Yeast Deletion Strains Research Articles

Genes required for survival in microgravity revealed by genome-wide yeast deletion collections cultured during spaceflight.

Spaceflight is a unique environment with profound effects on biological systems including tissue redistribution and musculoskeletal stresses. However, the more subtle biological effects of spaceflight on cells and organisms are difficult to measure in a systematic, unbiased manner. Here we test the utility of the molecularly barcoded yeast deletion collection to provide a quantitative assessment of the effects of microgravity on a model organism. We developed robust hardware to screen, in parallel, the complete collection of ~4800 homozygous and ~5900 heterozygous (including ~1100 single-copy deletions of essential genes) yeast deletion strains, each carrying unique DNA that acts as strain identifiers. We compared strain fitness for the homozygous and heterozygous yeast deletion collections grown in spaceflight and ground, as well as plus and minus hyperosmolar sodium chloride, providing a second additive stressor. The genome-wide sensitivity profiles obtained from these treatments were then queried for their similarity to a compendium of drugs whose effects on the yeast collection have been previously reported. We found that the effects of spaceflight have high concordance with the effects of DNA-damaging agents and changes in redox state, suggesting mechanisms by which spaceflight may negatively affect cell fitness.

Enhanced longevity by ibuprofen, conserved in multiple species, occurs in yeast through inhibition of tryptophan import.

The common non-steroidal anti-inflammatory drug ibuprofen has been associated with a reduced risk of some age-related pathologies. However, a general pro-longevity role for ibuprofen and its mechanistic basis remains unclear. Here we show that ibuprofen increased the lifespan of Saccharomyces cerevisiae, Caenorhabditis elegans and Drosophila melanogaster, indicative of conserved eukaryotic longevity effects. Studies in yeast indicate that ibuprofen destabilizes the Tat2p permease and inhibits tryptophan uptake. Loss of Tat2p increased replicative lifespan (RLS), but ibuprofen did not increase RLS when Tat2p was stabilized or in an already long-lived strain background impaired for aromatic amino acid uptake. Concomitant with lifespan extension, ibuprofen moderately reduced cell size at birth, leading to a delay in the G1 phase of the cell cycle. Similar changes in cell cycle progression were evident in a large dataset of replicatively long-lived yeast deletion strains. These results point to fundamental cell cycle signatures linked with longevity, implicate aromatic amino acid import in aging and identify a largely safe drug that extends lifespan across different kingdoms of life.

Cell Wall Perturbation Sensitizes Fungi to the Antimalarial Drug Chloroquine

Chloroquine (CQ) has been a mainstay of antimalarial drug treatment for several decades. Additional therapeutic actions of CQ have been described, including some reports of fungal inhibition. Here we investigated the action of CQ in fungi, including the yeast model Saccharomyces cerevisiae. A genomewide yeast deletion strain collection was screened against CQ, revealing that bck1Δ and slt2Δ mutants of the cell wall integrity pathway are CQ hypersensitive. This phenotype was rescued with sorbitol, consistent with cell wall involvement. The cell wall-targeting agent caffeine caused hypersensitivity to CQ, as did cell wall perturbation by sonication. The phenotypes were not caused by CQ-induced changes to cell wall components. Instead, CQ accumulated to higher levels in cells with perturbed cell walls: CQ uptake was 2- to 3-fold greater in bck1Δ and slt2Δ mutants than in wild-type yeast. CQ toxicity was synergistic with that of the major cell wall-targeting antifungal drug, caspofungin. The MIC of caspofungin against the yeast pathogen Candida albicans was decreased 2-fold by 250 μM CQ and up to 8-fold at higher CQ concentrations. Similar effects were seen in Candida glabrata and Aspergillus fumigatus. The results show that the cell wall is critical for CQ resistance in fungi and suggest that combination treatments with cell wall-targeting drugs could have potential for antifungal treatment.

Identification of Accessory Proteins Critical for the Epithelial Sodium Channel (ENaC)

The epithelial sodium channel (ENaC) is rate‐limiting for the reabsorption of sodium in the distal nephron of the kidneys. Genetic mutations in ENaC can cause diseases such as Liddle's syndrome and pseudohypoaldosteronism type 1, causing severe hypertension or hypotension, respectively. Accessory proteins including Nedd4, ubiquitin protein‐ligase, and a serum and glucocorticoid‐regulated kinase have been shown to regulate ENaC and additional proteins are likely to play an essential role in its proper function. In an effort to identify proteins necessary for proper assembly, localization, and function of ENaC, BY4742 yeast deletion strains were transformed with αENaC, which induces a salt‐sensitive phenotype, and yeast dilution pronging assays were performed. While five deletion strains showed no effect on ENaC function in yeast, three strains lacking scj1, jem1, or lhs1 showed loss of ENaC function while deletion of ypk1 seemed to enhance ENaC function. Localization studies of αENaC in these deletion strains were used to elucidate the role of each gene on ENaC surface expression. Funded by NIH (R15GM086798) and Welch Foundation (Dept Grant A10048).

Investigation of the functions of new genes involved in repair of DNA double‐strand breaks in Saccharomyces cerevisiae

Chromosomal DNA is constantly damaged by internal chemical and enzymatic processes and by external agents such as radiation and chemicals in the environment. Potentially lethal lesions induced by many agents are DNA double‐strand breaks (DSBs) within the chromosome. Repair of DSBs is critical for prevention of mutations and chromosome instability, which increase the risk of cancer and cell death. Eukaryotes repair these lesions via two conserved pathways, homologous recombination and nonhomologous end‐joining (NHEJ). The goal of this project is to improve our understanding of the genes and metabolic pathways required for efficient repair of broken DNA. Previous studies used a yeast deletion strain library to identify mutants that were unable to repair gamma radiation‐induced damage. In the current study, a subset of these mutants were characterized that cannot repair DNA DSBs induced by in vivo expression of EcoRI endonuclease or the chemicals methyl methanesulfonate (MMS) and bleomycin. Most of the 73 new genes identified in this work have not previously been associated with DNA repair. Forty‐one of the genes are known to affect nuclear processes. The functions of many of these genes in homologous recombination and maintenance of chromosome stability are being assessed using genetic and physical assays of DNA repair. This research is supported by an NIH AREA R15 grant to L.K. Lewis.

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.

Sequestration of Sup35 by Aggregates of huntingtin Fragments Causes Toxicity of [PSI+] Yeast

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

BackgroundChitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS.ResultsThree different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS.ConclusionsOverexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.

Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin.

SummaryWith renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited.

Regulatory Dynamics of the Transcriptional Network Controlling the Cold Shock Response in Saccharomyces cerevisiae

We are using mathematical modeling to describe the interactions between transcription factors that control the environmental stress response of cold shock in Saccharomyces cerevisiae. We inferred a network of twenty‐one transcription factors that regulate one another from timecourse microarray data from wild type yeast subjected to 15, 30, and 60 minutes of cold shock at 13°C. The concentrations of the individual transcription factors were modeled using ordinary differential equations, which we solved as a system to determine the dynamical behavior of the model. Each equation includes a production rate, a degradation rate (which we computed using published half‐life data), and weights that denote the influence of the connected transcription factors. We estimated parameter values using the DNA microarray data. Model/data comparisons were performed with least squares. Model predictions fit the data well within two standard deviations of the experimental error. We then compared simulated model data where the transcription factors Cin5, Gln3, Hmo1, and Zap1 were removed in silico with experimental microarray data from the corresponding yeast deletion strains. This revealed the relative influence of these transcription factors on the dynamics of the transcriptional response to cold shock in yeast. This work was supported by NSF award 0921038 (KDD, BGF, KS) and the McLaughlin Chair of Biology (KDD, NAR).

Identification of Assessory Proteins Needed for ENaC Assembly and Function

The epithelial sodium channel (ENaC) is a sodium transporting protein channel composed of three subunits, alpha, beta and gamma with a stoichiometric ratio of 1:1:1. ENaC regulates sodium reabsorption within the distal nephron and is essential for maintaining fluid homeostasis. ENaC dysfunction causes blood pressure disorders as seen with pseudohypoaldosteronism and Liddle's syndrome, which lead to hypotension and hypertension, respectively. Although there are several known activators and inhibitors of ENaC, including the sterol aldosterone and the peptide hormone arginine vasopressin, the role of other intracellular proteins affecting ENaC assembly is unclear. Our current studies utilize the yeast deletion strain library in conjunction with ENaC expression to identify accessory proteins that are crucial for ENaC assembly and function. Yeast strains expressing mouse ENaC demonstrate salt‐sensitivity (growth inhibition), while strains expressing ENaC and lacking SUR4, ERV1, EMP24 or ULA1 showed a reduction in salt‐sensitivity suggesting that they directly affect ENaC function or assembly. Supported by Research Corporation (CC6313) and NIH‐NIGMS (R15GM086798).

A Simple Yeast-Based Strategy to Identify Host Cellular Processes Targeted by Bacterial Effector Proteins

Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of Gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

A genome-wide screen identifies yeast genes required for protection against or enhanced cytotoxicity of the antimalarial drug quinine

Quinine is used in the treatment of Plasmodium falciparum severe malaria. However, both the drug's mode of action and mechanisms of resistance are still poorly understood and subject to debate. In an effort to clarify these questions, we used the yeast Saccharomyces cerevisiae as a model for pharmacological studies with quinine. Following on a previous work that examined the yeast genomic expression program in response to quinine, we now explore a genome-wide screen for altered susceptibility to quinine using the EUROSCARF collection of yeast deletion strains. We identified 279 quinine-susceptible strains, among which 112 conferred a hyper-susceptibility phenotype. The expression of these genes, mainly involved in carbohydrate metabolism, iron uptake and ion homeostasis functions, is required for quinine resistance in yeast. Sixty-two genes whose deletion leads to increased quinine resistance were also identified in this screen, including several genes encoding ribosome protein subunits. These well-known potential drug targets in Plasmodium are associated with quinine action for the first time in this study. The suggested involvement of phosphate signaling and transport in quinine tolerance was also studied, and activation of phosphate starvation-responsive genes was observed under a mild-induced quinine stress. Finally, P. falciparum homology searches were performed for a selected group of 41 genes. Thirty-two encoded proteins possess homologs in the parasite, including subunits of a parasitic vacuolar H(+)-ATPase complex, ion and phosphate importers, and several ribosome protein subunits, suggesting that the results obtained in yeast are good candidates to be transposed and explored in a P. falciparum context.

The resistance of the yeast Saccharomyces cerevisiae to the biocide polyhexamethylene biguanide: involvement of cell wall integrity pathway and emerging role for YAP1

BackgroundPolyhexamethylene biguanide (PHMB) is an antiseptic polymer that is mainly used for cleaning hospitals and pools and combating Acantamoeba infection. Its fungicide activity was recently shown by its lethal effect on yeasts that contaminate the industrial ethanol process, and on the PE-2 strain of Saccharomyces cerevisiae, one of the main fermenting yeasts in Brazil. This pointed to the need to know the molecular mechanism that lay behind the cell resistance to this compound. In this study, we examined the factors involved in PHMB-cell interaction and the mechanisms that respond to the damage caused by this interaction. To achieve this, two research strategies were employed: the expression of some genes by RT-qPCR and the analysis of mutant strains.ResultsCell Wall integrity (CWI) genes were induced in the PHMB-resistant Saccharomyces cerevisiae strain JP-1, although they are poorly expressed in the PHMB-sensitive Saccharomyces cerevisiae PE2 strain. This suggested that PHMB damages the glucan structure on the yeast cell wall. It was also confirmed by the observed sensitivity of the yeast deletion strains, Δslg1, Δrom2, Δmkk2, Δslt2, Δknr4, Δswi4 and Δswi4, which showed that the protein kinase C (PKC) regulatory mechanism is involved in the response and resistance to PHMB. The sensitivity of the Δhog1 mutant was also observed. Furthermore, the cytotoxicity assay and gene expression analysis showed that the part played by YAP1 and CTT1 genes in cell resistance to PHMB is unrelated to oxidative stress response. Thus, we suggested that Yap1p can play a role in cell wall maintenance by controlling the expression of the CWI genes.ConclusionThe PHMB treatment of the yeast cells activates the PKC1/Slt2 (CWI) pathway. In addition, it is suggested that HOG1 and YAP1 can play a role in the regulation of CWI genes.

FASTA barcodes: a simple method for the identification of yeast ORF deletions

A consortium of yeast geneticists have created -6000 individual ORF deletions, representing > 96% of the currently verified or predicted ORFs in S. cerevisiae. Importantly, molecular barcodes (each a unique 20 bp sequence termed either Uptag or Downtag) were used as identifiers for every ORF deletion. Microarray analyses of pooled yeast deletions has been used to identify thousands of genes involved in general fitness, haploinsufficiency, drug resistance and DNA damage repair. However, application of this powerful technology requires considerable expense, expertise and specialized equipment. While standard PCR techniques and specifically designed PCR primers can be used to confirm that a given ORF is in fact deleted, this procedure cannot be used to identify unknown deletions. In theory, every ORF deletion could be determined by barcode sequencing. However, neither a consolidated barcode database nor a reliable search engine is currently available for this purpose. To address this need, we have adapted a FASTA sequence program that utilizes the unique barcode database to allow users to identify individual ORF deletions, based upon simple sequencing reactions of PCR amplifications of either Uptag or Downtag barcodes. In silico and practical testing of this application reveals that it is an inexpensive, reliable and reproducible method for rapidly identifying unknown deletions. This approach allows laboratories to conduct small- or large-scale genetic screens with pooled yeast deletion strains and identify or verify any ORF deletion without the need for microarray technology.

Genomewide Profiling of Rapamycin Sensitivity in Saccharomyces cerevisiae on Synthetic Medium

The target of rapamycin (TOR) signaling pathway is a conserved pathway that regulates eukaryotic cell growth in response to environmental cues. Chemical genomic approaches that profile rapamycin sensitivity of yeast deletion strains have given insights into the function of TOR signaling pathway. In the present study, we analyzed the rapamycin sensitivity of yeast deletion library strains on synthetic medium. As a result, we identified 130 strains that are hypersensitive or resistant to rapamycin compared with wild-type cells. Among them, 36 genes are newly identified to be related to rapamycin sensitivity. Moreover, we found 16 strains that show alteration in rapamycin sensitivity between complex and synthetic media. We suggest that these genes may be involved in part of TOR signaling activities that is differentially regulated by media composition.

Identification of genes that enhance cellulase protein production in yeast

In order to enhance heterologous cellulase protein production in yeast, a plasmid harboring the endoglucanase gene from Clostridium thermocellum ( Ctcel8A) was used to systematically transform a homozygous diploid yeast deletion strain collection. We identified 55 deletion strains that exhibited enhanced endoglucanase activity compared with that of the wild-type strain. Genes disrupted in these strains were classified into the categories of transcription, translation, phospholipid synthesis, endosome/vacuole function, ER/Golgi function, nitrogen starvation response, and cytoskeleton. The vps3Δ and vps16Δ strains, which have deletion in genes encoding components of the class C core vacuole/endosome tethering (CORVET) complex, also exhibited enhanced β-glucosidase activity when Ctcel8A was heterologously expressed. Moreover, multiple gene deletion strains were constructed by using the vps3Δ strain. Endoglucanase activity of the resulting rav1Δ vps3Δ double deletion strain was exhibited higher than that of the rav1Δ or vps3Δ strains. Our genome-wide analyses using the yeast deletion strain collection identified useful genes that allow efficient expression of cellulase.

The Carboxyl-terminal End of Cox1 Is Required for Feedback Assembly Regulation of Cox1 Synthesis in Saccharomyces cerevisiae Mitochondria

Synthesis of the largest cytochrome c oxidase (CcO) subunit, Cox1, on yeast mitochondrial ribosomes is coupled to assembly of CcO. The translational activator Mss51 is sequestered in early assembly intermediate complexes by an interaction with Cox14 that depends on the presence of newly synthesized Cox1. If CcO assembly is prevented, the level of Mss51 available for translational activation is reduced. We deleted the C-terminal 11 or 15 residues of Cox1 by site-directed mutagenesis of mtDNA. Although these deletions did not prevent respiratory growth of yeast, they eliminated the assembly-feedback control of Cox1 synthesis. Furthermore, these deletions reduced the strength of the Mss51-Cox14 interaction as detected by co-immunoprecipitation, confirming the importance of the Cox1 C-terminal residues for Mss51 sequestration. We surveyed a panel of mutations that block CcO assembly for the strength of their effect on Cox1 synthesis, both by pulse labeling and expression of the ARG8(m) reporter fused to COX1. Deletion of the nuclear gene encoding Cox6, one of the first subunits to be added to assembling CcO, caused the most severe reduction in Cox1 synthesis. Deletion of the C-terminal 15 amino acids of Cox1 increased Cox1 synthesis in the presence of each of these mutations, except pet54. Our data suggest a novel activity of Pet54 required for normal synthesis of Cox1 that is independent of the Cox1 C-terminal end.

A ribosome-associating factor chaperones tail-anchored membrane proteins

Hundreds of proteins are post-translationally inserted into the endoplasmic reticulum (ER) membrane by a single carboxy-terminal transmembrane domain (TMD)1. During targeting through the cytosol, the hydrophobic TMD of these tail-anchored (TA) proteins requires constant chaperoning to prevent aggregation or inappropriate interactions. A central component of this targeting system is TRC40, a conserved cytosolic factor that recognizes the TMD of TA proteins and delivers them to the ER for insertion2-4. The mechanism that permits TRC40 to effectively find and capture its TA protein cargos in a highly crowded cytosol is unknown. Here, we identify a conserved three-protein complex composed of Bat3, TRC35, and Ubl4A that facilitates TA protein capture by TRC40. This Bat3 complex is recruited to ribosomes synthesizing membrane proteins, interacts with the TMDs of newly released TA proteins, and transfers them to TRC40 for targeting. Depletion of the Bat3 complex allows non-TRC40 factors to compete for TA proteins, explaining their mislocalization in the analogous yeast deletion strains5-7. Thus, the Bat3 complex acts as a TMD selective chaperone that effectively channels TA proteins to the TRC40 insertion pathway.

Global Protein Synthesis and Hypoxia Responses in Yeast

Oxygen is critical for the survival and development of many living organisms, particularly eukaryotes. Living organisms ranging from yeast to humans have developed sophisticated mechanisms to respond to the changes of oxygen level in the environment. We used the yeast Saccharomyces cerevisiae as a model system to study mechanisms of oxygen sensing and regulation. To identify potential global regulators mediating oxygen regulation and hypoxia responses, we initiated a genome‐wide screen for deletion mutants that enable cells to grow better under hypoxia. By visual screening, we initially identified 104 yeast deletion mutants that grew significantly better under hypoxia, compared to the wild type parent from a library of 4786 yeast deletion strains. Further quantitative measurements showed that six deletion mutants of genes encoding functions involved in ribosome biogenesis exhibit the highest growth enhancement under hypoxia. Experiments are underway to investigate the molecular mechanisms by which ribosome biogenesis and global protein synthesis promote cell growth under hypoxia. GM062246