Budding Yeast Saccharomyces Cerevisiae Research Articles

Response to sulfur in Schizosaccharomyces pombe

ABSTRACTSulfur is an essential component of various biologically important molecules, including methionine, cysteine and glutathione, and it is also involved in coping with oxidative and heavy metal stress. Studies using model organisms, including budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), have contributed not only to understanding various cellular processes but also to understanding the utilization and response mechanisms of each nutrient, including sulfur. Although fission yeast can use sulfate as a sulfur source, its sulfur metabolism pathway is slightly different from that of budding yeast because it does not have a trans-sulfuration pathway. In recent years, it has been found that sulfur starvation causes various cellular responses in S. pombe, including sporulation, cell cycle arrest at G2, chronological lifespan extension, autophagy induction and reduced translation. This MiniReview identifies two sulfate transporters in S. pombe, Sul1 (encoded by SPBC3H7.02) and Sul2 (encoded by SPAC869.05c), and summarizes the metabolic pathways of sulfur assimilation and cellular response to sulfur starvation. Understanding these responses, including metabolism and adaptation, will contribute to a better understanding of the various stress and nutrient starvation responses and chronological lifespan regulation caused by sulfur starvation.

Vps34 and TOR Kinases Coordinate HAC1 mRNA Translation in the Presence or Absence of Ire1-Dependent Splicing

In the budding yeast Saccharomyces cerevisiae , an mRNA, called HAC1 , exists in a translationally repressed form in the cytoplasm. Under conditions of cellular stress, such as when unfolded proteins accumulate inside the endoplasmic reticulum (ER), an RNase Ire1 removes an intervening sequence (intron) from the HAC1 mRNA by nonconventional cytosolic splicing.

Transcription at a Distance in the Budding Yeast Saccharomyces cerevisiae

Proper transcriptional regulation depends on the collaboration of multiple layers of control simultaneously. Cells tightly balance cellular resources and integrate various signaling inputs to maintain homeostasis during growth, development and stressors, among other signals. Many eukaryotes, including the budding yeast Saccharomyces cerevisiae, exhibit a non-random distribution of functionally related genes throughout their genomes. This arrangement coordinates the transcription of genes that are found in clusters, and can occur over long distances. In this work, we review the current literature pertaining to gene regulation at a distance in budding yeast.

Essential Oils of Rosmarinus officinalis L., Cymbopogon citratus (DC.) Stapf., and the phyto-compounds, delta-carene and alpha-pinene mediate cell cycle arrest at G2/M transition in budding yeast Saccharomyces cerevisiae

Ethnopharmacological relevance: Rosmarinus officinalis L. (Lamiaceae) is widely used in folk and traditional medicine for inflammation-related diseases due to its anti-oxidant and wound healing properties. Cymbopogon citratus (DC.) Stapf. is a widely distributed perennial herb belonging to the Poaceae family and has been extensively consumed for its traditional and ethnomedicinal, cosmetic, and nutritional effects for centuries.Aim of the study: Budding yeast is a valuable system to study cell-cycle regulation, which is the underlying defect in cancer cells. The aim of the present study was to characterize the antiproliferative effect of essential oil of Rosmarinus officinalis L. (rosemary) and Cymbopogon citratus (DC.) Stapf. (lemon grass) and their effect on cell cycle of Saccharomyces cerevisiae.Materials and methods: Rosmarinus officinalis L. and Cymbopogon citratus (DC.) Stapf. essential oil was extracted from leaves and characterized by GC–MS, TLC and FTIR. The antiproliferative activity of essential oils and their major compounds, delta-carene and α-pinene was studied by liquid growth assay. Microscopic examination of yeast cells was done to determine the budding index. Flowcytometry analysis of DNA content was analyzed to confirm the G2/M arrest of cell cycle of budding yeast.Results: Both the essential oils mediated a dose-dependent inhibition of yeast growth in liquid assays. Analysis of budding index and DNA content by flowcytometry revealed that R. officinalis L. and C. citratus (DC.) Stapf. essential oils mediate the arrest of yeast cells at G2/M phase of cell cycle. GC/MS analysis of R. officinalis oil revealed the presence of two major compounds, delta-carene (7.75%), and α-pinene (15.1%). Both delta-carene and α-pinene showed antiproliferative activity (92% and 95% of growth inhibition, respectively) and caused the arrest of yeast cell cycle at G2/M phase.Conclusions: The essential oils of R. officinalis and C. citratus, delta-carene and α-pinene can be validated in animal model for cell cycle inhibition and cancer therapeutics.

Involvement of a Multidrug Efflux Pump and Alterations in Cell Surface Structure in the Synergistic Antifungal Activity of Nagilactone E and Anethole against Budding Yeast Saccharomyces cerevisiae.

Nagilactone E, an antifungal agent derived from the root bark of Podocarpus nagi, inhibits 1,3-β glucan synthesis; however, its inhibitory activity is weak. Anethole, the principal component of anise oil, enhances the antifungal activity of nagilactone E. We aimed to determine the combinatorial effect and underlying mechanisms of action of nagilactone E and anethole against the budding yeast Saccharomyces cerevisiae. Analyses using gene-deficient strains showed that the multidrug efflux pump PDR5 is associated with nagilactone E resistance; its transcription was gradually restricted in cells treated with the drug combination for a prolonged duration but not in nagilactone-E-treated cells. Green-fluorescent-protein-tagged Pdr5p was intensively expressed and localized on the plasma membrane of nagilactone-E-treated cells but not in drug-combination-treated cells. Quick-freeze deep-etch electron microscopy revealed the smoothening of intertwined fiber structures on the cell surface of drug-combination-treated cells and spheroplasts, indicating a decline in cell wall components and loss of cell wall strength. Anethole enhanced the antifungal activity of nagilactone E by enabling its retention within cells, thereby accelerating cell wall damage. The combination of nagilactone E and anethole can be employed in clinical settings as an antifungal, as well as a food preservative to restrict food spoilage.

Hypoosmolality impedes cytoophidium integrity during nitrogen starvation.

CTP synthase (CTPS) cytoophidia have been found in many species over domains of life in the past 10 years, implying the evolutionary conservation of these structures. However, there are differences in cytoophidia between species. The difference in CTPS cytoophidium properties between budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe) inspires this research. We study the effects of culture environment on cytoophidia in S. cerevisiae by switching to the optimal medium for S. pombe. S. cerevisiae CTPS cytoophidium fragmentation and pseudohyphae formation are observed after treatment with S. pombe medium YES instead of S. cerevisiae medium YPD. By modifying the level of each ingredient of the media, we find that hypoosmolality impedes cytoophidium integrity during nitrogen starvation. Our study demonstrates the relationship between cytoophidium integrity and environmental stress, supporting the role of cytoophidia in stress resistance.

Yeast Lipid Extraction and Analysis by HPTLC.

The diversity of lipid structures, properties, and combinations in biological tissues makes their extraction and analysis an experimental challenge. Accordingly, even for one of the simplest single-cellular fungi, the budding yeast (Saccharomyces cerevisiae), numerous extraction and analysis protocols have been developed to separate and quantitate the different molecular lipid species. Among them, most are quite sophisticated and tricky to follow. Herein, we describe a yeast total lipids extraction procedure with a relatively good yield, which is appropriate for subsequent thin-layer chromatography (TLC) or liquid chromatography-mass (LC-MS) analysis. We then discuss the most widely used solvent systems to separate yeast phospholipids and neutral lipids by TLC. Finally, we describe an easy and rapid method for silica gel staining by a Coomassie Brilliant Blue-methanol mixture. The stained lipid species can then be quantitated using imaging software such as ImageJ. Overall, the methods described in this protocol are time-saving and novice-friendly.

A Yeast Mutant Screen Identifies TORC and Lys63 Polyubiquitination Pathway Genes among Determinants of Sensitivity to the Cancer Stem Cell-Specific Drug Salinomycin

The antibiotic salinomycin (SM) acts as a selective potassium ionophore. In budding yeast (Saccharomyces cerevisiae), we describe that the agent inhibits cell growth, elevates reactive oxygen species (ROS) levels and prominently causes mitochondrial damage, as revealed by the emergence of perpetually respiration-defective cells. The collection of systematic gene deletions in haploid yeast was screened to characterize genes whose deletion confers SM sensitivity or resistance if glycerol is provided as the only carbon source, thus requiring active respiration for growth. Mutants conferring the highest SM resistance were those of the Mms2-Ubi13 E2-ubiquitin conjugating enzyme (Lys63 polyubiquitination) and the TORC pathway, such as Sch9. Sch9 phosphorylation is reduced after SM treatment and, whereas initial SM-enhanced ROS levels are not diminished in the mutant, we suggest that a protective response is mounted in the absence of Sch9 that promotes mitochondrial stability under conditions of potassium ion loss. As indicated by other isolated mutants with altered SM sensitivity, levels and modifications of ribosomal proteins may also play a role in these responses. SM has attracted considerable attention due to its cancer stem-cell specific mode of action. Even if not all of its cancer stem cell targets may have an equivalent in yeast, these studies may suggest strategies for mitigating its side effects during treatment of cancer patients.

Construction of Tight Conditional Mutants Using the Improved Auxin-Inducible Degron (iAID) Method in the Budding Yeast Saccharomyces cerevisiae.

Conditional mutants, such as temperature-sensitive (ts) mutants, are effective tools for the analysis of essential genes. However, such mutants are frequently leaky. To overcome this problem, it is helpful to isolate a "tight" conditional mutant of a gene of interest, e.g., by using ubiquitin-mediated protein degradation to eliminate the gene product. One such strategy is the auxin-inducible degron (AID) system, which is easy to use because the simple addition of auxin can induce the degradation of a target protein. Sometimes, however, elimination of the target protein is not sufficient, and an AID mutant exhibits a "leaky" phenotype. To address this issue, the improved AID (iAID) system was developed. In this approach, transcriptional repression by the "Tet-OFF" promoter is combined with proteolytic elimination of the target protein by the AID system, yielding a much tighter mutant. Because simple addition of tetracycline is sufficient to repress the Tet-OFF promoter, the combination of Tet-OFF and AID maintains the ease of use of the original AID system. In this manuscript, we describe how to construct and use iAID mutants in the budding yeast Saccharomyces cerevisiae.

Targeted Integration of Complex Genetic Elements at Multi-Copy Loci by Golden Gate Assembly.

Budding yeast Saccharomyces cerevisiae has become a model eukaryotic microorganism for targeted genomic manipulation due to its efficient homologous recombination. A few genomic loci, including rDNA, Delta, and Ty1, can be utilized to introduce variable copies of genetic elements into the yeast genome. Here we describe a method that combines in vitro Golden Gate Assembly to assemble one or a complex genetic element in an orderly manner and then integrate it into predetermined multi-copy loci through homologous recombination. Different transformants may contain different copy numbers, which allows the selection of desired levels of target gene expression.

Transient intracellular acidification regulates the core transcriptional heat shock response.

Heat shock induces a conserved transcriptional program regulated by heat shock factor 1 (Hsf1) in eukaryotic cells. Activation of this heat shock response is triggered by heat-induced misfolding of newly synthesized polypeptides, and so has been thought to depend on ongoing protein synthesis. Here, using the budding yeast Saccharomyces cerevisiae, we report the discovery that Hsf1 can be robustly activated when protein synthesis is inhibited, so long as cells undergo cytosolic acidification. Heat shock has long been known to cause transient intracellular acidification which, for reasons which have remained unclear, is associated with increased stress resistance in eukaryotes. We demonstrate that acidification is required for heat shock response induction in translationally inhibited cells, and specifically affects Hsf1 activation. Physiological heat-triggered acidification also increases population fitness and promotes cell cycle reentry following heat shock. Our results uncover a previously unknown adaptive dimension of the well-studied eukaryotic heat shock response.

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

Effect of Stressors on the Carrying Capacity of Spatially Distributed Metapopulations.

Stressors such as antibiotics, herbicides, and pollutants are becoming increasingly common in the environment. The effects of stressors on populations are typically studied in homogeneous, nonspatial settings. However, most populations in nature are spatially distributed over environmentally heterogeneous landscapes with spatially restricted dispersal. Little is known about the effects of stressors in these more realistic settings. Here, we combine laboratory experiments with novel mathematical theory to rigorously investigate how a stressor's physiological effect and spatial distribution interact with dispersal to influence population dynamics. We prove mathematically that if a stressor increases the death rate and/or simultaneously decreases the population growth rate and yield, a homogeneous distribution of the stressor leads to a lower total population size than if the same amount of the stressor was heterogeneously distributed. We experimentally test this prediction on spatially distributed populations of budding yeast (Saccharomyces cerevisiae). We find that the antibiotic cycloheximide increases the yeast death rate but reduces the growth rate and yield. Consistent with our mathematical predictions, we observe that a homogeneous spatial distribution of cycloheximide minimizes the total equilibrium size of experimental metapopulations, with the magnitude of the effect depending predictably on the dispersal rate and the geographic pattern of antibiotic heterogeneity. Our study has implications for assessing the population risk posed by pollutants, antibiotics, and global change and for the rational design of strategies for employing toxins to control pathogens and pests.

Building blocks are synthesized on demand during the yeast cell cycle

For cells to replicate, a sufficient supply of biosynthetic precursors is needed, necessitating the concerted action of metabolism and protein synthesis during progressive phases of cell division. A global understanding of which biosynthetic processes are involved and how they are temporally regulated during replication is, however, currently lacking. Here, quantitative multiomics analysis is used to generate a holistic view of the eukaryal cell cycle, using the budding yeast Saccharomyces cerevisiae Protein synthesis and central carbon pathways such as glycolysis and amino acid metabolism are shown to synchronize their respective abundance profiles with division, with pathway-specific changes in metabolite abundance also being reflected by a relative increase in mitochondrial volume, as shown by quantitative fluorescence microscopy. These results show biosynthetic precursor production to be temporally regulated to meet phase-specific demands of eukaryal cell division.

Intracellular vesicle clusters are organelles that synthesize extracellular vesicle–associated cargo proteins in yeast

Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles-called intracellular vesicle clusters (IVCs)-serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.

Interaction between the yeast RAVE complex and Vph1-containing Vo sectors is a central glucose-sensitive interaction required for V-ATPase reassembly

The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1 Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE's role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.

The pseudosubstrate inhibitor Acm1 inhibits the anaphase-promoting complex/cyclosome by combining high-affinity activator binding with disruption of Doc1/Apc10 function

The anaphase-promoting complex/cyclosome (APC/C) is a large, multisubunit ubiquitin ligase involved in regulation of cell division. APC/C substrate specificity arises from binding of short degron motifs in its substrates to transient activator subunits, Cdc20 and Cdh1. The destruction box (D-box) is the most common APC/C degron and plays a crucial role in substrate degradation by linking the activator to the Doc1/Apc10 subunit of core APC/C to stabilize the active holoenzyme and promote processive ubiquitylation. Degrons are also employed as pseudosubstrate motifs by APC/C inhibitors, and pseudosubstrates must bind their cognate activators tightly to outcompete substrate binding while blocking their own ubiquitylation. Here we examined how APC/C activity is suppressed by the small pseudosubstrate inhibitor Acm1 from budding yeast (Saccharomyces cerevisiae). Mutation of a conserved D-box converted Acm1 into an efficient ABBA (cyclin A, BubR1, Bub1, Acm1) motif-dependent APC/CCdh1 substrate in vivo, suggesting that this D-box somehow inhibits APC/C. We then identified a short conserved sequence at the C terminus of the Acm1 D-box that was necessary and sufficient for APC/C inhibition. In several APC/C substrates, the corresponding D-box region proved to be important for their degradation despite poor sequence conservation, redefining the D-box as a 12-amino acid motif. Biochemical analysis suggested that the Acm1 D-box extension inhibits reaction processivity by perturbing the normal interaction with Doc1/Apc10. Our results reveal a simple, elegant mode of pseudosubstrate inhibition that combines high-affinity activator binding with specific disruption of Doc1/Apc10 function in processive ubiquitylation.

Impact of curcumin on replicative and chronological aging in the Saccharomyces cerevisiae yeast

Curcumin is a biologically active compound of vegetable origin which has a hormetic effect. Pro-health and anti-aging properties of curcumin have been known for years. The main benefit of curcumin is thought to be its anti-oxidative action. Despite vast amount of data confirming age-delaying activity of curcumin in various groups of organisms, so far little has been discovered about curcumin’s impact on cell aging in the experimental model of the Saccharomyces cerevisiae budding yeast. We have been able to demonstrate that curcumin significantly increases oxidative stress and accelerates replicative and chronological aging of yeast cells devoid of anti-oxidative protection (with SOD1 and SOD2 gene deletion) and deprived of DNA repair mechanisms (RAD52). Interestingly, curcumin delays aging, probably through hormesis, of the wild-type strain BY4741.

A Validated Set of Fluorescent-Protein-Based Markers for Major Organelles in Yeast (Saccharomyces cerevisiae)

ABSTRACTEukaryotic cells share a basic scheme of internal organization featuring membrane-based organelles. The use of fluorescent proteins (FPs) greatly facilitated live-cell imaging of organelle dynamics and protein trafficking. One major limitation of this approach is that the fusion of an FP to a target protein can and often does compromise the function of the target protein and alter its subcellular localization. The optimization process to obtain a desirable fusion construct can be time-consuming or even unsuccessful. In this work, we set out to provide a validated set of FP-based markers for major organelles in the budding yeast (Saccharomyces cerevisiae). Out of over 160 plasmids constructed, we present a final set of 42 plasmids, the recommendations for which are backed up by meticulous evaluations. The tool set includes three colors (green, red, and blue) and covers the endoplasmic reticulum (ER), nucleus, Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, and lipid droplets. The fidelity of the markers was established by systematic cross-comparison and quantification. Functional assays were performed to examine the impact of marker expression on the secretory pathway, endocytic pathway, and metabolic activities of mitochondria and peroxisomes. Concomitantly, our work constitutes a reassessment of organelle identities in this model organism. Our data support the recognition that “late Golgi” and “early endosomes,” two seemingly distinct terms, denote the same compartment in yeast. Conversely, all other organelles can be visually separated from each other at the resolution of conventional light microscopy, and quantification results justify their classification as distinct entities.