Yeast Genetics Research Articles

Yeast R2TP Interacts with Extended Termini of Client Protein Nop58p

The AAA + ATPase R2TP complex facilitates assembly of a number of ribonucleoprotein particles (RNPs). Although the architecture of R2TP is known, its molecular basis for acting upon multiple RNPs remains unknown. In yeast, the core subunit of the box C/D small nucleolar RNPs, Nop58p, is the target for R2TP function. In the recently observed U3 box C/D snoRNP as part of the 90 S small subunit processome, the unfolded regions of Nop58p are observed to form extensive interactions, suggesting a possible role of R2TP in stabilizing the unfolded region of Nop58p prior to its assembly. Here, we analyze the interaction between R2TP and a Maltose Binding Protein (MBP)-fused Nop58p by biophysical and yeast genetics methods. We present evidence that R2TP interacts largely with the unfolded termini of Nop58p. Our results suggest a general mechanism for R2TP to impart specificity by recognizing unfolded regions in its clients.

Effect of BRCA1 missense variants on gene reversion in DNA double-strand break repair mutants and cell cycle-arrested cells of Saccharomyces cerevisiae.

Evaluation of the functional impact of germline BRCA1 variants that are likely to be associated to breast and ovarian cancer could help to investigate the mechanism of BRCA1 tumorigenesis. Expression of pathogenic BRCA1 missense variants increased homologous recombination (HR) and gene reversion (GR) in yeast. We thought to exploit yeast genetics to shed light on BRCA1-induced genome instability and tumorigenesis. We determined the effect on GR of several neutral and pathogenic BRCA1 variants in the yeast strain RSY6wt and its isogenic DSB repair mutants, such as mre11∆, rad50∆ and rad51∆. In the RSY6wt, four out of five pathogenic and two out of six neutral variants significantly increased GR; rad51∆ strain, the pathogenic variants C61G and A1708E induced a weak but significant increase in GR. On the other hand, in rad50∆ mutant expressing the pathogenic variants localised at the BRCT domain, a further GR increase was seen. The neutral variant N132K and the VUS A1789T induced a weak GR increase in mre11∆ mutant. Thus, BRCA1 missense variants require specific genetic functions and presumably induced GR by different mechanisms. As DNA repair is regulated by cell cycle, we determined the effect on GR of BRCA1 variants in cell cycle-arrested RSYwt cells. GR is highly BRCA1-inducible in S-phase-arrested cells as compared to G1 or G2. Sequence analysis of genomic DNA from ILV1 revertant clones showed that BRCA1-induced ilv1-92 reversion by base substitution when GR is at least 6-fold over the control. Our study demonstrated that BRCA1 may interfere with yeast DNA repair functions that are active in S-phase causing high level of GR. In addition, we confirmed here that yeast could be a reliable model to investigate the mechanism and genetic requirements of BRCA1-induced genome instability. Finally, developing yeast-based assays to characterise BRCA1 missense variants could be useful to design more precise therapies.

MSphere of Influence: the Power of Yeast Genetics Still Going Strong!

Felipe Santiago-Tirado studies the cell biology of cryptococcal infections. In this mSphere of Influence article, he reflects on how the papers "Systematic Genetic Analysis of Virulence in the Human Fungal Pathogen Cryptococcus neoformans" (https://doi.org/10.1016/j.cell.2008.07.046) and "Unraveling the Biology of a Fungal Meningitis Pathogen Using Chemical Genetics" (https://doi.org/10.1016/j.cell.2014.10.044) by the Noble and Madhani groups influenced his thinking by showcasing the various modern applications of yeast genetics in an organism where genetic manipulation was difficult.

Temperature-sensitive cytoophidium assembly in Schizosaccharomyces pombe

The metabolic enzyme CTP synthase (CTPS) is able to compartmentalize into filaments, termed cytoophidia, in a variety of organisms including bacteria, budding yeast, fission yeast, fruit flies and mammals. A previous study in budding yeast shows that the filament-forming process of CTPS is not sensitive to temperature shift. Here we study CTPS filamentation in the fission yeast Schizosaccharomyces pombe. To our surprise, we find that both the length and the occurrence of cytoophidia in S. pombe decrease upon cold shock or heat shock. The temperature-dependent changes of cytoophidia are fast and reversible. Taking advantage of yeast genetics, we demonstrate that heat-shock proteins are required for cytoophidium assembly in S. pombe. Temperature sensitivity of cytoophidia makes S. pombe an attractive model system for future investigations of this novel membraneless organelle.

Progress in the genomics and genome-wide study of sake yeast.

Completion of the whole genome sequence of a laboratory yeast strain Saccharomyces cerevisiae in 1996 ushered in the development of genome-wide experimental tools and accelerated subsequent genetic study of S. cerevisiae. The study of sake yeast also shared the benefit of such tools as DNA microarrays, gene disruption-mutant collections, and others. Moreover, whole genome analysis of representative sake yeast strain Kyokai no. 7 was performed in the late 2000s, and enabled comparative genomics between sake yeast and laboratory yeast, resulting in some notable finding for of sake yeast genetics. Development of next-generation DNA sequencing and bioinformatics also drastically changed the field of the genetics, including for sake yeast. Genomics and the genome-wide study of sake yeast have progressed under these circumstances during the last two decades, and are summarized in this article. Abbreviations: AFLP: amplified fragment length polymorphism; CGH: comparative genomic hybridization; CNV: copy number variation; DMS: dimethyl succinate; DSW: deep sea water; LOH: loss of heterozygosity; NGS: next generation sequencer; QTL: quantitative trait loci; QTN: quantitative trait nucleotide; SAM: S-adenosyl methionine; SNV: single nucleotide variation.

Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background.

Here we report a new plasmid shuffle vector for forcing budding yeast (Saccharomyces cerevisiae) to incorporate a new genetic pathway in place of a native pathway – even an essential one – while maintaining low false positive rates (less than 1 in 108 per cell). This plasmid, dubbed “Superloser,” was designed with reduced sequence similarity to commonly used yeast plasmids (i.e., pRS400 series) to limit recombination, a process that in our experience leads to retention of the yeast gene(s) instead of the desired gene(s). In addition, Superloser utilizes two orthogonal copies of the counter-selectable marker URA3 to reduce spontaneous 5-fluoroorotic acid resistance. Finally, the CEN/ARS sequence is fused to the GAL1-10 promoter, which disrupts plasmid segregation in the presence of the sugar galactose, causing Superloser to rapidly be removed from a population of cells. We show one proof-of-concept shuffling experiment: swapping yeast’s core histones out for their human counterparts. Superloser is especially useful for forcing yeast to use highly unfavorable genes, such as human histones, as it enables plating a large number of cells (1.4x109) on a single 10 cm petri dish while maintaining a very low background. Therefore, Superloser is a useful tool for yeast geneticists to effectively shuffle low viability genes and/or pathways in yeast that may arise in as few as 1 in 108 cells.

Humanized yeast genetic interaction mapping predicts synthetic lethal interactions of FBXW7 in breast cancer

BackgroundSynthetic lethal interactions (SLIs) that occur between gene pairs are exploited for cancer therapeutics. Studies in the model eukaryote yeast have identified ~ 550,000 negative genetic interactions that have been extensively studied, leading to characterization of novel pathways and gene functions. This resource can be used to predict SLIs that can be relevant to cancer therapeutics.MethodsWe used patient data to identify genes that are down-regulated in breast cancer. InParanoid orthology mapping was performed to identify yeast orthologs of the down-regulated genes and predict their corresponding SLIs in humans. The predicted network graphs were drawn with Cytoscape. CancerRXgene database was used to predict drug response.ResultsHarnessing the vast available knowledge of yeast genetics, we generated a Humanized Yeast Genetic Interaction Network (HYGIN) for 1009 human genes with 10,419 interactions. Through the addition of patient-data from The Cancer Genome Atlas (TCGA), we generated a breast cancer specific subnetwork. Specifically, by comparing 1009 genes in HYGIN to genes that were down-regulated in breast cancer, we identified 15 breast cancer genes with 130 potential SLIs. Interestingly, 32 of the 130 predicted SLIs occurred with FBXW7, a well-known tumor suppressor that functions as a substrate-recognition protein within a SKP/CUL1/F-Box ubiquitin ligase complex for proteasome degradation. Efforts to validate these SLIs using chemical genetic data predicted that patients with loss of FBXW7 may respond to treatment with drugs like Selumitinib or Cabozantinib.ConclusionsThis study provides a patient-data driven interpretation of yeast SLI data. HYGIN represents a novel strategy to uncover therapeutically relevant cancer drug targets and the yeast SLI data offers a major opportunity to mine these interactions.

A role for the Saccharomyces cerevisiae ABCF protein New1 in translation termination/recycling.

Translation is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related protein, New1, is encoded by a non-essential gene with cold sensitivity and ribosome assembly defect knock-out phenotypes. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3′-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination or ribosome recycling.

CRISPR screening using an expanded toolkit of autophagy reporters identifies TMEM41B as a novel autophagy factor.

The power of forward genetics in yeast is the foundation on which the field of autophagy research firmly stands. Complementary work on autophagy in higher eukaryotes has revealed both the deep conservation of this process, as well as novel mechanisms by which autophagy is regulated in the context of development, immunity, and neuronal homeostasis. The recent emergence of new clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based technologies has begun facilitating efforts to define novel autophagy factors and pathways by forward genetic screening in mammalian cells. Here, we set out to develop an expanded toolkit of autophagy reporters amenable to CRISPR/Cas9 screening. Genome-wide screening of our reporters in mammalian cells recovered virtually all known autophagy-related (ATG) factors as well as previously uncharacterized factors, including vacuolar protein sorting 37 homolog A (VPS37A), transmembrane protein 251 (TMEM251), amyotrophic lateral sclerosis 2 (ALS2), and TMEM41B. To validate this data set, we used quantitative microscopy and biochemical analyses to show that 1 novel hit, TMEM41B, is required for phagophore maturation. TMEM41B is an integral endoplasmic reticulum (ER) membrane protein distantly related to the established autophagy factor vacuole membrane protein 1 (VMP1), and our data show that these two factors play related, albeit not fully overlapping, roles in autophagosome biogenesis. In sum, our work uncovers new ATG factors, reveals a malleable network of autophagy receptor genetic interactions, and provides a valuable resource (http://crispr.deniclab.com) for further mining of novel autophagy mechanisms.

Yeast-Based Screens to Target Alpha-Synuclein Toxicity.

The budding yeast Saccharomyces cerevisiae (S. cerevisiae) has been a remarkable experimental model for the discovery of fundamental biological processes. The high degree of conservation of cellular and molecular processes between the budding yeast and higher eukaryotes has made it a valuable system for the investigation of the molecular mechanisms behind various types of devastating human pathologies. Genetic screens in yeast provided important insight into the toxic mechanisms associated with the accumulation of misfolded proteins. Thus, using yeast genetics and high-throughput screens, novel molecular targets with therapeutic potential have been identified. Here, we describe a yeast screen protocol for the identification of genetic modifiers of alpha-synuclein (aSyn) toxicity, thereby accelerating the identification of novel potential targets for intervention in Parkinson's disease (PD) and other synucleinopathies.

Notch signaling governs the expression of glypican Dally to define the stem cell niche.

ABSTRACTExtracellular glypicans play pivotal roles in organogenesis, stem cell maintenance and cancer development. However, the growth phenotypes associated with different levels of glypican are not consistent in development or tumorigenesis. This requires clarification on how the spatial patterns of glypican relate to the distribution of signaling molecules in different cellular contexts, and how glypican expression is regulated. We have previously reported that Dally, one of the glypican members in Drosophila, is required in the niche for the maintenance of germline stem cells (GSCs) via short-range BMP signaling in ovary. However, the regulatory mechanism of glypican pattern in the ovarian stem cell niche remains elusive. Our current data demonstrate that the Notch pathway is genetically upstream of Dally and its function to maintain GSCs relies on Dally expression. Combining yeast and fruit fly genetics, we illustrate that Dally is under the transcriptional control of Notch signaling via the transcription factor Su(H). Further, we assayed human glypicans and disease-associated variants in Drosophila ovary, which can serve as an effective system to evaluate the structure–function relationship of human homologs.

Ribosome biogenesis factor Ltv1 chaperones the assembly of the small subunit head.

The correct assembly of ribosomes from ribosomal RNAs (rRNAs) and ribosomal proteins (RPs) is critical, as indicated by the diseases caused by RP haploinsufficiency and loss of RP stoichiometry in cancer cells. Nevertheless, how assembly of each RP is ensured remains poorly understood. We use yeast genetics, biochemistry, and structure probing to show that the assembly factor Ltv1 facilitates the incorporation of Rps3, Rps10, and Asc1/RACK1 into the small ribosomal subunit head. Ribosomes from Ltv1-deficient yeast have substoichiometric amounts of Rps10 and Asc1 and show defects in translational fidelity and ribosome-mediated RNA quality control. These defects provide a growth advantage under some conditions but sensitize the cells to oxidative stress. Intriguingly, relative to glioma cell lines, breast cancer cells have reduced levels of LTV1 and produce ribosomes lacking RPS3, RPS10, and RACK1. These data describe a mechanism to ensure RP assembly and demonstrate how cancer cells circumvent this mechanism to generate diverse ribosome populations that can promote survival under stress.

Genetics is the logic of life (at least of mine).

I retrace my path from math to medicine to biochemistry to yeast genetics, my focus on infectious diseases of yeast and finally prions. My discovery of yeast prions relied on my particular focus on the logical relations of non-chromosomal genetic elements and the chromosomal genes involved in their propagation and expression. Pursuing an understanding of yeast prions involved structural biology based on genetics, solid-state NMR, population genetics and more genetics.

Identification of Changing Ribosome Protein Compositions using Mass Spectrometry.

The regulatory role of the ribosome in gene expression has come into sharper focus. It has been proposed that ribosomes are dynamic complexes capable of changing their protein composition in response to environmental stimuli. MS is applied to identify quantitative changes in the protein composition of S. cerevisiae 80S ribosomes in response to different environmental stimuli. Using quantitative MS, it is found that the paralog yeast ribosomal proteins RPL8A (eL8A) and RPL8B (eL8B) change their relative proportions in the 80S ribosome when yeast is switched from growth in glucose to glycerol. By using yeast genetics and polysome profiling, it is shown that yeast ribosomes containing either RPL8A or RPL8B are not functionally interchangeable. The quantitative proteomic data support the hypothesis that ribosomes are dynamic complexes that alter their composition and functional activity in response to changes in growth or environmental conditions.

Pursuing the Secrets of Histone Proteins: An Amazing Journey with a Remarkable Supporting Cast

Pursuing the Secrets of Histone Proteins: An Amazing Journey with a Remarkable Supporting Cast

Starmerella bombicola, an industrially relevant, yet fundamentally underexplored yeast.

In this review, we focus on one of the most important microbial producers of biosurfactants, Starmerella bombicola. Emphasis is laid on the discovery, taxonomy, habitat, cellular characteristics, biochemistry and genetics of this non-pathogenic yeast. Biosurfactants are natural surface-active compounds produced by several types of microorganisms and have been considered an interesting alternative to synthetic surfactants. The sophorolipids produced by S. bombicola are promising biosurfactants, with application potential in food, pharmaceutical, cosmetic and cleaning industries. The fundamental knowledge described in this review is of crucial interest to optimize production of these promising compounds. Furthermore, it can be translated to produce novel non-native bioactive molecules with S. bombicola, and to deepen fundamental knowledge on other non-conventional yeast species and in the end to broaden their application potential as well.

Fluorinated Phenylalanine Precursor Resistance in Yeast

Development of a counter-selection method for phenylalanine auxotrophy could be a useful tool in the repertoire of yeast genetics. Fluorinated and sulfurated precursors of phenylalanine were tested for toxicity in Saccharomyces cerevisiae. One such precursor, 4-fluorophenylpyruvate (FPP), was found to be toxic to several strains from the Saccharomyces and Candida genera. Toxicity was partially dependent on ARO8 and ARO9, and correlated with a strain’s ability to convert FPP into 4-fluorophenylalanine (FPA). Thus, strains with deletions in ARO8 and ARO9, having a mild phenylalanine auxotrophy, could be separated from a culture of wild-type strains using FPP. Tetrad analysis suggests FPP resistance in one strain is due to two genes. Strains resistant to FPA have previously been shown to exhibit increased phenylethanol production. However, FPP resistant isolates did not follow this trend. These results suggest that FPP could effectively be used for counter-selection but not for enhanced phenylethanol production.

Rapid Nuclear Exclusion of Hcm1 in Aging Saccharomyces cerevisiae Leads to Vacuolar Alkalization and Replicative Senescence.

The yeast, Saccharomyces cerevisiae, like other higher eukaryotes, undergo a finite number of cell divisions before exiting the cell cycle due to the effects of aging. Here, we show that yeast aging begins with the nuclear exclusion of Hcm1 in young cells, resulting in loss of acidic vacuoles. Autophagy is required for healthy aging in yeast, with proteins targeted for turnover by autophagy directed to the vacuole. Consistent with this, vacuolar acidity is necessary for vacuolar function and yeast longevity. Using yeast genetics and immunofluorescence microscopy, we confirm that vacuolar acidity plays a critical role in cell health and lifespan, and is potentially maintained by a series of Forkhead Box (Fox) transcription factors. An interconnected transcriptional network involving the Fox proteins (Fkh1, Fkh2 and Hcm1) are required for transcription of v-ATPase subunits and vacuolar acidity. As cells age, Hcm1 is rapidly excluded from the nucleus in young cells, blocking the expression of Hcm1 targets (Fkh1 and Fkh2), leading to loss of v-ATPase gene expression, reduced vacuolar acidification, increased α-syn-GFP vacuolar accumulation, and finally, diminished replicative lifespan (RLS). Loss of vacuolar acidity occurs about the same time as Hcm1 nuclear exclusion and is conserved; we have recently demonstrated that lysosomal alkalization similarly contributes to aging in C. elegans following a transition from progeny producing to post-reproductive life. Our data points to a molecular mechanism regulating vacuolar acidity that signals the end of RLS when acidification is lost.

Protein methylation and translation: Role of lysine modification on the function of yeast elongation factor 1 alpha

Proteins of the translational apparatus, including ribosomal proteins, release factors, and elongation factors, are known to be modified by a large number of methyltransferases in eukaryotic cells. The majority of these methylation reactions occurs at lysine residues. To date, 12 protein lysine methyltransferase involved in these reactions (Efm1–7; Rkm 1–5) have been identified in the yeast Saccharomyces cerevisiae. Of these 12, 5 appear to be specific to elongation factor 1 alpha (EF1A)(Efm1, Efm4–7). Each methyltransferase modifies a specific lysine residue to give mono‐, di‐, and tri‐methyllysine products. Two of these enzymes (Efm4 and Efm5) are highly conserved in nature while the others appear to be fungal specific.In S. cerevisiae the functional implications of lysine methylation are mostly unknown. EF1A's primary function, with its GTP cofactor, is to deliver amino‐acyl tRNA to the A site of the ribosome during translation. Once the correct tRNA is positioned, a conformational change results in the hydrolysis of GTP and its release from the ribosome for another cycle. Studies have also shown the involvement of EF1A in the assembly of ribosomes.Using yeast genetics we have assessed the biological relevance of EF1A lysine methylation on its translational roles using two approaches. The first approach uses site directed mutagenesis to mutate four of the most conserved methylated lysine sites to arginine residues. The second approach involves combinatorial knockouts of the genes that code for each of the five methyltransferases. In each approach, we have then used functional translation assays in yeast to evaluate the physiological impact of the loss of EF1A methylations. We have found that inactivation of multiple methyltransferases can cause a significant reduction of translational fidelity. Additionally, we have shown that the ribosomal assembly function of EF1A does not appear to be affected by the loss of methyltransferase activity. These studies should illuminate why one protein appears to need five different methyltransferases for its functions.Support or Funding InformationThis work is funded by the National Science Foundation Grant MCB‐1714569 and NSF‐LSAMP Bridge to Doctorate Fellowship.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Some assembly required: Contributions of Tom Stevens' lab to the V-ATPase field.

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V-ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V-ATPase proton pumps and led to the discovery of protein splicing of the V-ATPase catalytic subunit. The Vma- phenotype, characteristic of loss-of-V-ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V-ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V-ATPase assembly factors and helped unravel the complex pathways for V-ATPase assembly. In later years, exploration of the evolutionary history of several V-ATPase subunits provided new information about the enzyme's structure and function. This review highlights V-ATPase work in the Stevens' lab between 1987 and 2017.