Prepared yeast samples were irradiated to doses from 0.5 to 8 Gy using a 137Cs source, and a dose response curve was developed that showed a linear relationship of dose with changes in impedance measurements. Fading of the impedance signal was also investigated, and it was shown that there was no noticeable fading of the impedance signal over a period of 7 mo. Finally, the lowest detectable limit measured using this methodology was determined to be 300 mGy. This work presents an alternative retrospective dosimetry technique that can be used at a high scale and low cost following large-scale radiological accidents.

Integrated Transcriptomics and Metabolomics Analysis Reveal the Regulatory Mechanisms Underlying Sodium Butyrate-Induced Carotenoid Biosynthesis in Rhodotorula glutinis.

Sodium butyrate (SB) is a histone deacetylase inhibitor that can induce changes in gene expression and secondary metabolite titers by inhibiting histone deacetylation. Our preliminary analysis also indicated that SB significantly enhanced the biosynthesis of carotenoids in the Rhodotorula glutinis strain YM25079, although the underlying regulatory mechanisms remained unclear. Based on an integrated analysis of transcriptomics and metabolomics, this study revealed changes in cell membrane stability, DNA and protein methylation levels, amino acid metabolism, and oxidative stress in the strain YM25079 under SB exposure. Among them, the upregulation of oxidative stress may be a contributing factor for the increase in carotenoid biosynthesis, subsequently enhancing the strain resistance to oxidative stress and maintaining the membrane fluidity and function for normal cell growth. To summarize, our results showed that SB promoted carotenoid synthesis in the Rhodotorula glutinis strain YM25079 and increased the levels of the key metabolites and regulators involved in the stress response of yeast cells. Additionally, epigenetic modifiers were applied to produce fungal carotenoid, providing a novel and promising strategy for the biosynthesis of yeast-based carotenoids.

The cAMP-PKA signalling crosstalks with CWI and HOG-MAPK pathways in yeast cell response to osmotic and thermal stress.

The yeast Saccharomyces cerevisiae is widely used in food and non-food industries. During industrial fermentation yeast strains are exposed to fluctuations in oxygen concentration, osmotic pressure, pH, ethanol concentration, nutrient availability and temperature. Fermentation performance depends on the ability of the yeast strains to adapt to these changes. Suboptimal conditions trigger responses to the external stimuli to allow homeostasis to be maintained. Stress-specific signalling pathways are activated to coordinate changes in transcription, translation, protein function, and metabolic fluxes while a transient arrest of growth and cell cycle progression occur. cAMP-PKA, HOG-MAPK and CWI signalling pathways are turned on during stress response. Comprehension of the mechanisms involved in the responses and in the adaptation to these stresses during fermentation is key to improving this industrial process. The scope of this review is to outline the advancement of knowledge about the cAMP-PKA signalling and the crosstalk of this pathway with the CWI and HOG-MAPK cascades in response to the environmental challenges heat and hyperosmotic stress.

The Reaction of the Yeast Saccharomyces cerevisiae to Contamination of the Medium with Aflatoxins B2 and G1, Ochratoxin A and Zearalenone in Aerobic Cultures.

The mechanisms by which yeast cells respond to environmental stress include the production of heat shock proteins (HSPs) and the reduction of oxidative stress. The response of yeast exposed to aflatoxins B2+G1 (AFB2+G1), ochratoxin A (OTA), and zearalenone (ZEA) in aerobic conditions was studied. After 72 h of yeast cultivation in media contaminated with mycotoxins, the growth of yeast biomass, the level of malondialdehyde, and the activity of superoxide dismutase, glutathione S-transferase and glutathione peroxidase were examined; the expression profile of the following heat shock proteins was also determined: HSP31, HSP40, HSP60, HSP70, and HSP104. It was demonstrated that at the tested concentrations, both AFB2+G1 and ZEA inhibited yeast biomass growth. OTA at a concentration of 8.4 [µg/L] raised the MDA level. Intensified lipoperoxidation and increased activity of SOD and GPx were observed, regardless of the level of contamination with ZEA (300 µg/L or 900 µg/L). Increased contamination with AFB2+G1 and OTA caused an increase in the production of most HSPs tested (HSP31, HSP40, HSP70, HSP104). ZEA contamination in the used concentration ranges reduced the production of HSP31. The response of yeast cells to the presence of mycotoxin as a stressor resulted in the expression of certain HSPs, but the response was not systematic, which was manifested in different profiles of protein expression depending on the mycotoxin used. The tested mycotoxins influenced the induction of oxidative stress in yeast cells to varying degrees, which resulted in the activation of mainly SOD without GST mobilization or with a small involvement of GPx.

The yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.

SO2-резистентность как критерий отбора штаммов Saccharomyces cerevisiae для органического виноделия

Sulfur dioxide is a popular conserving agent and antioxidant in winemaking. Unfortunately, it is bad for human health. Some yeast strains can reduce the dose of sulfur dioxide. Such yeasts should have good fermentation activity and dominate when inoculated into grape must. In addition, it should not synthesize sulfur dioxide and SO2-binding substances. The synthesis of sulfur dioxide and carbonyl compounds by yeast is related to the mechanisms of sulfur dioxide detoxification. The research objective was to study the relationship between the resistance of yeast to sulfur dioxide and its ability to synthesize sulfur dioxide and acetaldehyde during growth. 
 The study featured 17 yeast strains of the genus Saccharomyces. The yeasts were cultivated on grape must in a CGQ device until the stationary growth phase. The concentration of free and bound forms of sulfur dioxide was determined by titration, while that of aldehydes was determined by bisulfite method. The sulfite resistance of strains was measured ¬¬by the growth response of yeast cells to sulfur dioxide using CGQ technology. 
 Yeast strains differed in the degree of sulfur dioxide resistance. The samples were divided according to the increase in the lag phase time: by ≥ 8 h (sensitive), by 2–6 h, without changes (resistant). At Wilks L = 0.228 and α = 0.05, the sensitive cultures in a SO2-free medium had the highest value of minimal generation time in the exponential growth phase (5.3 ± 2.1 h). The resistant samples demonstrated the highest synthesis of acetaldehyde (54.7 ± 11.1 mg/L) and sulfur dioxide (21.0 ± 10.3 mg/L). The second group cultures had the lowest content of SO2-bound forms in the medium (10.9 ± 4.2 mg/L) and were in an interposition in terms of other indicators. 
 The time it takes a yeast strain to adapt to sulfur dioxide can be used as a parameter for the primary culture selection in eco-winemaking. According to the physiological and biochemical profile, the resistant strains can be recommended for the production of SO2-low wines, while the samples from the second test group proved optimal for organic wines. Further research will expand the range of yeast strains and their indicators.

Response of Saccharomyces cerevisiae var. diastaticus to nerol: Evaluation of antifungal potential by inhibitory effect and proteome analyses

Saccharomyces cerevisiae var. diastaticus (S. diastaticus) is a major spoilage yeast in brewing. In the present research, the antifungal properties of nerol and the proteome response of S. diastaticus were studied. Results showed nerol can inhibit cell budding and delay yeast fermentation in a dose-depended manner. After 3 d of treatment with 0.25 mg·mL−1 nerol, intracellular ROS levels increased 1.66-fold (P < 0.01), and the cells with damaged membrane increased to 23.2 %. Quantitative proteomic profiles utilizing a capillary-HPLC-MS/MS technology revealed that proteins involved in the metabolism of fermentable sugars were up-regulated in S. diastaticus cells treated with nerol, indicating nerol treatment altered the metabolite pattern of fermentable sugars. Proteins associated with the cell membrane biogenesis, heat shock proteins, amino acid biosynthesis, and glutathione metabolism were similarly up-regulated. These findings revealed the mechanism of nerol-induced yeast cell damage as well as the detoxification response of yeast cells.

Neomycin Interferes with Phosphatidylinositol-4,5-Bisphosphate at the Yeast Plasma Membrane and Activates the Cell Wall Integrity Pathway

The cell wall integrity pathway (CWI) is a MAPK-mediated signaling route essential for yeast cell response to cell wall damage, regulating distinct aspects of fungal physiology. We have recently proven that the incorporation of a genetic circuit that operates as a signal amplifier into this pathway allows for the identification of novel elements involved in CWI signaling. Here, we show that the strong growth inhibition triggered by pathway hyperactivation in cells carrying the “Integrity Pathway Activation Circuit” (IPAC) also allows the easy identification of new stimuli. By using the IPAC, we have found various chemical agents that activate the CWI pathway, including the aminoglycoside neomycin. Cells lacking key components of this pathway are sensitive to this antibiotic, due to the disruption of signaling upon neomycin stimulation. Neomycin reduces both phosphatidylinositol-4,5-bisphosphate (PIP2) availability at the plasma membrane and myriocin-induced TORC2-dependent Ypk1 phosphorylation, suggesting a strong interference with plasma membrane homeostasis, specifically with PIP2. The neomycin-induced transcriptional profile involves not only genes related to stress and cell wall biogenesis, but also to amino acid metabolism, reflecting the action of this antibiotic on the yeast ribosome.

SLX5 deletion confers tolerance to oxidative stress in Saccharomyces cerevisiae.

Slx5, a subunit of a SUMO-targeted ubiquitin ligase (STUbL) in yeast, has been implicated in maintenance of genomic stability. SUMOylation is an important post-translational modification involved in the regulation of several important cellular processes and cellular response to various environmental stressors. Oxidative stress occurs when production of reactive oxygen species (ROS) exceeds the antioxidant defense capacity of the cell. Elevated ROS levels cause oxidative damage to important cellular macromolecules such as DNA, lipids, and proteins, which is associated with several diseases. Herein, we investigated the role of Slx5 in oxidative stress tolerance in Saccharomyces cerevisiae. We show that deletion of SLX5 increases survival of yeast cells in response to H2O2-induced oxidative stress in a cell cycle independent manner. Accumulation of intracellular ROS as well as DNA and lipid damages were reduced; expressions of antioxidant defense mechanism-related genes were increased in slx5Δ cells compared to wild type (WT) under oxidative stress. We also show that slx5Δ cells have increased intracellular ROS levels and oxidative damage to DNA and lipids compared to WT in the absence of oxidative stress. Thus, our data together suggest that an adaptive stress induced by SLX5 deletion increases tolerance to oxidative stress in slx5∆ cells.

Functional verification and screening of protein interacting with the slPHB3

ABSTRACT slPHB3 was cloned from Salix linearistipularis, the amino acid sequence blast and phylogenetic tree analysis showed that slPHB3 has the most similarity with PHB3 from Populus trichocarpa using DNAMAN software and MEGA7 software. RT-qPCR results confirmed that the expression of slPHB3 was induced obviously under stress treatments. The growth of recombinant yeast cells was better than that of the control group under the stress treatment, indicating that slPHB3 may be involved in the stress response of yeast cells. The transgenic tobacco was treated with different concentrations of NaCl, NaHCO3 and H2O2, fresh weigh of overexpression tobacco were heavier than wild-types. The results showed that transgenic tobacco was more tolerant to salt and oxidation than wild-type tobacco. Expression of important genes including NHX1 and SOS1 in salt stress response pathways are steadily higher in overexpression tobacco than that in wild-types. We identified 17 proteins interacting with slPHB3 by yeast two-hybrid technique, most of these proteins were relation to the stresses. The salt tolerance of slPHB3 expressing yeast and slPHB3 overexpressing plants were better than that of the control. Ten stress-related proteins may interact with slPHB3, which preliminarily indicated that slPHB3 had a certain response relationship with salt stress. The study of slPHB3 under abiotic stress can improve our understanding of PHB3 gene function.

Rad53 checkpoint kinase regulation of DNA replication fork rate via Mrc1 phosphorylation.

The Rad53 DNA checkpoint protein kinase plays multiple roles in the budding yeast cell response to DNA replication stress. Key amongst these is its enigmatic role in safeguarding DNA replication forks. Using DNA replication reactions reconstituted with purified proteins, we show Rad53 phosphorylation of Sld3/7 or Dbf4-dependent kinase blocks replication initiation whilst phosphorylation of Mrc1 or Mcm10 slows elongation. Mrc1 phosphorylation is necessary and sufficient to slow replication forks in complete reactions; Mcm10 phosphorylation can also slow replication forks, but only in the absence of unphosphorylated Mrc1. Mrc1 stimulates the unwinding rate of the replicative helicase, CMG, and Rad53 phosphorylation of Mrc1 prevents this. We show that a phosphorylation-mimicking Mrc1 mutant cannot stimulate replication in vitro and partially rescues the sensitivity of a rad53 null mutant to genotoxic stress in vivo. Our results show that Rad53 protects replication forks in part by antagonising Mrc1 stimulation of CMG unwinding.

Impact of Lignocellulose Pretreatment By-Products on S. cerevisiae Strain Ethanol Red Metabolism during Aerobic and An-aerobic Growth.

Understanding the specific response of yeast cells to environmental stress factors is the starting point for selecting the conditions of adaptive culture in order to obtain a yeast line with increased resistance to a given stress factor. The aim of the study was to evaluate the specific cellular response of Saccharomyces cerevisiae strain Ethanol Red to stress caused by toxic by-products generated during the pretreatment of lignocellulose, such as levulinic acid, 5-hydroxymethylfurfural, furfural, ferulic acid, syringaldehyde and vanillin. The presence of 5-hydroxymethylfurfural at the highest analyzed concentration (5704.8 ± 249.3 mg/L) under aerobic conditions induced the overproduction of ergosterol and trehalose. On the other hand, under anaerobic conditions (during the alcoholic fermentation), a decrease in the biosynthesis of these environmental stress indicators was observed. The tested yeast strain was able to completely metabolize 5-hydroxymethylfurfural, furfural, syringaldehyde and vanillin, both under aerobic and anaerobic conditions. Yeast cells reacted to the presence of furan aldehydes by overproducing Hsp60 involved in the control of intracellular protein folding. The results may be helpful in optimizing the process parameters of second-generation ethanol production, in order to reduce the formation and toxic effects of fermentation inhibitors.

Time-dependent re-organization of biological processes by the analysis of the dynamic transcriptional response of yeast cells to doxorubicin.

Doxorubicin is an efficient chemotherapeutic reagent in the treatment of a variety of cancers. However, its underlying molecular mechanism is not fully understood and several severe side effects limit its application. In this study, the dynamic transcriptomic response of Saccharomyces cerevisiae cells to a doxorubicin pulse in a chemostat system was investigated to reveal the underlying molecular mechanism of this drug. The clustering of differentially and significantly expressed genes (DEGs) indicated that the response of yeast cells to doxorubicin is time dependent and may be classified as short-term, mid-term and long-term responses. The cells have started to reorganize their response after the first minute following the injection of the pulse. A modified version of Weighted Gene Co-expression Network Analysis (WGCNA) was used to cluster the positively correlated co-expression profiles, and functional enrichment analysis of these clusters was carried out. DNA replication and DNA repair processes were significantly affected and induced 60 minutes after exposure to doxorubicin. The response to oxidative stress was not identified as a significant term. A transcriptional re-organization of the metabolic pathways seems to be an early event and persists afterwards. The present study reveals for the first time that the RNA surveillance pathway, which is a post-transcriptional regulatory pathway, may be implicated in the short-term reaction of yeast cells to doxorubicin. Integration with regulome revealed the dynamic re-organization of the transcriptomic landscape. Fhl1p, Mbp1p, and Mcm1p were identified as primary regulatory factors responsible for tuning the differentially expressed genes.

Anticipatory Stress Responses and Immune Evasion in Fungal Pathogens.

In certain niches, microbes encounter environmental challenges that are temporally linked. In such cases, microbial fitness is enhanced by the evolution of anticipatory responses where the initial challenge simultaneously activates pre-emptive protection against the second impending challenge. The accumulation of anticipatory responses in domesticated yeasts, which have been termed 'adaptive prediction', has led to the emergence of 'core stress responses' that provide stress cross-protection. Protective anticipatory responses also seem to be common in fungal pathogens of humans. These responses reflect the selective pressures that these fungi have faced relatively recently in their evolutionary history. Consequently, some pathogens have evolved 'core environmental responses' which exploit host signals to trigger immune evasion strategies that protect them against imminent immune attack.

Insights Into the Mechanism of Anticancer Drug Imatinib Revealed Through Multi-Omic Analyses in Yeast.

Imatinib mesylate is a receptor tyrosine kinase inhibitor drug with broad applications in cancer therapeutics, for example, in chronic myeloid leukemia and gastrointestinal stromal tumors. In this study, new multi-omics findings in yeast on the mechanism of imatinib are reported, using the model organism Saccharomyces cerevisiae. Whole-genome analysis of the transcriptional response of yeast cells following long-term exposure to imatinib, flux-balance analysis (FBA), and modular analysis of protein/protein interaction network consisting of proteins encoded by differentially expressed genes (DEGs) were performed. DEGs indicated that carbon, nitrogen, starch, sucrose, glyoxylate/dicarboxylate metabolism, valine and leucine degradation, and tricarboxylic acid cycle (TCA) were significantly upregulated. By contrast, ribosome biogenesis, pentose/glucuronate interconversion, tryptophan/pyruvate metabolic pathways, and meiosis were significantly downregulated. FBA revealed that a large set of metabolic pathways was altered by imatinib to compensate cancer-associated metabolic changes. Integration of transcriptome and interactome (protein/protein interactions) data helped to identify the core regulatory genes and pathways through elucidation of the active subnetworks. It appears that imatinib may also contribute to antitumoral immune response in the tumor microenvironment and most of the metabolic rearrangements are at posttranscriptional level. Furthermore, additional support for possible contribution of thiamine/pyridoxal phosphate biosynthesis and mitogen-activated protein kinase pathway to drug resistance is noted. This report advances multi-omics understanding of the mechanism of imatinib, and by extension, offers new molecular avenues toward precision medicine and discovery of novel drug targets in cancer therapeutics.

Stiffness of brewers’ yeast under ethanol stress investigated by atomic force microscopy

Yeast cell exposed under ethanol environment changes the mechanical properties, in addition to physiological properties including the ability of growth and ethanol tolerance. However, it is unclear how the temporal variation in ethanol concentration, CEtOH, occurring during brewing, influences the mechanical properties of yeast cells. Here, we investigated the stiffness of brewers’ yeast cells in response to the change in CEtOH using atomic force microscopy (AFM). We found that as CEtOH was abruptly changed into a critical value, the survival rate of cells was drastically decreased while cells rather survived as CEtOH was gradually increased toward the critical value for a few days. Furthermore, our AFM measurements showed that cells that acquired the ethanol tolerance significantly decreased their stiffness. The result indicated an intimate relationship between cell stiffness and ethanol tolerance that depends on the pathway of changing ethanol environments.

Monitoring the Functionality and Stress Response of Yeast Cells Using Flow Cytometry.

Throughout fermentation, yeast faces continuously changing medium conditions and reacts by adapting its metabolism. The adaptation is a critical process and is dependent on the accurate functioning of the cell. A stable membrane potential, which is, among other roles, responsible for protecting the yeast from low pH, is an important attribute for evaluating functionality. Other factors are storage products such as glycogen, trehalose, and neutral lipids, as well as mitochondrial activity and the integrity of the DNA. These parameters can be complemented by the analysis of viability, cell cycle, intracellular pH, and reactive oxygen species in the cell. The correlation of all these factors provides valuable information for evaluating the performance of a yeast population during fermentation. In order to demonstrate the analytical capabilities of flow cytometry, a Saccharomyces cerevisiae yeast strain was observed in a modified growth medium for 384 h (16 days). The results confirm observations made with other methods and reports from the literature. However, with flow cytometry, it is possible to gain deeper insight into stress response and adaptation behavior of yeast at a cellular level. The causality from the formation of oxygen-radicals to cell death, for example, can be shown, as well as the dependency of the intracellular pH on the stability of the membrane. The proposed bio-monitoring system has the potential to provide applicable information as a process control tool for wineries.

Rpn4 and proteasome-mediated yeast resistance to ethanol includes regulation of autophagy.

Distilled spirits production using Saccharomyces cerevisiae requires understanding of the mechanisms of yeast cell response to alcohol stress. Reportedly, specific mutations in genes of the ubiquitin-proteasome system, e.g., RPN4, may result in strains exhibiting hyper-resistance to different alcohols. To study the Rpn4-dependent yeast response to short-term ethanol exposure, we performed a comparative analysis of the wild-type (WT) strain, strain with RPN4 gene deletion (rpn4-Δ), and a mutant strain with decreased proteasome activity and consequent Rpn4 accumulation due to PRE1 deregulation (YPL). The stress resistance tests demonstrated an increased sensitivity of mutant strains to ethanol compared with WT. Comparative proteomics analysis revealed significant differences in molecular responses to ethanol between these strains. GO analysis of proteins upregulated in WT showed enrichments represented by oxidative and heat responses, protein folding/unfolding, and protein degradation. Enrichment of at least one of these responses was not observed in the mutant strains. Moreover, activity of autophagy was not increased in the RPN4 deletion strain upon ethanol stress which agrees with changes in mRNA levels of ATG7 and PRB1 genes of the autophagy system. Activity of the autophagic system was clearly induced and accompanied with PRB1 overexpression in the YPL strain upon ethanol stress. We demonstrated that Rpn4 stabilization contributes to the PRB1 upregulation. CRISPR-Cas9-mediated repression of PACE-core Rpn4 binding sites in the PRB1 promoter inhibits PRB1 induction in the YPL strain upon ethanol treatment and results in YPL hypersensitivity to ethanol. Our data suggest that Rpn4 affects the autophagic system activity upon ethanol stress through the PRB1 regulation. These findings can be a basis for creating genetically modified yeast strains resistant to high levels of alcohol, being further used for fermentation in ethanol production.