Nonessential Yeast Research Articles

Assignment of unimodal probability distribution models for quantitative morphological phenotyping

BackgroundCell morphology is a complex and integrative readout, and therefore, an attractive measurement for assessing the effects of genetic and chemical perturbations to cells. Microscopic images provide rich information on cell morphology; therefore, subjective morphological features are frequently extracted from digital images. However, measured datasets are fundamentally noisy; thus, estimation of the true values is an ultimate goal in quantitative morphological phenotyping. Ideal image analyses require precision, such as proper probability distribution analyses to detect subtle morphological changes, recall to minimize artifacts due to experimental error, and reproducibility to confirm the results.ResultsHere, we present UNIMO (UNImodal MOrphological data), a reliable pipeline for precise detection of subtle morphological changes by assigning unimodal probability distributions to morphological features of the budding yeast cells. By defining the data type, followed by validation using the model selection method, examination of 33 probability distributions revealed nine best-fitting probability distributions. The modality of the distribution was then clarified for each morphological feature using a probabilistic mixture model. Using a reliable and detailed set of experimental log data of wild-type morphological replicates, we considered the effects of confounding factors. As a result, most of the yeast morphological parameters exhibited unimodal distributions that can be used as basic tools for powerful downstream parametric analyses. The power of the proposed pipeline was confirmed by reanalyzing morphological changes in non-essential yeast mutants and detecting 1284 more mutants with morphological defects compared with a conventional approach (Box–Cox transformation). Furthermore, the combined use of canonical correlation analysis permitted global views on the cellular network as well as new insights into possible gene functions.ConclusionsBased on statistical principles, we showed that UNIMO offers better predictions of the true values of morphological measurements. We also demonstrated how these concepts can provide biologically important information. This study draws attention to the necessity of employing a proper approach to do more with less.

Saccharomyces cerevisiae Ecm2 Modulates the Catalytic Steps of pre-mRNA Splicing.

Genetic, biochemical, and structural studies have elucidated the molecular basis for spliceosome catalysis. Splicing is RNA catalyzed and the essential snRNA and protein factors are well-conserved. However, little is known about how nonessential components of the spliceosome contribute to the reaction and modulate the activities of the fundamental core machinery. Ecm2 is a nonessential yeast splicing factor that is a member of the Prp19-related complex of proteins. Cryo-electron microscopy (cryo-EM) structures have revealed that Ecm2 binds the U6 snRNA and is entangled with Cwc2, a factor previously found to promote a catalytically active conformation of the spliceosome. These structures also indicate that Ecm2 and the U2 snRNA likely form a transient interaction during 5′ splice site (SS) cleavage. We have characterized genetic interactions between ECM2 and alleles of splicing factors that alter the catalytic steps in splicing. In addition, we have studied how loss of ECM2 impacts splicing of pre-mRNAs containing nonconsensus or competing SS. Our results show that ECM2 functions during the catalytic stages of splicing. Our data are consistent with Ecm2 facilitating the formation and stabilization of the first-step catalytic site, promoting second-step catalysis, and permitting alternate 5′ SS usage. We propose that Cwc2 and Ecm2 can each fine-tune the spliceosome active site in unique ways. Their interaction network may act as a conduit through which splicing of certain pre-mRNAs, such as those containing weak or alternate splice sites, can be regulated.

Cross-Species Complementation of Nonessential Yeast Genes Establishes Platforms for Testing Inhibitors of Human Proteins.

Cross-species complementation can be used to generate humanized yeast, which is a valuable resource with which to model and study human biology. Humanized yeast can be used as an in vivo platform to screen for chemical inhibition of human protein drug targets. To this end, we report the systematic complementation of nonessential yeast genes implicated in chromosome instability (CIN) with their human homologs. We identified 20 human-yeast complementation pairs that are replaceable in 44 assays that test rescue of chemical sensitivity and/or CIN defects. We selected a human-yeast pair (hFEN1/yRAD27), which is frequently overexpressed in cancer and is an anticancer therapeutic target, to perform in vivo inhibitor assays using a humanized yeast cell-based platform. In agreement with published in vitro assays, we demonstrate that HU-based PTPD is a species-specific hFEN1 inhibitor. In contrast, another reported hFEN1 inhibitor, the arylstibonic acid derivative NSC-13755, was determined to have off-target effects resulting in a synthetic lethal phenotype with yRAD27-deficient strains. Our study expands the list of human-yeast complementation pairs to nonessential genes by defining novel cell-based assays that can be utilized as a broad resource to study human drug targets.

Early-onset torsion dystonia: a novel high-throughput yeast genetic screen for factors modifying protein levels of torsinAΔE.

ABSTRACTDystonia is the third most common movement disorder, but its diagnosis and treatment remain challenging. One of the most severe types of dystonia is early-onset torsion dystonia (EOTD). The best studied and validated EOTD-associated mutation, torsinAΔE, is a deletion of a C-terminal glutamate residue in the AAA+ ATPase torsinA. TorsinA appears to be an endoplasmic reticulum (ER)/nuclear envelope chaperone with multiple roles in the secretory pathway and in determining subcellular architecture. Many functions are disabled in the torsinAΔE variant, and torsinAΔE is also less stable than wild-type torsinA and is a substrate for ER-associated degradation. Nevertheless, the molecular factors involved in the biogenesis and degradation of torsinA and torsinAΔE have not been fully explored. To identify conserved cellular factors that can alter torsinAΔE protein levels, we designed a new high-throughput, automated, genome-wide screen utilizing our validated Saccharomyces cerevisiae torsinA expression system. By analyzing the yeast non-essential gene deletion collection, we identified 365 deletion strains with altered torsinAΔE steady-state levels. One notable hit was EUG1, which encodes a member of the protein disulfide isomerase family (PDIs). PDIs reside in the ER and catalyze the formation of disulfide bonds, mediate protein quality control and aid in nascent protein folding. We validated the role of select human PDIs in torsinA biogenesis in mammalian cells and found that overexpression of PDIs reduced the levels of torsinA and torsinAΔE. Together, our data report the first genome-wide screen to identify cellular factors that alter expression levels of the EOTD-associated protein torsinAΔE. More generally, the identified hits help in dissecting the cellular machinery involved in folding and degrading a torsinA variant, and constitute potential therapeutic factors for EOTD. This screen can also be readily adapted to identify factors impacting the levels of any protein of interest, considerably expanding the applicability of yeast in both basic and applied research.

Leveraging DNA damage response signaling to identify yeast genes controlling genome stability.

Oncogenesis frequently is accompanied by rampant genome instability, which fuels genetic heterogeneity and resistance to targeted cancer therapy. We have developed an approach that allows precise, quantitative measurement of genome instability in high-throughput format in the Saccharomyces cerevisiae model system. Our approach takes advantage of the strongly DNA damage-inducible gene RNR3, in conjunction with the reporter synthetic genetic array methodology, to infer mutants exhibiting genome instability by assaying for increased Rnr3 abundance. We screen for genome instability across a set of ~1000 essential and ~4200 nonessential mutant yeast alleles in untreated conditions and in the presence of the DNA-damaging agent methylmethane sulfonate. Our results provide broad insights into the cellular processes and pathways required for genome maintenance. Through comparison with existing genome instability screens, we isolated 130 genes that had not previously been linked to genome maintenance, 51% of which have human homologs. Several of these homologs are associated with a genome instability phenotype in human cells or are causally mutated in cancer. A comprehensive understanding of the processes required to prevent genome instability will facilitate a better understanding of its sources in oncogenesis.

Chemical genomics approach to identify genes associated with sensitivity to rapamycin in the fission yeast Schizosaccharomyces pombe.

Rapamycin and its derivatives have now emerged as an attractive therapeutic strategy with both immunosuppressant and antitumor properties. In addition, rapamycin has been proposed as a calorie restriction mimetic to extend the life span of various organisms. The fission yeast Schizosaccharomyces pombe (S. pombe) serves as a valuable genetic model system to study the mechanism(s) of drug action as well as to determine genetic contexts associated with drug sensitivity or resistance. Here, we identified genes that when deleted modulate the rapamycin-sensitive strains in S. pombe. We carried out a chemical genomics screen for rapamycin-sensitive mutants using the genome-deletion library which covers 95.3% of all nonessential fission yeast genes and confirmed 59 genes to be rapamycin sensitive. Gene Ontology (GO) enrichment analysis showed that strains sensitive to rapamycin are highly enriched in processes regulating tRNA modification and mitochondria as well as other ontologies, including cellular metabolic process, chromatin organization, cell cycle, signaling, translation, transport and other cellular processes. Analysis also showed that components of the Elongator complex are overrepresented in the sensitive strains. Here, the data obtained will provide valuable information for speculation on the actions of rapamycin as well as on TORC signaling, thereby presenting a strategy to enhance sensitivity to rapamycin.

Rapid identification of mRNA processing defects with a novel single-cell yeast reporter

It has become increasingly evident that gene expression processes in eukaryotes involve communication and coordination between many complex, independent macromolecular machines. To query these processes and to explore the potential relationships between them in the budding yeast Saccharomyces cerevisiae, we designed a versatile reporter using multicolor high-throughput flow cytometry. Due to its design, this single reporter exhibits a distinctive signature for many defects in gene expression including transcription, histone modification, pre-mRNA splicing, mRNA export, nonsense-mediated decay, and mRNA degradation. Analysis of the reporter in 4967 nonessential yeast genes revealed striking phenotypic overlaps between chromatin remodeling, histone modification, and pre-mRNA splicing. Additionally, we developed a copper-inducible reporter, with which we demonstrate that 5-fluorouracil mimics the mRNA decay phenotype of cells lacking the 3'-5' exonuclease Rrp6p. Our reporter is capable of performing high-throughput, rapid, and large-scale screens to identify and characterize genetic and chemical perturbations of the major eukaryotic gene expression processes.

Systematic screen for mutants resistant to TORC1 inhibition in fission yeast reveals genes involved in cellular ageing and growth

SummaryTarget of rapamycin complex 1 (TORC1), which controls growth in response to nutrients, promotes ageing in multiple organisms. The fission yeast Schizosaccharomyces pombe emerges as a valuable genetic model system to study TORC1 function and cellular ageing. Here we exploited the combinatorial action of rapamycin and caffeine, which inhibit fission yeast growth in a TORC1-dependent manner. We screened a deletion library, comprising ∼84% of all non-essential fission yeast genes, for drug-resistant mutants. This screen identified 33 genes encoding functions such as transcription, kinases, mitochondrial respiration, biosynthesis, intra-cellular trafficking, and stress response. Among the corresponding mutants, 5 showed shortened and 21 showed increased maximal chronological lifespans; 15 of the latter mutants showed no further lifespan increase with rapamycin and might thus represent key targets downstream of TORC1. We pursued the long-lived sck2 mutant with additional functional analyses, revealing that the Sck2p kinase functions within the TORC1 network and is required for normal cell growth, global protein translation, and ribosomal S6 protein phosphorylation in a nutrient-dependent manner. Notably, slow cell growth was associated with all long-lived mutants while oxidative-stress resistance was not.

Vanillin Inhibits Translation and Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae: Application and Validation of High-Content, Image-Based Profiling

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

A protocol for screening of autophagy regulatory genes applying cell sorting and next generation sequencing technologies

Autophagy, as a conserved pathway in eukaryotic cells, plays a critical role in maintaining cellular homeostasis by delivering cytoplasmic materials to lysosomes/vacuoles for degradation. Abnormal autophagy activities are associated with several human diseases, such as diabetes, neurodegenerative diseases, and cancers. These observation leads to the possibilities of manipulating autophagy for therapeutic applications. To achieve this purpose, detailed information on the molecular mechanisms of autophagy regulation is essential. In this study, we have developed a protocol to screen for more selective autophagy regulatory genes based on cell image analysis, which is compliable to cell sorter application. We constructed a plasmid for expressing peroxisome‐targeted pH‐sensitive green fluorescent proteins in Saccharomyces cerevisiae. The plasmid was introduced into yeast deletion mutant pools and pexophagy‐deficient cells with high fluorescent signals after induction of pexophagy was sorted out. The identities of the mutated genes of those selected cells were determined by next generation sequencing of the gene barcodes. Our results proved it is able to distinguish atg mutants from wild types cells. We are currently characterizing the isolated autophagy mutants from the nonessential yeast gene deletion mutant pool. Grant support: NSC 101–2311‐B‐002–005

Drug Uptake, Lipid Rafts, and Vesicle Trafficking Modulate Resistance to an Anticancer Lysophosphatidylcholine Analogue in Yeast

The ether-phospholipid edelfosine, a prototype antitumor lipid (ATL), kills yeast cells and selectively kills several cancer cell types. To gain insight into its mechanism of action, we performed chemogenomic screens in the Saccharomyces cerevisiae gene-deletion strain collection, identifying edelfosine-resistant mutants. LEM3, AGP2, and DOC1 genes were required for drug uptake. Edelfosine displaced the essential proton pump Pma1p from rafts, inducing its internalization into the vacuole. Additional ATLs, including miltefosine and perifosine, also displaced Pma1p from rafts to the vacuole, suggesting that this process is a major hallmark of ATL cytotoxicity in yeast. Radioactive and synthetic fluorescent edelfosine analogues accumulated in yeast plasma membrane rafts and subsequently the endoplasmic reticulum. Although both edelfosine and Pma1p were initially located at membrane rafts, internalization of the drug toward endoplasmic reticulum and Pma1p to the vacuole followed different routes. Drug internalization was not dependent on endocytosis and was not critical for yeast cytotoxicity. However, mutants affecting endocytosis, vesicle sorting, or trafficking to the vacuole, including the retromer and ESCRT complexes, prevented Pma1p internalization and were edelfosine-resistant. Our data suggest that edelfosine-induced cytotoxicity involves raft reorganization and retromer- and ESCRT-mediated vesicular transport and degradation of essential raft proteins leading to cell death. Cytotoxicity of ATLs is mainly dependent on the changes they induce in plasma membrane raft-located proteins that lead to their internalization and subsequent degradation. Edelfosine toxicity can be circumvented by inactivating genes that then result in the recycling of internalized cell-surface proteins back to the plasma membrane.

Actin Dosage Lethality Screening in Yeast Mediated by Selective Ploidy Ablation Reveals Links to Urmylation/Wobble Codon Recognition and Chromosome Stability

The actin cytoskeleton exists in a dynamic equilibrium with monomeric and filamentous states of its subunit protein actin. The spatial and temporal regulation of actin dynamics is critical to the many functions of actin. Actin levels are remarkably constant, suggesting that cells have evolved to function within a narrow range of actin concentrations. Here we report the results of screens in which we have increased actin levels in strains deleted for the ~4800 nonessential yeast genes using a technical advance called selective ploidy ablation. We detected 83 synthetic dosage interactions with actin, 78 resulted in reduced growth, whereas in 5 cases overexpression of actin suppressed the growth defects caused by the deleted genes. The genes were highly enriched in several classes, including transfer RNA wobble uridine modification, chromosome stability and segregation, cell growth, and cell division. We show that actin overexpression sequesters a limited pool of eEF1A, a bifunctional protein involved in aminoacyl-transfer RNA recruitment to the ribosome and actin filament cross-linking. Surprisingly, the largest class of genes is involved in chromosome stability and segregation. We show that actin mutants have chromosome segregation defects, suggesting a possible role in chromosome structure and function. Monomeric actin is a core component of the INO80 and SWR chromatin remodeling complexes and the NuA4 histone modification complex, and our results suggest these complexes may be sensitive to actin stoichiometry. We propose that the resulting effects on chromatin structure can lead to synergistic effects on chromosome stability in strains lacking genes important for chromosome maintenance.

Identification of a novel deletion mutant strain in Saccharomyces cerevisiae that results in a microsatellite instability phenotype

The DNA mismatch repair (MMR) pathway corrects specific types of DNA replication errors that affect microsatellites and thus is critical for maintaining genomic integrity. The genes of the MMR pathway are highly conserved across different organisms. Likewise, defective MMR function universally results in microsatellite instability (MSI) which is a hallmark of certain types of cancer associated with the Mendelian disorder hereditary nonpolyposis colorectal cancer. (Lynch syndrome). To identify previously unrecognized deleted genes or loci that can lead to MSI, we developed a functional genomics screen utilizing a plasmid containing a microsatellite sequence that is a host spot for MSI mutations and the comprehensive homozygous diploid deletion mutant resource for Saccharomyces cerevisiae. This pool represents a collection of non-essential homozygous yeast diploid (2N) mutants in which there are deletions for over four thousand yeast open reading frames (ORFs). From our screen, we identified a deletion mutant strain of the PAU24 gene that leads to MSI. In a series of validation experiments, we determined that this PAU24 mutant strain had an increased MSI-specific mutation rate in comparison to the original background wildtype strain, other deletion mutants and comparable to a MMR mutant involving the MLH1 gene. Likewise, in yeast strains with a deletion of PAU24, we identified specific de novo indel mutations that occurred within the targeted microsatellite used for this screen.

The Role of the Parkinson's Disease Gene PARK9 in Essential Cellular Pathways and the Manganese Homeostasis Network in Yeast

YPK9 (Yeast PARK9; also known as YOR291W) is a non-essential yeast gene predicted by sequence to encode a transmembrane P-type transport ATPase. However, its substrate specificity is unknown. Mutations in the human homolog of YPK9, ATP13A2/PARK9, have been linked to genetic forms of early onset parkinsonism. We previously described a strong genetic interaction between Ypk9 and another Parkinson's disease (PD) protein α-synuclein in multiple model systems, and a role for Ypk9 in manganese detoxification in yeast. In humans, environmental exposure to toxic levels of manganese causes a syndrome similar to PD and is thus an environmental risk factor for the disease. How manganese contributes to neurodegeneration is poorly understood. Here we describe multiple genome-wide screens in yeast aimed at defining the cellular function of Ypk9 and the mechanisms by which it protects cells from manganese toxicity. In physiological conditions, we found that Ypk9 genetically interacts with essential genes involved in cellular trafficking and the cell cycle. Deletion of Ypk9 sensitizes yeast cells to exposure to excess manganese. Using a library of non-essential gene deletions, we screened for additional genes involved in tolerance to excess manganese exposure, discovering several novel pathways involved in manganese homeostasis. We defined the dependence of the deletion strain phenotypes in the presence of manganese on Ypk9, and found that Ypk9 deletion modifies the manganese tolerance of only a subset of strains. These results confirm a role for Ypk9 in manganese homeostasis and illuminates cellular pathways and biological processes in which Ypk9 likely functions.

Genomic phenotyping of the essential and non-essential yeast genome detects novel pathways for alkylation resistance

Genomic phenotyping of the essential and non-essential yeast genome detects novel pathways for alkylation resistance

Genomic phenotyping of the essential and non-essential yeast genome detects novel pathways for alkylation resistance

BackgroundA myriad of new chemicals has been introduced into our environment and exposure to these agents can damage cells and induce cytotoxicity through different mechanisms, including damaging DNA directly. Analysis of global transcriptional and phenotypic responses in the yeast S. cerevisiae provides means to identify pathways of damage recovery upon toxic exposure.ResultsHere we present a phenotypic screen of S. cerevisiae in liquid culture in a microtiter format. Detailed growth measurements were analyzed to reveal effects on ~5,500 different haploid strains that have either non-essential genes deleted or essential genes modified to generate unstable transcripts. The pattern of yeast mutants that are growth-inhibited (compared to WT cells) reveals the mechanisms ordinarily used to recover after damage. In addition to identifying previously-described DNA repair and cell cycle checkpoint deficient strains, we also identified new functional groups that profoundly affect MMS sensitivity, including RNA processing and telomere maintenance.ConclusionsWe present here a data-driven method to reveal modes of toxicity of different agents that impair cellular growth. The results from this study complement previous genomic phenotyping studies as we have expanded the data to include essential genes and to provide detailed mutant growth analysis for each individual strain. This eukaryotic testing system could potentially be used to screen compounds for toxicity, to identify mechanisms of toxicity, and to reduce the need for animal testing.

Cell Cycle-dependent Phosphorylation and Ubiquitination of a G Protein α Subunit

A diverse array of external stimuli, including most hormones and neurotransmitters, bind to cell surface receptors that activate G proteins. Mating pheromones in yeast Saccharomyces cerevisiae activate G protein-coupled receptors and initiate events leading to cell cycle arrest in G(1) phase. Here, we show that the Gα subunit (Gpa1) is phosphorylated and ubiquitinated in response to changes in the cell cycle. We systematically screened 109 gene deletion strains representing the non-essential yeast kinome and identified a single kinase gene, ELM1, as necessary and sufficient for Gpa1 phosphorylation. Elm1 is expressed in a cell cycle-dependent manner, primarily at S and G(2)/M. Accordingly, phosphorylation of Gpa1 in G(2)/M phase leads to polyubiquitination in G(1) phase. These findings demonstrate that Gpa1 is dynamically regulated. More broadly, they reveal how G proteins can simultaneously regulate, and become regulated by, progression through the cell cycle.

Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis

We developed a procedure for automated confocal microscopy to image the effect of the non-essential yeast gene deletion set on the localisation of the plasma membrane GFP-labelled protein Mrh1p-GFP. To achieve this it was necessary to devise an expression system expressing Redstar2 RFP-fluorescence specifically in the nucleus, mCherry RFP at a lower intensity in the cytoplasm and Mrh1p-GFP in the plasma membrane. This fluorescence labelling scheme utilising specifically designed image analysis scripts allowed automated segmentation of the cells into sub-regions comprising nuclei, cytoplasm and cell-surface. From this high-throughput high content screening approach we were able to determine that gene deletions including emc1Δ, emc2Δ, emc3Δ, emc4Δ, emc5Δ and emc6Δ, caused intracellular mislocalisation at the ER of a plasma membrane protein Mrh1p-GFP. CPY processing patterns were unaffected in these mutants and collectively our data suggest a transport role for the EMC genes within the early secretory pathway. HAC1 is central to the unfolded protein response (UPR) and in its absence, i.e. the absence of UPR, emc1Δ-, emc3Δ-, emc4Δ-, emc5Δ-hac1Δ double mutants were specifically hypersensitive to ER-stress (tunicamycin) lending credence to the usefulness of the high content microscope screening for discovery of functional effects of single mutants.

Genome-wide screen for inositol auxotrophy in Saccharomyces cerevisiae implicates lipid metabolism in stress response signaling

Inositol auxotrophy (Ino(-) phenotype) in budding yeast has classically been associated with misregulation of INO1 and other genes involved in lipid metabolism. To identify all non-essential yeast genes that are necessary for growth in the absence of inositol, we carried out a genome-wide phenotypic screening for deletion mutants exhibiting Ino(-) phenotypes under one or more growth conditions. We report the identification of 419 genes, including 385 genes not previously reported, which exhibit this phenotype when deleted. The identified genes are involved in a wide range of cellular processes, but are particularly enriched in those affecting transcription, protein modification, membrane trafficking, diverse stress responses, and lipid metabolism. Among the Ino(-) mutants involved in stress response, many exhibited phenotypes that are strengthened at elevated temperature and/or when choline is present in the medium. The role of inositol in regulation of lipid metabolism and stress response signaling is discussed.

Identification of Genes Required for Maximal Tolerance to High-Glucose Concentrations, as Those Present in Industrial Alcoholic Fermentation Media, Through a Chemogenomics Approach

Chemogenomics, the study of genomic responses to chemical compounds, has the potential to elucidate the basis of cellular resistance to those chemicals. This knowledge can be applied to improve the performance of strains of industrial interest. In this study, a collection of approximately 5,000 haploid single deletion mutants of Saccharomyces cerevisiae in which each nonessential yeast gene was individually deleted, was screened for strains with increased susceptibility toward stress induced by high-glucose concentration (30% w/v), one of the main stresses occurring during industrial alcoholic fermentation processes aiming the production of alcoholic beverages or bio-ethanol. Forty-four determinants of resistance to high-glucose stress were identified. The most significant Gene Ontology (GO) terms enriched in this dataset are vacuolar organization, late endosome to vacuole transport, and regulation of transcription. Clustering the identified resistance determinants by their known physical and genetic interactions further highlighted the importance of nutrient metabolism control in this context. A concentration of 30% (w/v) of glucose was found to perturb vacuolar function, by reducing cell ability to maintain the physiological acidification of the vacuolar lumen. This stress also affects the active rate of proton efflux through the plasma membrane. Based on results of published studies, the present work revealed shared determinants of yeast resistance to high-glucose and ethanol stresses, including genes involved in vacuolar function, cell wall biogenesis (ANP1), and in the transcriptional control of nutrient metabolism (GCN4 and GCR1), with possible impact on the design of more robust strains to be used in industrial alcoholic fermentation processes.