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Sample records for budding yeast reveals

  1. Genetically Engineered Transvestites Reveal Novel Mating Genes in Budding Yeast

    PubMed Central

    Huberman, Lori B.; Murray, Andrew W.

    2013-01-01

    Haploid budding yeast has two mating types, defined by the alleles of the MAT locus, MATa and MATα. Two haploid cells of opposite mating types mate by signaling to each other using reciprocal pheromones and receptors, polarizing and growing toward each other, and eventually fusing to form a single diploid cell. The pheromones and receptors are necessary and sufficient to define a mating type, but other mating-type-specific proteins make mating more efficient. We examined the role of these proteins by genetically engineering “transvestite” cells that swap the pheromone, pheromone receptor, and pheromone processing factors of one mating type for another. These cells mate with each other, but their mating is inefficient. By characterizing their mating defects and examining their transcriptomes, we found Afb1 (a-factor barrier), a novel MATα-specific protein that interferes with a-factor, the pheromone secreted by MATa cells. Strong pheromone secretion is essential for efficient mating, and the weak mating of transvestites can be improved by boosting their pheromone production. Synthetic biology can characterize the factors that control efficiency in biological processes. In yeast, selection for increased mating efficiency is likely to have continually boosted pheromone levels and the ability to discriminate between partners who make more and less pheromone. This discrimination comes at a cost: weak mating in situations where all potential partners make less pheromone. PMID:24121774

  2. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

    PubMed

    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  3. Screening the Budding Yeast Genome Reveals Unique Factors Affecting K2 Toxin Susceptibility

    PubMed Central

    Servienė, Elena; Lukša, Juliana; Orentaitė, Irma

    2012-01-01

    Background Understanding how biotoxins kill cells is of prime importance in biomedicine and the food industry. The budding yeast (S. cerevisiae) killers serve as a convenient model to study the activity of biotoxins consistently supplying with significant insights into the basic mechanisms of virus-host cell interactions and toxin entry into eukaryotic target cells. K1 and K2 toxins are active at the cell wall, leading to the disruption of the plasma membrane and subsequent cell death by ion leakage. K28 toxin is active in the cell nucleus, blocking DNA synthesis and cell cycle progression, thereby triggering apoptosis. Genome-wide screens in the budding yeast S. cerevisiae identified several hundred effectors of K1 and K28 toxins. Surprisingly, no such screen had been performed for K2 toxin, the most frequent killer toxin among industrial budding yeasts. Principal Findings We conducted several concurrent genome-wide screens in S. cerevisiae and identified 332 novel K2 toxin effectors. The effectors involved in K2 resistance and hypersensitivity largely map in distinct cellular pathways, including cell wall and plasma membrane structure/biogenesis and mitochondrial function for K2 resistance, and cell wall stress signaling and ion/pH homeostasis for K2 hypersensitivity. 70% of K2 effectors are different from those involved in K1 or K28 susceptibility. Significance Our work demonstrates that despite the fact that K1 and K2 toxins share some aspects of their killing strategies, they largely rely on different sets of effectors. Since the vast majority of the host factors identified here is exclusively active towards K2, we conclude that cells have acquired a specific K2 toxin effectors set. Our work thus indicates that K1 and K2 have elaborated different biological pathways and provides a first step towards the detailed characterization of K2 mode of action. PMID:23227207

  4. Improved statistical analysis of budding yeast TAG microarrays revealed by defined spike-in pools.

    PubMed

    Peyser, Brian D; Irizarry, Rafael A; Tiffany, Carol W; Chen, Ou; Yuan, Daniel S; Boeke, Jef D; Spencer, Forrest A

    2005-09-15

    Saccharomyces cerevisiae knockout collection TAG microarrays are an emergent platform for rapid, genome-wide functional characterization of yeast genes. TAG arrays report abundance of unique oligonucleotide 'TAG' sequences incorporated into each deletion mutation of the yeast knockout collection, allowing measurement of relative strain representation across experimental conditions for all knockout mutants simultaneously. One application of TAG arrays is to perform genome-wide synthetic lethality screens, known as synthetic lethality analyzed by microarray (SLAM). We designed a fully defined spike-in pool to resemble typical SLAM experiments and performed TAG microarray hybridizations. We describe a method for analyzing two-color array data to efficiently measure the differential knockout strain representation across two experimental conditions, and use the spike-in pool to show that the sensitivity and specificity of this method exceed typical current approaches.

  5. A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

    PubMed Central

    Hoggard, Timothy; Shor, Erika; Müller, Carolin A.; Nieduszynski, Conrad A.; Fox, Catherine A.

    2013-01-01

    Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time. PMID:24068963

  6. Mitochondrial network size scaling in budding yeast.

    PubMed

    Rafelski, Susanne M; Viana, Matheus P; Zhang, Yi; Chan, Yee-Hung M; Thorn, Kurt S; Yam, Phoebe; Fung, Jennifer C; Li, Hao; Costa, Luciano da F; Marshall, Wallace F

    2012-11-01

    Mitochondria must grow with the growing cell to ensure proper cellular physiology and inheritance upon division. We measured the physical size of mitochondrial networks in budding yeast and found that mitochondrial network size increased with increasing cell size and that this scaling relation occurred primarily in the bud. The mitochondria-to-cell size ratio continually decreased in aging mothers over successive generations. However, regardless of the mother's age or mitochondrial content, all buds attained the same average ratio. Thus, yeast populations achieve a stable scaling relation between mitochondrial content and cell size despite asymmetry in inheritance.

  7. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

    PubMed Central

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  8. Energy Landscape Reveals That the Budding Yeast Cell Cycle Is a Robust and Adaptive Multi-stage Process

    PubMed Central

    Lv, Cheng; Li, Xiaoguang; Li, Fangting; Li, Tiejun

    2015-01-01

    Quantitatively understanding the robustness, adaptivity and efficiency of cell cycle dynamics under the influence of noise is a fundamental but difficult question to answer for most eukaryotic organisms. Using a simplified budding yeast cell cycle model perturbed by intrinsic noise, we systematically explore these issues from an energy landscape point of view by constructing an energy landscape for the considered system based on large deviation theory. Analysis shows that the cell cycle trajectory is sharply confined by the ambient energy barrier, and the landscape along this trajectory exhibits a generally flat shape. We explain the evolution of the system on this flat path by incorporating its non-gradient nature. Furthermore, we illustrate how this global landscape changes in response to external signals, observing a nice transformation of the landscapes as the excitable system approaches a limit cycle system when nutrients are sufficient, as well as the formation of additional energy wells when the DNA replication checkpoint is activated. By taking into account the finite volume effect, we find additional pits along the flat cycle path in the landscape associated with the checkpoint mechanism of the cell cycle. The difference between the landscapes induced by intrinsic and extrinsic noise is also discussed. In our opinion, this meticulous structure of the energy landscape for our simplified model is of general interest to other cell cycle dynamics, and the proposed methods can be applied to study similar biological systems. PMID:25794282

  9. Single-cell phenomics in budding yeast

    PubMed Central

    Ohya, Yoshikazu; Kimori, Yoshitaka; Okada, Hiroki; Ohnuki, Shinsuke

    2015-01-01

    The demand for phenomics, a high-dimensional and high-throughput phenotyping method, has been increasing in many fields of biology. The budding yeast Saccharomyces cerevisiae, a unicellular model organism, provides an invaluable system for dissecting complex cellular processes using high-resolution phenotyping. Moreover, the addition of spatial and temporal attributes to subcellular structures based on microscopic images has rendered this cell phenotyping system more reliable and amenable to analysis. A well-designed experiment followed by appropriate multivariate analysis can yield a wealth of biological knowledge. Here we review recent advances in cell imaging and illustrate their broad applicability to eukaryotic cells by showing how these techniques have advanced our understanding of budding yeast. PMID:26543200

  10. Synchronization of the Budding Yeast Saccharomyces cerevisiae.

    PubMed

    Foltman, Magdalena; Molist, Iago; Sanchez-Diaz, Alberto

    2016-01-01

    A number of model organisms have provided the basis for our understanding of the eukaryotic cell cycle. These model organisms are generally much easier to manipulate than mammalian cells and as such provide amenable tools for extensive genetic and biochemical analysis. One of the most common model organisms used to study the cell cycle is the budding yeast Saccharomyces cerevisiae. This model provides the ability to synchronise cells efficiently at different stages of the cell cycle, which in turn opens up the possibility for extensive and detailed study of mechanisms regulating the eukaryotic cell cycle. Here, we describe methods in which budding yeast cells are arrested at a particular phase of the cell cycle and then released from the block, permitting the study of molecular mechanisms that drive the progression through the cell cycle.

  11. Interaction between bud-site selection and polarity-establishment machineries in budding yeast

    PubMed Central

    Wu, Chi-Fang; Savage, Natasha S.; Lew, Daniel J.

    2013-01-01

    Saccharomyces cerevisiae yeast cells polarize in order to form a single bud in each cell cycle. Distinct patterns of bud-site selection are observed in haploid and diploid cells. Genetic approaches have identified the molecular machinery responsible for positioning the bud site: during bud formation, specific locations are marked with immobile landmark proteins. In the next cell cycle, landmarks act through the Ras-family GTPase Rsr1 to promote local activation of the conserved Rho-family GTPase, Cdc42. Additional Cdc42 accumulates by positive feedback, creating a concentrated patch of GTP-Cdc42, which polarizes the cytoskeleton to promote bud emergence. Using time-lapse imaging and mathematical modelling, we examined the process of bud-site establishment. Imaging reveals unexpected effects of the bud-site-selection system on the dynamics of polarity establishment, raising new questions about how that system may operate. We found that polarity factors sometimes accumulate at more than one site among the landmark-specified locations, and we suggest that competition between clusters of polarity factors determines the final location of the Cdc42 cluster. Modelling indicated that temporally constant landmark-localized Rsr1 would weaken or block competition, yielding more than one polarity site. Instead, we suggest that polarity factors recruit Rsr1, effectively sequestering it from other locations and thereby terminating landmark activity. PMID:24062579

  12. Cell Polarization and Cytokinesis in Budding Yeast

    PubMed Central

    Bi, Erfei; Park, Hay-Oak

    2012-01-01

    Asymmetric cell division, which includes cell polarization and cytokinesis, is essential for generating cell diversity during development. The budding yeast Saccharomyces cerevisiae reproduces by asymmetric cell division, and has thus served as an attractive model for unraveling the general principles of eukaryotic cell polarization and cytokinesis. Polarity development requires G-protein signaling, cytoskeletal polarization, and exocytosis, whereas cytokinesis requires concerted actions of a contractile actomyosin ring and targeted membrane deposition. In this chapter, we discuss the mechanics and spatial control of polarity development and cytokinesis, emphasizing the key concepts, mechanisms, and emerging questions in the field. PMID:22701052

  13. Combined Transcriptomics and Chemical-Genetics Reveal Molecular Mode of Action of Valproic acid, an Anticancer Molecule using Budding Yeast Model

    PubMed Central

    Golla, Upendarrao; Joseph, Deepthi; Tomar, Raghuvir Singh

    2016-01-01

    Valproic acid (VA) is a pharmacologically important histone deacetylase inhibitor that recently garnered attention as an anticancer agent. Since the molecular mechanisms behind the multiple effects of VA are unclear, this study was aimed to unravel the comprehensive cellular processes affected by VA and its molecular targets in vivo using budding yeast as a model organism. Interestingly, genome-wide transcriptome analysis of cells treated with VA showed differential regulation of 30% of the genome. Functional enrichment analysis of VA transcriptome evidenced alteration of various cellular processes including cell cycle, cell wall biogenesis, DNA repair, ion homeostasis, metabolism, stress response, transport and ribosomal biogenesis, etc. Moreover, our genetic screening analysis revealed VA molecular targets belonging to oxidative and osmotic stress, DNA repair, cell wall integrity, and iron homeostasis. Further, our results demonstrated the activation of mitogen-activated protein kinases (MAPKs) Hog1 (p38) and Slt2 (p44/42) upon VA treatment. Our results also exhibited that VA acts through alteration of mitochondrial, ER architecture and functions. Especially, VA effects were neutralized in cells lacking lipid particles. Altogether, our results deciphered the novel molecular insights and mechanistic links to strengthen our knowledge on diverse cellular effects of VA along with its probable therapeutic targets and detoxification approaches. PMID:27734932

  14. The Inside-Out Mechanism of Dicers from Budding Yeasts

    SciTech Connect

    Weinberg, David E.; Nakanishi, Kotaro; Patel, Dinshaw J.; Bartel, David P.

    2011-09-20

    The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeasts. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural molecular rulers and imparts substrate preferences with ramifications for biological function.

  15. The Inside-Out Mechanism of Dicers from Budding Yeasts

    SciTech Connect

    D Weinberg; K Nakanishi; D Patel; D Bartel

    2011-12-31

    The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeasts. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural molecular rulers and imparts substrate preferences with ramifications for biological function.

  16. Calling Card Analysis in Budding Yeast.

    PubMed

    Mayhew, David; Mitra, Robi D

    2016-02-01

    Calling card analysis is a high-throughput method for identifying the genomic binding sites of multiple transcription factors in a single experiment in budding yeast. By tagging a DNA-binding protein with a targeting domain that directs the insertion of the Ty5 retrotransposon, the genomic binding sites for that transcription factor are marked. The transposition locations are then identified en masse by Illumina sequencing. The calling card protocol allows for simultaneous analysis of multiple transcription factors. By cloning barcodes into the Ty5 transposon, it is possible to pair a unique barcode with every transcription factor in the experiment. The method presented here uses expression of transcription factors from their native loci; however, it can also be altered to measure binding sites of transcription factors overexpressed from a plasmid. PMID:26832687

  17. Electrochemical Regulation of Budding Yeast Polarity

    PubMed Central

    Piel, Matthieu; Chang, Fred; Minc, Nicolas

    2014-01-01

    Cells are naturally surrounded by organized electrical signals in the form of local ion fluxes, membrane potential, and electric fields (EFs) at their surface. Although the contribution of electrochemical elements to cell polarity and migration is beginning to be appreciated, underlying mechanisms are not known. Here we show that an exogenous EF can orient cell polarization in budding yeast (Saccharomyces cerevisiae) cells, directing the growth of mating projections towards sites of hyperpolarized membrane potential, while directing bud emergence in the opposite direction, towards sites of depolarized potential. Using an optogenetic approach, we demonstrate that a local change in membrane potential triggered by light is sufficient to direct cell polarization. Screens for mutants with altered EF responses identify genes involved in transducing electrochemical signals to the polarity machinery. Membrane potential, which is regulated by the potassium transporter Trk1p, is required for polarity orientation during mating and EF response. Membrane potential may regulate membrane charges through negatively charged phosphatidylserines (PSs), which act to position the Cdc42p-based polarity machinery. These studies thus define an electrochemical pathway that directs the orientation of cell polarization. PMID:25548923

  18. ¹¹³Cd NMR experiments reveal an unusual metal cluster in the solution structure of the yeast splicing protein Bud31p.

    PubMed

    van Roon, Anne-Marie M; Yang, Ji-Chun; Mathieu, Daniel; Bermel, Wolfgang; Nagai, Kiyoshi; Neuhaus, David

    2015-04-13

    Establishing the binding topology of structural zinc ions in proteins is an essential part of their structure determination by NMR spectroscopy. Using (113)Cd NMR experiments with (113)Cd-substituted samples is a useful approach but has previously been limited mainly to very small protein domains. Here we used (113)Cd NMR spectroscopy during structure determination of Bud31p, a 157-residue yeast protein containing an unusual Zn3Cys9 cluster, demonstrating that recent hardware developments make this approach feasible for significantly larger systems.

  19. Measuring mitotic spindle dynamics in budding yeast

    NASA Astrophysics Data System (ADS)

    Plumb, Kemp

    In order to carry out its life cycle and produce viable progeny through cell division, a cell must successfully coordinate and execute a number of complex processes with high fidelity, in an environment dominated by thermal noise. One important example of such a process is the assembly and positioning of the mitotic spindle prior to chromosome segregation. The mitotic spindle is a modular structure composed of two spindle pole bodies, separated in space and spanned by filamentous proteins called microtubules, along which the genetic material of the cell is held. The spindle is responsible for alignment and subsequent segregation of chromosomes into two equal parts; proper spindle positioning and timing ensure that genetic material is appropriately divided amongst mother and daughter cells. In this thesis, I describe fluorescence confocal microscopy and automated image analysis algorithms, which I have used to observe and analyze the real space dynamics of the mitotic spindle in budding yeast. The software can locate structures in three spatial dimensions and track their movement in time. By selecting fluorescent proteins which specifically label the spindle poles and cell periphery, mitotic spindle dynamics have been measured in a coordinate system relevant to the cell division. I describe how I have characterised the accuracy and precision of the algorithms by simulating fluorescence data for both spindle poles and the budding yeast cell surface. In this thesis I also describe the construction of a microfluidic apparatus that allows for the measurement of long time-scale dynamics of individual cells and the development of a cell population. The tools developed in this thesis work will facilitate in-depth quantitative analysis of the non-equilibrium processes in living cells.

  20. The essential function of Rrs1 in ribosome biogenesis is conserved in budding and fission yeasts.

    PubMed

    Wan, Kun; Kawara, Haruka; Yamamoto, Tomoyuki; Kume, Kazunori; Yabuki, Yukari; Goshima, Tetsuya; Kitamura, Kenji; Ueno, Masaru; Kanai, Muneyoshi; Hirata, Dai; Funato, Kouichi; Mizuta, Keiko

    2015-09-01

    The Rrs1 protein plays an essential role in the biogenesis of 60S ribosomal subunits in budding yeast (Saccharomyces cerevisiae). Here, we examined whether the fission yeast (Schizosaccharomyces pombe) homologue of Rrs1 also plays a role in ribosome biogenesis. To this end, we constructed two temperature-sensitive fission yeast strains, rrs1-D14/22G and rrs1-L51P, which had amino acid substitutions corresponding to those of the previously characterized budding yeast rrs1-84 (D22/30G) and rrs1-124 (L61P) strains, respectively. The fission yeast mutants exhibited severe defects in growth and 60S ribosomal subunit biogenesis at high temperatures. In addition, expression of the Rrs1 protein of fission yeast suppressed the growth defects of the budding yeast rrs1 mutants at high temperatures. Yeast two-hybrid analyses revealed that the interactions of Rrs1 with the Rfp2 and Ebp2 proteins were conserved in budding and fission yeasts. These results suggest that the essential function of Rrs1 in ribosome biogenesis may be conserved in budding and fission yeasts.

  1. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    NASA Astrophysics Data System (ADS)

    Shibata, M.; Torigoe, M.; Matsumoto, Y.; Yamamoto, M.; Takizawa, N.; Hada, Y.; Mori, Y.; Takarabe, K.; Ono, F.

    2014-05-01

    Our studies on the tolerance of plants and animals against very high pressure of several GPa have been extended to a smaller sized fungus, the budding yeast Saccharomyces cerevisiae. Several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate, and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar. It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for longer than 12 h were found dead. The high pressure tolerance of budding yeast is a little weaker than that of tardigrades.

  2. Characterization of Septin Ultrastructure in Budding Yeast Using Electron Tomography

    PubMed Central

    Bertin, Aurélie; Nogales, Eva

    2015-01-01

    Summary Septins are essential for the completion of cytokinesis. In budding yeast, Saccharomyces cerevisiae, septins are located at the bud neck during mitosis and are closely connected to the inner plasma membrane. In vitro, yeast septins have been shown to self-assemble into a variety of filamentous structures, including rods, paired filaments, bundles and rings [1–3]. Using electron tomography of freeze-substituted section and cryo-electron tomography of frozen sections, we determined the three dimensional organization of the septin cytoskeleton in dividing budding yeast with molecular resolution [4,5]. Here we describe the detailed procedures used for our characterization of the septin cellular ultrastructure. PMID:26519309

  3. Post-transcriptional regulation in budding yeast meiosis.

    PubMed

    Jin, Liang; Neiman, Aaron M

    2016-05-01

    The precise regulation of gene expression is essential for developmental processes in eukaryotic organisms. As an important post-transcriptional regulatory point, translational control is complementary to transcriptional regulation. Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental process controlled by a well-studied transcriptional cascade that drives the cell through the events of DNA replication, meiotic chromosome segregation, and spore assembly. Recent studies have revealed that as cells begin the meiotic divisions, translational regulation of gene expression fine tunes this transcriptional cascade. The significance and mechanisms of this translational regulation are beginning to emerge. These studies may also provide insights into translational regulation in germ cell development of multicellular organisms.

  4. Mechanical feedback stabilizes budding yeast morphogenesis

    NASA Astrophysics Data System (ADS)

    Banavar, Samhita; Trogdon, Michael; Petzold, Linda; Campas, Otger

    Walled cells have the ability to remodel their shape while sustaining an internal turgor pressure that can reach values up to 10 atmospheres. This requires a tight and simultaneous regulation of cell wall assembly and mechanochemistry, but the underlying mechanisms by which this is achieved remain unclear. Using the growth of mating projections in budding yeast (S. cerevisiae) as a motivating example, we have developed a theoretical description that couples the mechanics of cell wall expansion and assembly via a mechanical feedback. In the absence of a mechanical feedback, cell morphogenesis is inherently unstable. The presence of a mechanical feedback stabilizes changes in cell shape and growth, and provides a mechanism to prevent cell lysis in a wide range of conditions. We solve for the dynamics of the system and obtain the different dynamical regimes. In particular, we show that several parameters affect the stability of growth, including the strength of mechanical feedback in the system. Finally, we compare our results to existing experimental data.

  5. Budding yeast for budding geneticists: a primer on the Saccharomyces cerevisiae model system.

    PubMed

    Duina, Andrea A; Miller, Mary E; Keeney, Jill B

    2014-05-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.

  6. Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

    PubMed Central

    Duina, Andrea A.; Miller, Mary E.; Keeney, Jill B.

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans. PMID:24807111

  7. Molecular cytotoxicity mechanisms of allyl alcohol (acrolein) in budding yeast.

    PubMed

    Golla, Upendarrao; Bandi, Goutham; Tomar, Raghuvir S

    2015-06-15

    Allyl alcohol (AA) is one of the environmental pollutants used as a herbicide and industrial chemical. AA undergoes enzymatic oxidation in vivo to form Acrolein (Acr), a highly reactive and ubiquitous environmental toxicant. The exposure to AA/Acr has detrimental effects on cells and is highly fatal. In corroboration to the current literature describing AA/Acr toxicity, this study aimed to investigate the molecular cytotoxicity mechanisms of AA/Acr using budding yeast as a eukaryotic model organism. Genome-wide transcriptome analysis of cells treated with a sublethal dose of AA (0.4 mM) showed differential regulation of approximately 30% of the yeast genome. Functional enrichment analysis of the AA transcriptome revealed that genes belong to diverse cellular processes including the cell cycle, DNA damage repair, metal homeostasis, stress response genes, ribosomal biogenesis, metabolism, meiosis, ubiquitination, cell morphogenesis, and transport. Moreover, we have identified novel molecular targets of AA/Acr through genetic screening, which belongs to oxidative stress, DNA damage repair, iron homeostasis, and cell wall integrity. This study also demonstrated the epigenetic basis of AA/Acr toxicity mediated through histone tails and chromatin modifiers. Interestingly, our study disclosed the use of pyrazole and ethanol as probable antidotes for AA intoxication. For the first time, this study also demonstrated the reproductive toxicity of AA/Acr using the yeast gametogenesis (spermatogenesis) model. Altogether, this study unravels the molecular mechanisms of AA/Acr cytotoxicity and facilitates the prediction of biomarkers for toxicity assessment and therapeutic approaches. PMID:25919230

  8. Tolerance of budding yeast Saccharomyces cerevisiae to ultra high pressure

    NASA Astrophysics Data System (ADS)

    Ono, Fumihisa; Shibata, Michiko; Torigoe, Motoki; Matsumoto, Yuta; Yamamoto, Shinsuke; Takizawa, Noboru; Hada, Yoshio; Mori, Yoshihisa; Takarabe, Kenichi

    2013-06-01

    In our previous studies on the tolerance of small plants and animals to the high hydrostatic pressure of 7.5 GPa, it was shown that all the living samples could be borne at this high pressure, which is more than one order of magnitude higher than the proteinic denaturation pressure. To make this inconsistency clear, we have extended these studies to a smaller sized fungus, budding yeast Saccharomyces cerevisiae. A several pieces of budding yeast (dry yeast) were sealed in a small teflon capsule with a liquid pressure medium fluorinate (PC72, Sumitomo 3M), and exposed to 7.5 GPa by using a cubic anvil press. The pressure was kept constant for various duration of time from 2 to 24 h. After the pressure was released, the specimens were brought out from the teflon capsule, and they were cultivated on a potato dextrose agar (PDA). It was found that the budding yeast exposed to 7.5 GPa for up to 6 h showed multiplication. However, those exposed to 7.5 GPa for 12 and 24 h were found dead. The high pressure tolerance of budding yeast is weaker than that of tardigrades.

  9. Computational model of polarized actin cables and cytokinetic actin ring formation in budding yeast

    PubMed Central

    Tang, Haosu; Bidone, Tamara C.

    2015-01-01

    The budding yeast actin cables and contractile ring are important for polarized growth and division, revealing basic aspects of cytoskeletal function. To study these formin-nucleated structures, we built a 3D computational model with actin filaments represented as beads connected by springs. Polymerization by formins at the bud tip and bud neck, crosslinking, severing, and myosin pulling, are included. Parameter values were estimated from prior experiments. The model generates actin cable structures and dynamics similar to those of wild type and formin deletion mutant cells. Simulations with increased polymerization rate result in long, wavy cables. Simulated pulling by type V myosin stretches actin cables. Increasing the affinity of actin filaments for the bud neck together with reduced myosin V pulling promotes the formation of a bundle of antiparallel filaments at the bud neck, which we suggest as a model for the assembly of actin filaments to the contractile ring. PMID:26538307

  10. Polarity-Driven Geometrical Cluster Growth Model of Budding Yeast

    NASA Astrophysics Data System (ADS)

    Cabral, Reniel B.; Lim, May T.

    We present a polarity-driven activator-inhibitor model of budding yeast in a two-dimensional medium wherein impeding metabolites secretion (or growth inhibitors) and growth directionality are determined by the local nutrient level. We found that colony size and morphological features varied with nutrient concentration. A branched-type morphology is associated with high impeding metabolite concentration together with a high fraction of distal budding, while opposite conditions (low impeding metabolite concentration, high fraction of proximal budding) promote Eden-type patterns. Increasing the anisotropy factor (or polarity) produced other spatial patterns akin to the electrical breakdown under varying electric field. Rapid changes in the colony morphology, which we conjecture to be equivalent to a transition from an inactive quiescent state to an active budding state, appeared when nutrients were limited.

  11. Actin and Endocytosis in Budding Yeast

    PubMed Central

    Goode, Bruce L.; Eskin, Julian A.; Wendland, Beverly

    2015-01-01

    Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed. PMID:25657349

  12. Asymmetric nucleosomes flank promoters in the budding yeast genome.

    PubMed

    Ramachandran, Srinivas; Zentner, Gabriel E; Henikoff, Steven

    2015-03-01

    Nucleosomes in active chromatin are dynamic, but whether they have distinct structural conformations is unknown. To identify nucleosomes with alternative structures genome-wide, we used H4S47C-anchored cleavage mapping, which revealed that 5% of budding yeast (Saccharomyces cerevisiae) nucleosome positions have asymmetric histone-DNA interactions. These asymmetric interactions are enriched at nucleosome positions that flank promoters. Micrococcal nuclease (MNase) sequence-based profiles of asymmetric nucleosome positions revealed a corresponding asymmetry in MNase protection near the dyad axis, suggesting that the loss of DNA contacts around H4S47 is accompanied by protection of the DNA from MNase. Chromatin immunoprecipitation mapping of selected nucleosome remodelers indicated that asymmetric nucleosomes are bound by the RSC chromatin remodeling complex, which is required for maintaining nucleosomes at asymmetric positions. These results imply that the asymmetric nucleosome-RSC complex is a metastable intermediate representing partial unwrapping and protection of nucleosomal DNA on one side of the dyad axis during chromatin remodeling.

  13. Budding yeast colony growth study based on circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, Devi; Khotimah, S. N.; Viridi, S.

    2016-08-01

    Yeast colony growth can be modelled by using circular granular cells, which can grow and produce buds. The bud growth angle can be set to regulate cell budding pattern. Cohesion force, contact force and Stokes force were adopted to accommodate the behaviour and interactions among cells. Simulation steps are divided into two steps, the explicit step is due to cell growing and implicit step for the cell rearrangement. Only in explicit step that time change was performed. In this study, we examine the influence of cell diameter growth time and reproduction time combination toward the growth of cell number and colony formation. We find a commutative relation between the cell diameter growth time and reproduction time to the specific growth rate. The greater value of the multiplication of the parameters, the smaller specific growth rate is obtained. It also shows a linear correlation between the specific growth rate and colony diameter growth rate.

  14. Mitochondrial inheritance in budding yeasts: towards an integrated understanding.

    PubMed

    Solieri, Lisa

    2010-11-01

    Recent advances in yeast mitogenomics have significantly contributed to our understanding of the diversity of organization, structure and topology in the mitochondrial genome of budding yeasts. In parallel, new insights on mitochondrial DNA (mtDNA) inheritance in the model organism Saccharomyces cerevisiae highlighted an integrated scenario where recombination, replication and segregation of mtDNA are intricately linked to mitochondrial nucleoid (mt-nucleoid) structure and organelle sorting. In addition to this, recent discoveries of bifunctional roles of some mitochondrial proteins have interesting implications on mito-nuclear genome interactions and the relationship between mtDNA inheritance, yeast fitness and speciation. This review summarizes the current knowledge on yeast mitogenomics, mtDNA inheritance with regard to mt-nucleoid structure and organelle dynamics, and mito-nuclear genome interactions.

  15. The fascinating and secret wild life of the budding yeast S. cerevisiae

    PubMed Central

    Liti, Gianni

    2015-01-01

    The budding yeast Saccharomyces cerevisiae has been used in laboratory experiments for over a century and has been instrumental in understanding virtually every aspect of molecular biology and genetics. However, it wasn't until a decade ago that the scientific community started to realise how little was known about this yeast's ecology and natural history, and how this information was vitally important for interpreting its biology. Recent large-scale population genomics studies coupled with intensive field surveys have revealed a previously unappreciated wild lifestyle of S. cerevisiae outside the restrictions of human environments and laboratories. The recent discovery that Chinese isolates harbour almost twice as much genetic variation as isolates from the rest of the world combined suggests that Asia is the likely origin of the modern budding yeast. DOI: http://dx.doi.org/10.7554/eLife.05835.001 PMID:25807086

  16. Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Higuchi-Sanabria, Ryo; Charalel, Joseph K; Viana, Matheus P; Garcia, Enrique J; Sing, Cierra N; Koenigsberg, Andrea; Swayne, Theresa C; Vevea, Jason D; Boldogh, Istvan R; Rafelski, Susanne M; Pon, Liza A

    2016-03-01

    Higher-functioning mitochondria that are more reduced and have less ROS are anchored in the yeast bud tip by the Dsl1-family protein Mmr1p. Here we report a role for mitochondrial fusion in bud-tip anchorage of mitochondria. Fluorescence loss in photobleaching (FLIP) and network analysis experiments revealed that mitochondria in large buds are a continuous reticulum that is physically distinct from mitochondria in mother cells. FLIP studies also showed that mitochondria that enter the bud can fuse with mitochondria that are anchored in the bud tip. In addition, loss of fusion and mitochondrial DNA (mtDNA) by deletion of mitochondrial outer or inner membrane fusion proteins (Fzo1p or Mgm1p) leads to decreased accumulation of mitochondria at the bud tip and inheritance of fitter mitochondria by buds compared with cells with no mtDNA. Conversely, increasing the accumulation and anchorage of mitochondria in the bud tip by overexpression of MMR1 results in inheritance of less-fit mitochondria by buds and decreased replicative lifespan and healthspan. Thus quantity and quality of mitochondrial inheritance are ensured by two opposing processes: bud-tip anchorage by mitochondrial fusion and Mmr1p, which favors bulk inheritance; and quality control mechanisms that promote segregation of fitter mitochondria to the bud. PMID:26764088

  17. Novel features of ARS selection in budding yeast Lachancea kluyveri

    PubMed Central

    2011-01-01

    Background The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. Results In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. Conclusions Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs. PMID:22204614

  18. Saccharomyces Genome Database: the genomics resource of budding yeast

    PubMed Central

    Cherry, J. Michael; Hong, Eurie L.; Amundsen, Craig; Balakrishnan, Rama; Binkley, Gail; Chan, Esther T.; Christie, Karen R.; Costanzo, Maria C.; Dwight, Selina S.; Engel, Stacia R.; Fisk, Dianna G.; Hirschman, Jodi E.; Hitz, Benjamin C.; Karra, Kalpana; Krieger, Cynthia J.; Miyasato, Stuart R.; Nash, Rob S.; Park, Julie; Skrzypek, Marek S.; Simison, Matt; Weng, Shuai; Wong, Edith D.

    2012-01-01

    The Saccharomyces Genome Database (SGD, http://www.yeastgenome.org) is the community resource for the budding yeast Saccharomyces cerevisiae. The SGD project provides the highest-quality manually curated information from peer-reviewed literature. The experimental results reported in the literature are extracted and integrated within a well-developed database. These data are combined with quality high-throughput results and provided through Locus Summary pages, a powerful query engine and rich genome browser. The acquisition, integration and retrieval of these data allow SGD to facilitate experimental design and analysis by providing an encyclopedia of the yeast genome, its chromosomal features, their functions and interactions. Public access to these data is provided to researchers and educators via web pages designed for optimal ease of use. PMID:22110037

  19. Saccharomyces Genome Database: the genomics resource of budding yeast.

    PubMed

    Cherry, J Michael; Hong, Eurie L; Amundsen, Craig; Balakrishnan, Rama; Binkley, Gail; Chan, Esther T; Christie, Karen R; Costanzo, Maria C; Dwight, Selina S; Engel, Stacia R; Fisk, Dianna G; Hirschman, Jodi E; Hitz, Benjamin C; Karra, Kalpana; Krieger, Cynthia J; Miyasato, Stuart R; Nash, Rob S; Park, Julie; Skrzypek, Marek S; Simison, Matt; Weng, Shuai; Wong, Edith D

    2012-01-01

    The Saccharomyces Genome Database (SGD, http://www.yeastgenome.org) is the community resource for the budding yeast Saccharomyces cerevisiae. The SGD project provides the highest-quality manually curated information from peer-reviewed literature. The experimental results reported in the literature are extracted and integrated within a well-developed database. These data are combined with quality high-throughput results and provided through Locus Summary pages, a powerful query engine and rich genome browser. The acquisition, integration and retrieval of these data allow SGD to facilitate experimental design and analysis by providing an encyclopedia of the yeast genome, its chromosomal features, their functions and interactions. Public access to these data is provided to researchers and educators via web pages designed for optimal ease of use. PMID:22110037

  20. A comprehensive model to predict mitotic division in budding yeasts

    PubMed Central

    Sutradhar, Sabyasachi; Yadav, Vikas; Sridhar, Shreyas; Sreekumar, Lakshmi; Bhattacharyya, Dibyendu; Ghosh, Santanu Kumar; Paul, Raja; Sanyal, Kaustuv

    2015-01-01

    High-fidelity chromosome segregation during cell division depends on a series of concerted interdependent interactions. Using a systems biology approach, we built a robust minimal computational model to comprehend mitotic events in dividing budding yeasts of two major phyla: Ascomycota and Basidiomycota. This model accurately reproduces experimental observations related to spindle alignment, nuclear migration, and microtubule (MT) dynamics during cell division in these yeasts. The model converges to the conclusion that biased nucleation of cytoplasmic microtubules (cMTs) is essential for directional nuclear migration. Two distinct pathways, based on the population of cMTs and cortical dyneins, differentiate nuclear migration and spindle orientation in these two phyla. In addition, the model accurately predicts the contribution of specific classes of MTs in chromosome segregation. Thus we present a model that offers a wider applicability to simulate the effects of perturbation of an event on the concerted process of the mitotic cell division. PMID:26310442

  1. A comprehensive model to predict mitotic division in budding yeasts.

    PubMed

    Sutradhar, Sabyasachi; Yadav, Vikas; Sridhar, Shreyas; Sreekumar, Lakshmi; Bhattacharyya, Dibyendu; Ghosh, Santanu Kumar; Paul, Raja; Sanyal, Kaustuv

    2015-11-01

    High-fidelity chromosome segregation during cell division depends on a series of concerted interdependent interactions. Using a systems biology approach, we built a robust minimal computational model to comprehend mitotic events in dividing budding yeasts of two major phyla: Ascomycota and Basidiomycota. This model accurately reproduces experimental observations related to spindle alignment, nuclear migration, and microtubule (MT) dynamics during cell division in these yeasts. The model converges to the conclusion that biased nucleation of cytoplasmic microtubules (cMTs) is essential for directional nuclear migration. Two distinct pathways, based on the population of cMTs and cortical dyneins, differentiate nuclear migration and spindle orientation in these two phyla. In addition, the model accurately predicts the contribution of specific classes of MTs in chromosome segregation. Thus we present a model that offers a wider applicability to simulate the effects of perturbation of an event on the concerted process of the mitotic cell division.

  2. Actin Depolymerization Drives Actomyosin Ring Contraction during Budding Yeast Cytokinesis

    PubMed Central

    Pinto, Inês Mendes; Rubinstein, Boris; Kucharavy, Andrei; Unruh, Jay R.; Li, Rong

    2012-01-01

    SUMMARY Actin filaments and myosin-II are evolutionarily conserved force generating components of the contractile ring during cytokinesis. Here we show that in budding yeast actin filament depolymerization plays a major role in actomyosin ring constriction. Cofilin mutation or chemically stabilizing actin filaments attenuates actomyosin ring constriction. Deletion of myosin-II motor domain or the myosin regulatory light chain reduced the contraction rate and also the rate of actin depolymerization in the ring. We constructed a quantitative microscopic model of actomyosin ring constriction via filament sliding driven by both actin depolymerization and myosin-II motor activity. Model simulations based on experimental measurements supports the notion that actin depolymerization is the predominant mechanism for ring constriction. The model predicts invariability of total contraction time irrespective of the initial ring size as originally reported for C elegans embryonic cells. This prediction was validated in yeast cells of different sizes due to having different ploidies. PMID:22698284

  3. Replication-Associated Recombinational Repair: Lessons from Budding Yeast

    PubMed Central

    Bonner, Jaclyn N.; Zhao, Xiaolan

    2016-01-01

    Recombinational repair processes multiple types of DNA lesions. Though best understood in the repair of DNA breaks, recombinational repair is intimately linked to other situations encountered during replication. As DNA strands are decorated with many types of blocks that impede the replication machinery, a great number of genomic regions cannot be duplicated without the help of recombinational repair. This replication-associated recombinational repair employs both the core recombination proteins used for DNA break repair and the specialized factors that couple replication with repair. Studies from multiple organisms have provided insights into the roles of these specialized factors, with the findings in budding yeast being advanced through use of powerful genetics and methods for detecting DNA replication and repair intermediates. In this review, we summarize recent progress made in this organism, ranging from our understanding of the classical template switch mechanisms to gap filling and replication fork regression pathways. As many of the protein factors and biological principles uncovered in budding yeast are conserved in higher eukaryotes, these findings are crucial for stimulating studies in more complex organisms. PMID:27548223

  4. Cdc28 Activates Exit from Mitosis in Budding Yeast

    PubMed Central

    Rudner, Adam D.; Hardwick, Kevin G.; Murray, Andrew W.

    2000-01-01

    The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast. PMID:10871278

  5. Programmed Cell Death Initiation and Execution in Budding Yeast

    PubMed Central

    Strich, Randy

    2015-01-01

    Apoptosis or programmed cell death (PCD) was initially described in metazoans as a genetically controlled process leading to intracellular breakdown and engulfment by a neighboring cell . This process was distinguished from other forms of cell death like necrosis by maintenance of plasma membrane integrity prior to engulfment and the well-defined genetic system controlling this process. Apoptosis was originally described as a mechanism to reshape tissues during development. Given this context, the assumption was made that this process would not be found in simpler eukaryotes such as budding yeast. Although basic components of the apoptotic pathway were identified in yeast, initial observations suggested that it was devoid of prosurvival and prodeath regulatory proteins identified in mammalian cells. However, as apoptosis became extensively linked to the elimination of damaged cells, key PCD regulatory proteins were identified in yeast that play similar roles in mammals. This review highlights recent discoveries that have permitted information regarding PCD regulation in yeast to now inform experiments in animals. PMID:26272996

  6. A nutrient dependant switch explains mutually exclusive existence of meiosis and mitosis initiation in budding yeast.

    PubMed

    Wannige, C T; Kulasiri, D; Samarasinghe, S

    2014-01-21

    Nutrients from living environment are vital for the survival and growth of any organism. Budding yeast diploid cells decide to grow by mitosis type cell division or decide to create unique, stress resistant spores by meiosis type cell division depending on the available nutrient conditions. To gain a molecular systems level understanding of the nutrient dependant switching between meiosis and mitosis initiation in diploid cells of budding yeast, we develop a theoretical model based on ordinary differential equations (ODEs) including the mitosis initiator and its relations to budding yeast meiosis initiation network. Our model accurately and qualitatively predicts the experimentally revealed temporal variations of related proteins under different nutrient conditions as well as the diverse mutant studies related to meiosis and mitosis initiation. Using this model, we show how the meiosis and mitosis initiators form an all-or-none type bistable switch in response to available nutrient level (mainly nitrogen). The transitions to and from meiosis or mitosis initiation states occur via saddle node bifurcation. This bidirectional switch helps the optimal usage of available nutrients and explains the mutually exclusive existence of meiosis and mitosis pathways.

  7. The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription.

    PubMed

    Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco

    2014-09-01

    Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity.

  8. The yeast prefoldin-like URI-orthologue Bud27 associates with the RSC nucleosome remodeler and modulates transcription

    PubMed Central

    Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; Martínez-Fernández, Verónica; Fernández-Pevida, Antonio; Cuevas-Bermúdez, Abel; Martín-Expósito, Manuel; Chávez, Sebastián; de la Cruz, Jesús; Navarro, Francisco

    2014-01-01

    Bud27, the yeast orthologue of human URI/RMP, is a member of the prefoldin-like family of ATP-independent molecular chaperones. It has recently been shown to mediate the assembly of the three RNA polymerases in an Rpb5-dependent manner. In this work, we present evidence of Bud27 modulating RNA pol II transcription elongation. We show that Bud27 associates with RNA pol II phosphorylated forms (CTD-Ser5P and CTD-Ser2P), and that its absence affects RNA pol II occupancy of transcribed genes. We also reveal that Bud27 associates in vivo with the Sth1 component of the chromatin remodeling complex RSC and mediates its association with RNA pol II. Our data suggest that Bud27, in addition of contributing to Rpb5 folding within the RNA polymerases, also participates in the correct assembly of other chromatin-associated protein complexes, such as RSC, thereby modulating their activity. PMID:25081216

  9. Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae.

    PubMed

    Wu, Ziyun; Song, Lixia; Liu, Shao Quan; Huang, Dejian

    2014-10-01

    Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans.

  10. The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

    PubMed Central

    2013-01-01

    The propagation of all organisms depends on the accurate and orderly segregation of chromosomes in mitosis and meiosis. Budding yeast has long served as an outstanding model organism to identify the components and underlying mechanisms that regulate chromosome segregation. This review focuses on the kinetochore, the macromolecular protein complex that assembles on centromeric chromatin and maintains persistent load-bearing attachments to the dynamic tips of spindle microtubules. The kinetochore also serves as a regulatory hub for the spindle checkpoint, ensuring that cell cycle progression is coupled to the achievement of proper microtubule–kinetochore attachments. Progress in understanding the composition and overall architecture of the kinetochore, as well as its properties in making and regulating microtubule attachments and the spindle checkpoint, is discussed. PMID:23908374

  11. Polarization of Diploid Daughter Cells Directed by Spatial Cues and GTP Hydrolysis of Cdc42 in Budding Yeast

    PubMed Central

    Narayan, Monisha; Chou, Ching-Shan; Park, Hay-Oak

    2013-01-01

    Cell polarization occurs along a single axis that is generally determined by a spatial cue. Cells of the budding yeast exhibit a characteristic pattern of budding, which depends on cell-type-specific cortical markers, reflecting a genetic programming for the site of cell polarization. The Cdc42 GTPase plays a key role in cell polarization in various cell types. Although previous studies in budding yeast suggested positive feedback loops whereby Cdc42 becomes polarized, these mechanisms do not include spatial cues, neglecting the normal patterns of budding. Here we combine live-cell imaging and mathematical modeling to understand how diploid daughter cells establish polarity preferentially at the pole distal to the previous division site. Live-cell imaging shows that daughter cells of diploids exhibit dynamic polarization of Cdc42-GTP, which localizes to the bud tip until the M phase, to the division site at cytokinesis, and then to the distal pole in the next G1 phase. The strong bias toward distal budding of daughter cells requires the distal-pole tag Bud8 and Rga1, a GTPase activating protein for Cdc42, which inhibits budding at the cytokinesis site. Unexpectedly, we also find that over 50% of daughter cells lacking Rga1 exhibit persistent Cdc42-GTP polarization at the bud tip and the distal pole, revealing an additional role of Rga1 in spatiotemporal regulation of Cdc42 and thus in the pattern of polarized growth. Mathematical modeling indeed reveals robust Cdc42-GTP clustering at the distal pole in diploid daughter cells despite random perturbation of the landmark cues. Moreover, modeling predicts different dynamics of Cdc42-GTP polarization when the landmark level and the initial level of Cdc42-GTP at the division site are perturbed by noise added in the model. PMID:23437206

  12. A critical three-way junction is conserved in budding yeast and vertebrate telomerase RNAs

    PubMed Central

    Brown, Yogev; Abraham, Mira; Pearl, Sivan; Kabaha, Majdi M.; Elboher, Elhanan; Tzfati, Yehuda

    2007-01-01

    The telomerase ribonucleoprotein copies a short template within its integral RNA moiety onto eukaryotic chromosome ends, compensating for incomplete replication and degradation. Non-template regions of telomerase RNA (TER) are also crucial for telomerase function, yet they are highly divergent in sequence among species and their roles are largely unclear. Using both phylogenetic and mutational analyses, we predicted secondary structures for TERs from Kluyveromyces budding yeast species. A comparison of these secondary structure models with the published model for the Saccharomyces cerevisiae TER reveals a common arrangement into three long arms, a templating domain in the center and several conserved elements in the same positions within the structure. One of them, a three-way junction element, is highly conserved in budding yeast TERs. This element also shows sequence and structure similarity to the critical CR4-CR5 activating domain of vertebrate TERs. Mutational analysis in Kluyveromyces lactis confirmed that this element, and in particular the residues conserved across yeast and vertebrates, is critical for telomerase action both in vivo and in vitro. These findings demonstrate that despite the extreme divergence of TER sequences from different organisms, they do share conserved elements, which presumably carry out common roles in telomerase function. PMID:17855392

  13. Fission Yeast Hotspot Sequence Motifs Are Also Active in Budding Yeast

    PubMed Central

    Steiner, Walter W.; Steiner, Estelle M.

    2012-01-01

    In most organisms, including humans, meiotic recombination occurs preferentially at a limited number of sites in the genome known as hotspots. There has been substantial progress recently in elucidating the factors determining the location of meiotic recombination hotspots, and it is becoming clear that simple sequence motifs play a significant role. In S. pombe, there are at least five unique sequence motifs that have been shown to produce hotspots of recombination, and it is likely that there are more. In S. cerevisiae, simple sequence motifs have also been shown to produce hotspots or show significant correlations with hotspots. Some of the hotspot motifs in both yeasts are known or suspected to bind transcription factors (TFs), which are required for the activity of those hotspots. Here we show that four of the five hotspot motifs identified in S. pombe also create hotspots in the distantly related budding yeast S. cerevisiae. For one of these hotspots, M26 (also called CRE), we identify TFs, Cst6 and Sko1, that activate and inhibit the hotspot, respectively. In addition, two of the hotspot motifs show significant correlations with naturally occurring hotspots. The conservation of these hotspots between the distantly related fission and budding yeasts suggests that these sequence motifs, and others yet to be discovered, may function widely as hotspots in many diverse organisms. PMID:23300865

  14. Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

    PubMed Central

    Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

    2016-01-01

    The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

  15. Using dielectrophoresis to study the dynamic response of single budding yeast cells to Lyticase.

    PubMed

    Tang, Shi-Yang; Yi, Pyshar; Soffe, Rebecca; Nahavandi, Sofia; Shukla, Ravi; Khoshmanesh, Khashayar

    2015-05-01

    Budding yeast cells are quick and easy to grow and represent a versatile model of eukaryotic cells for a variety of cellular studies, largely because their genome has been widely studied and links can be drawn with higher eukaryotes. Therefore, the efficient separation, immobilization, and conversion of budding yeasts into spheroplast or protoplast can provide valuable insight for many fundamentals investigations in cell biology at a single cell level. Dielectrophoresis, the induced motion of particles in non-uniform electric fields, possesses a great versatility for manipulation of cells in microfluidic platforms. Despite this, dielectrophoresis has been largely utilized for studying of non-budding yeast cells and has rarely been used for manipulation of budding cells. Here, we utilize dielectrophoresis for studying the dynamic response of budding cells to different concentrations of Lyticase. This involves separation of the budding yeasts from a background of non-budding cells and their subsequent immobilization onto the microelectrodes at desired densities down to single cell level. The immobilized yeasts are then stimulated with Lyticase to remove the cell wall and convert them into spheroplasts, in a highly dynamic process that depends on the concentration of Lyticase. We also introduce a novel method for immobilization of the cell organelles released from the lysed cells by patterning multi-walled carbon nanotubes (MWCNTs) between the microelectrodes. PMID:25701421

  16. Evidence for cohesin sliding along budding yeast chromosomes

    PubMed Central

    Ocampo-Hafalla, Maria; Muñoz, Sofía; Samora, Catarina P.; Uhlmann, Frank

    2016-01-01

    The ring-shaped cohesin complex is thought to topologically hold sister chromatids together from their synthesis in S phase until chromosome segregation in mitosis. How cohesin stably binds to chromosomes for extended periods, without impeding other chromosomal processes that also require access to the DNA, is poorly understood. Budding yeast cohesin is loaded onto DNA by the Scc2–Scc4 cohesin loader at centromeres and promoters of active genes, from where cohesin translocates to more permanent places of residence at transcription termination sites. Here we show that, at the GAL2 and MET17 loci, pre-existing cohesin is pushed downstream along the DNA in response to transcriptional gene activation, apparently without need for intermittent dissociation or reloading. We observe translocation intermediates and find that the distribution of most chromosomal cohesin is shaped by transcription. Our observations support a model in which cohesin is able to slide laterally along chromosomes while maintaining topological contact with DNA. In this way, stable cohesin binding to DNA and enduring sister chromatid cohesion become compatible with simultaneous underlying chromosomal activities, including but maybe not limited to transcription. PMID:27278645

  17. A simple biophysical model emulates budding yeast chromosome condensation

    PubMed Central

    Cheng, Tammy MK; Heeger, Sebastian; Chaleil, Raphaël AG; Matthews, Nik; Stewart, Aengus; Wright, Jon; Lim, Carmay; Bates, Paul A; Uhlmann, Frank

    2015-01-01

    Mitotic chromosomes were one of the first cell biological structures to be described, yet their molecular architecture remains poorly understood. We have devised a simple biophysical model of a 300 kb-long nucleosome chain, the size of a budding yeast chromosome, constrained by interactions between binding sites of the chromosomal condensin complex, a key component of interphase and mitotic chromosomes. Comparisons of computational and experimental (4C) interaction maps, and other biophysical features, allow us to predict a mode of condensin action. Stochastic condensin-mediated pairwise interactions along the nucleosome chain generate native-like chromosome features and recapitulate chromosome compaction and individualization during mitotic condensation. Higher order interactions between condensin binding sites explain the data less well. Our results suggest that basic assumptions about chromatin behavior go a long way to explain chromosome architecture and are able to generate a molecular model of what the inside of a chromosome is likely to look like. DOI: http://dx.doi.org/10.7554/eLife.05565.001 PMID:25922992

  18. Kinetic Analysis of a Molecular Model of the Budding Yeast Cell Cycle

    PubMed Central

    Chen, Katherine C.; Csikasz-Nagy, Attila; Gyorffy, Bela; Val, John; Novak, Bela; Tyson, John J.

    2000-01-01

    The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1–3 and Clb1–6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling “Start” (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and “Finish” (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. PMID:10637314

  19. Microtubule dynamics from mating through the first zygotic division in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Maddox, P; Chin, E; Mallavarapu, A; Yeh, E; Salmon, E D; Bloom, K

    1999-03-01

    We have used time-lapse digital imaging microscopy to examine cytoplasmic astral microtubules (Mts) and spindle dynamics during the mating pathway in budding yeast Saccharomyces cerevisiae. Mating begins when two cells of opposite mating type come into proximity. The cells arrest in the G1 phase of the cell cycle and grow a projection towards one another forming a shmoo projection. Imaging of microtubule dynamics with green fluorescent protein (GFP) fusions to dynein or tubulin revealed that the nucleus and spindle pole body (SPB) became oriented and tethered to the shmoo tip by a Mt-dependent search and capture mechanism. Dynamically unstable astral Mts were captured at the shmoo tip forming a bundle of three or four astral Mts. This bundle changed length as the tethered nucleus and SPB oscillated toward and away from the shmoo tip at growth and shortening velocities typical of free plus end astral Mts (approximately 0.5 micrometer/min). Fluorescent fiduciary marks in Mt bundles showed that Mt growth and shortening occurred primarily at the shmoo tip, not the SPB. This indicates that Mt plus end assembly/disassembly was coupled to pushing and pulling of the nucleus. Upon cell fusion, a fluorescent bar of Mts was formed between the two shmoo tip bundles, which slowly shortened (0.23 +/- 0.07 micrometer/min) as the two nuclei and their SPBs came together and fused (karyogamy). Bud emergence occurred adjacent to the fused SPB approximately 30 min after SPB fusion. During the first mitosis, the SPBs separated as the spindle elongated at a constant velocity (0.75 micrometer/min) into the zygotic bud. There was no indication of a temporal delay at the 2-micrometer stage of spindle morphogenesis or a lag in Mt nucleation by replicated SPBs as occurs in vegetative mitosis implying a lack of normal checkpoints. Thus, the shmoo tip appears to be a new model system for studying Mt plus end dynamic attachments and much like higher eukaryotes, the first mitosis after haploid

  20. Interorganelle interactions and inheritance patterns of nuclei and vacuoles in budding yeast meiosis.

    PubMed

    Tsai, I-Ting; Lin, Jyun-Liang; Chiang, Yi-Hsuan; Chuang, Yu-Chien; Liang, Shu-Shan; Chuang, Chi-Ning; Huang, Tzyy-Nan; Wang, Ting-Fang

    2014-02-01

    Many of the mechanisms by which organelles are inherited by spores during meiosis are not well understood. Dramatic chromosome motion and bouquet formation are evolutionarily conserved characteristics of meiotic chromosomes. The budding yeast bouquet genes (NDJ1, MPS3, CSM4) mediate these movements via telomere attachment to the nuclear envelope (NE). Here, we report that during meiosis the NE is in direct contact with vacuoles via nucleus-vacuole junctions (NVJs). We show that in meiosis NVJs are assembled through the interaction of the outer NE-protein Nvj1 and the vacuolar membrane protein Vac8. Notably, NVJs function as diffusion barriers that exclude the nuclear pore complexes, the bouquet protein Mps3 and NE-tethered telomeres from the outer nuclear membrane and nuclear ER, resulting in distorted NEs during early meiosis. An increase in NVJ area resulting from Nvj1-GFP overexpression produced a moderate bouquet mutant-like phenotype in wild-type cells. NVJs, as the vacuolar contact sites of the nucleus, were found to undergo scission alongside the NE during meiotic nuclear division. The zygotic NE and NVJs were partly segregated into 4 spores. Lastly, new NVJs were also revealed to be synthesized de novo to rejoin the zygotic NE with the newly synthesized vacuoles in the mature spores. In conclusion, our results revealed that budding yeast nuclei and vacuoles exhibit dynamic interorganelle interactions and different inheritance patterns in meiosis, and also suggested that nvj1Δ mutant cells may be useful to resolve the technical challenges pertaining to the isolation of intact nuclei for the biochemical study of meiotic nuclear proteins.

  1. Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast.

    PubMed

    Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R; Pon, Liza A

    2016-01-01

    Maintenance and regulation of proper mitochondrial dynamics and functions are necessary for cellular homeostasis. Numerous diseases, including neurodegeneration and muscle myopathies, and overall cellular aging are marked by declining mitochondrial function and subsequent loss of multiple other cellular functions. For these reasons, optimized protocols are needed for visualization and quantification of mitochondria and their function and fitness. In budding yeast, mitochondria are intimately associated with the actin cytoskeleton and utilize actin for their movement and inheritance. This chapter describes optimal approaches for labeling mitochondria and the actin cytoskeleton in living budding yeast cells, for imaging the labeled cells, and for analyzing the resulting images. PMID:26498778

  2. Study of budding yeast colony formation and its characterizations by using circular granular cell

    NASA Astrophysics Data System (ADS)

    Aprianti, D.; Haryanto, F.; Purqon, A.; Khotimah, S. N.; Viridi, S.

    2016-03-01

    Budding yeast can exhibit colony formation in solid substrate. The colony of pathogenic budding yeast can colonize various surfaces of the human body and medical devices. Furthermore, it can form biofilm that resists drug effective therapy. The formation of the colony is affected by the interaction between cells and with its growth media. The cell budding pattern holds an important role in colony expansion. To study this colony growth, the molecular dynamic method was chosen to simulate the interaction between budding yeast cells. Every cell was modelled by circular granular cells, which can grow and produce buds. Cohesion force, contact force, and Stokes force govern this model to mimic the interaction between cells and with the growth substrate. Characterization was determined by the maximum (L max) and minimum (L min) distances between two cells within the colony and whether two lines that connect the two cells in the maximum and minimum distances intersect each other. Therefore, it can be recognized the colony shape in circular, oval, and irregular shapes. Simulation resulted that colony formation are mostly in oval shape with little branch. It also shows that greater cohesion strength obtains more compact colony formation.

  3. Cell Biology of Yeast Zygotes, from Genesis to Budding

    PubMed Central

    Tartakoff, Alan M.

    2015-01-01

    The zygote is the essential intermediate that allows interchange of nuclear, mitochondrial and cytosolic determinants between cells. Zygote formation in S. cerevisiae is accomplished by mechanisms that are not characteristic of mitotic cells. These include shifting the axis of growth away from classical cortical landmarks, dramatically reorganizing the cell cortex, remodeling the cell wall in preparation for cell fusion, fusing with an adjacent partner, accomplishing nuclear fusion, orchestrating two steps of septin morphogenesis that account for a delay in fusion of mitochondria, and implementing new norms for bud site selection. This essay emphasizes the sequence of dependent relationships that account for this progression from cell encounters through to zygote budding. It briefly summarizes classical studies of signal transduction and polarity specification and then focuses on downstream events. PMID:25862405

  4. Cell biology of yeast zygotes, from genesis to budding.

    PubMed

    Tartakoff, Alan M

    2015-07-01

    The zygote is the essential intermediate that allows interchange of nuclear, mitochondrial and cytosolic determinants between cells. Zygote formation in Saccharomyces cerevisiae is accomplished by mechanisms that are not characteristic of mitotic cells. These include shifting the axis of growth away from classical cortical landmarks, dramatically reorganizing the cell cortex, remodeling the cell wall in preparation for cell fusion, fusing with an adjacent partner, accomplishing nuclear fusion, orchestrating two steps of septin morphogenesis that account for a delay in fusion of mitochondria, and implementing new norms for bud site selection. This essay emphasizes the sequence of dependent relationships that account for this progression from cell encounters through zygote budding. It briefly summarizes classical studies of signal transduction and polarity specification and then focuses on downstream events. PMID:25862405

  5. Evidence for widespread adaptive evolution of gene expression in budding yeast.

    PubMed

    Fraser, Hunter B; Moses, Alan M; Schadt, Eric E

    2010-02-16

    Changes in gene expression have been proposed to underlie many, or even most, adaptive differences between species. Despite the increasing acceptance of this view, only a handful of cases of adaptive gene expression evolution have been demonstrated. To address this discrepancy, we introduce a simple test for lineage-specific selection on gene expression. Applying the test to genome-wide gene expression data from the budding yeast Saccharomyces cerevisiae, we find that hundreds of gene expression levels have been subject to lineage-specific selection. Comparing these findings with independent population genetic evidence of selective sweeps suggests that this lineage-specific selection has resulted in recent sweeps at over a hundred genes, most of which led to increased transcript levels. Examination of the implicated genes revealed a specific biochemical pathway--ergosterol biosynthesis--where the expression of multiple genes has been subject to selection for reduced levels. In sum, these results suggest that adaptive evolution of gene expression is common in yeast, that regulatory adaptation can occur at the level of entire pathways, and that similar genome-wide scans may be possible in other species, including humans.

  6. Distinct roles for Khd1p in the localization and expression of bud-localized mRNAs in yeast

    PubMed Central

    Hasegawa, Yuko; Irie, Kenji; Gerber, André P.

    2008-01-01

    The RNA-binding protein Khd1p (KH-domain protein 1) is required for efficient localization of ASH1 mRNA to the bud-tip, probably acting as a translational repressor during mRNA transport in yeast. Here, we have systematically examined Khd1p mRNA targets and colocalization with known bud-tip-localized mRNAs in vivo. Affinity purification and DNA microarray analysis of Khd1p-associated mRNAs revealed hundreds of potential mRNAs targets, many of them encoding membrane-associated proteins. The putative targets include the messages for MID2, MTL1, WSC2, SRL1, EGT2, CLB2, ASH1, and Khd1p colocalizes with these mRNAs at the bud-tip. The combination of bioinformatics, RNA localization, and in vitro RNA-binding assays revealed that Khd1p binds to CNN repeats in coding regions of mRNA targets. Among the proteins encoded by previously known bud-tip-localized mRNAs, only Mtl1p levels were decreased in khd1Δ mutant cells, whereas Ash1p and Srl1p were reduced in cells overexpressing KHD1. Hence, Khd1p differentially affects gene expression possibly due to combinatorial arrangement with additional factors reflecting the redundant structure of post-transcriptional regulatory systems. PMID:18805955

  7. Cytoskeletal impairment during isoamyl alcohol-induced cell elongation in budding yeast

    PubMed Central

    Murata, Wakae; Kinpara, Satoko; Kitahara, Nozomi; Yamaguchi, Yoshihiro; Ogita, Akira; Tanaka, Toshio; Fujita, Ken-ichi

    2016-01-01

    Isoamyl alcohol (IAA) induces pseudohyphae including cell elongation in the budding yeast Saccharomyces cerevisiae. Detailed regulation of microtubules and actin in developmental transition during cell elongation is poorly understood. Here, we show that although IAA did not affect the intracellular actin level, it reduced the levels of both α- and β-tubulins. In budding yeast, cytoplasmic microtubules are linked to actin via complexes consisting of at least Kar9, Bim1, and Myo2, and reach from the spindle pole body to the cortical attachment site at the bud tip. However, IAA did not affect migration of Myo2 to the bud tip and kept Kar9 in the interior portion of the cell. In addition, bud elongation was observed in Kar9-overexpressing cells in the absence of IAA. These results indicate that impairment of the link between cytoplasmic microtubules and actin is possibly involved in the lowered interaction of Myo2 with Kar9. Our study might explain the reason for delayed cell cycle during IAA-induced cell elongation. PMID:27507042

  8. Effects of the F-actin inhibitor latrunculin A on the budding yeast Saccharomyces cerevisiae.

    PubMed

    Kopecká, Marie; Yamaguchi, Masashi; Kawamoto, Susumu

    2015-07-01

    Our basic cell biology research was aimed at investigating the effect on eukaryotic cells of the sudden loss of the F-actin cytoskeleton. Cells treated with latrunculin A (LA) in yeast extract peptone dextrose (YEPD) medium were examined using phase-contrast and fluorescent microscopy, freeze-substitution, transmission and scanning electron microscopy, counted using a Bürker chamber and their absorbance measured. The cells responded to the presence of LA, an F-actin inhibitor, with the disappearance of actin patches, actin cables and actin rings. This resulted in the formation of larger spherical cells with irregular morphology in the cell walls and ultrastructural disorder of the cell organelles and secretory vesicles. Instead of buds, LA-inhibited cells formed only 'table-mountain-like' wide flattened swellings without apical growth with a thinner glucan cell-wall layer containing β-1,3-glucan microfibrils. The LA-inhibited cells lysed. Actin cables and patches were required for bud formation and bud growth. In addition, actin patches were required for the formation of β-1,3-glucan microfibrils in the bud cell wall. LA has fungistatic, fungicidal and fungilytic effects on the budding yeast Saccharomyces cerevisiae.

  9. The anillin-related region of Bud4 is the major functional determinant for Bud4's function in septin organization during bud growth and axial bud site selection in budding yeast.

    PubMed

    Wu, Huan; Guo, Jia; Zhou, Ya-Ting; Gao, Xiang-Dong

    2015-03-01

    The anillin-related protein Bud4 of Saccharomyces cerevisiae is required for axial bud site selection by linking the axial landmark to the septins, which localize at the mother bud neck. Recent studies indicate that Bud4 plays a role in septin organization during cytokinesis. Here we show that Bud4 is also involved in septin organization during bud growth prior to cytokinesis, as bud4Δ shs1Δ cells displayed an elongated bud morphology and defective septin organization at 18°C. Bud4 overexpression also affected septin organization during bud growth in shs1Δ cells at 30°C. Bud4 was previously thought to associate with the septins via its central region, while the C-terminal anillin-related region was not involved in septin association. Surprisingly, we found that the central region of Bud4 alone targets to the bud neck throughout the cell cycle, unlike full-length Bud4, which localizes to the bud neck only during G2/M phase. We identified the anillin-related region to be a second targeting domain that cooperates with the central region for proper septin association. In addition, the anillin-related region could largely mediate Bud4's function in septin organization during bud growth and bud site selection. We show that this region interacts with the C terminus of Bud3 and the two segments depend on each other for association with the septins. Moreover, like the bud4Δ mutant, the bud3Δ mutant genetically interacts with shs1Δ and cdc12-6 mutants in septin organization, suggesting that Bud4 and Bud3 may cooperate in septin organization during bud growth. These observations provide new insights into the interaction of Bud4 with the septins and Bud3.

  10. Replication origins and timing of temporal replication in budding yeast: how to solve the conundrum?

    PubMed

    Barberis, Matteo; Spiesser, Thomas W; Klipp, Edda

    2010-05-01

    Similarly to metazoans, the budding yeast Saccharomyces cereviasiae replicates its genome with a defined timing. In this organism, well-defined, site-specific origins, are efficient and fire in almost every round of DNA replication. However, this strategy is neither conserved in the fission yeast Saccharomyces pombe, nor in Xenopus or Drosophila embryos, nor in higher eukaryotes, in which DNA replication initiates asynchronously throughout S phase at random sites. Temporal and spatial controls can contribute to the timing of replication such as Cdk activity, origin localization, epigenetic status or gene expression. However, a debate is going on to answer the question how individual origins are selected to fire in budding yeast. Two opposing theories were proposed: the "replicon paradigm" or "temporal program" vs. the "stochastic firing". Recent data support the temporal regulation of origin activation, clustering origins into temporal blocks of early and late replication. Contrarily, strong evidences suggest that stochastic processes acting on origins can generate the observed kinetics of replication without requiring a temporal order. In mammalian cells, a spatiotemporal model that accounts for a partially deterministic and partially stochastic order of DNA replication has been proposed. Is this strategy the solution to reconcile the conundrum of having both organized replication timing and stochastic origin firing also for budding yeast? In this review we discuss this possibility in the light of our recent study on the origin activation, suggesting that there might be a stochastic component in the temporal activation of the replication origins, especially under perturbed conditions. PMID:21037857

  11. Proliferation enhancement of budding yeast and mammalian cells with periodic oxygen radical treatment

    NASA Astrophysics Data System (ADS)

    Mori, Yosuke; Kobayashi, Jun; Murata, Tomiyasu; Hahizume, Hiroshi; Hori, Masaru; Ito, Masafumi

    2015-09-01

    Recently, nonequilibrium atmospheric-pressure plasmas have been intensively studied for biological applications. However, the each effect of species in plasmas to biological tissue has not been clarified yet because various factors exist in the plasmas. Accordingly, we have studied effects of atomic oxygen dose on cell growth such as budding yeast and mouse NIH3T3 fibroblasts of mammalian cells. Both of cells were suspended with PBS, and treated using oxygen radical source. In order to prevent the radicals from reacting with the ambient air, the treatment region was surrounded by a plastic cover and purged with Ar. The proliferative effect of 15 % was observed at the O3Pj dose of around 1 . 0 ×1017 cm-3 in NIH3T3 cells as well as in yeast cells. Moreover, periodic oxygen treatment enhanced the effect in budding yeast cells. The best interval of periodic oxygen radical treatment was around 2 hours, which is almost the same period as that of their cell cycle. With the optimum interval time, we have investigated the effect of the number of the treatments. As the number of treatments increases, the growth rate of budding yeast cells was gradually enhanced and saturated at thrice treatments. This work was partly supported by JSPS KAKENHI Grant Numbers 26286072 and project for promoting Research Center in Meijo University.

  12. Tracking Diacylglycerol and Phosphatidic Acid Pools in Budding Yeast

    PubMed Central

    Ganesan, Suriakarthiga; Shabits, Brittney N.; Zaremberg, Vanina

    2015-01-01

    Phosphatidic acid (PA) and diacylglycerol (DAG) are key signaling molecules and important precursors for the biosynthesis of all glycerolipids found in eukaryotes. Research conducted in the model organism Saccharomyces cerevisiae has been at the forefront of the identification of the enzymes involved in the metabolism and transport of PA and DAG. Both these lipids can alter the local physical properties of membranes by introducing negative curvature, but the anionic nature of the phosphomonoester headgroup in PA sets it apart from DAG. As a result, the mechanisms underlying PA and DAG interaction with other lipids and proteins are notoriously different. This is apparent from the analysis of the protein domains responsible for recognition and binding to each of these lipids. We review the current evidence obtained using the PA-binding proteins and domains fused to fluorescent proteins for in vivo tracking of PA pools in yeast. In addition, we present original results for visualization of DAG pools in yeast using the C1 domain from mammalian PKCδ. An emerging first cellular map of the distribution of PA and DAG pools in actively growing yeast is discussed. PMID:27081314

  13. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism.

  14. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast.

    PubMed

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R; Drubin, David G

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin-Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  15. Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast

    PubMed Central

    Miao, Yansong; Han, Xuemei; Zheng, Liangzhen; Xie, Ying; Mu, Yuguang; Yates, John R.; Drubin, David G.

    2016-01-01

    Actin cables, composed of actin filament bundles nucleated by formins, mediate intracellular transport for cell polarity establishment and maintenance. We previously observed that metaphase cells preferentially promote actin cable assembly through cyclin-dependent kinase 1 (Cdk1) activity. However, the relevant metaphase Cdk1 targets were not known. Here we show that the highly conserved actin filament crosslinking protein fimbrin is a critical Cdk1 target for actin cable assembly regulation in budding yeast. Fimbrin is specifically phosphorylated on threonine 103 by the metaphase cyclin–Cdk1 complex, in vivo and in vitro. On the basis of conformational simulations, we suggest that this phosphorylation stabilizes fimbrin's N-terminal domain, and modulates actin filament binding to regulate actin cable assembly and stability in cells. Overall, this work identifies fimbrin as a key target for cell cycle regulation of actin cable assembly in budding yeast, and suggests an underlying mechanism. PMID:27068241

  16. A budding yeast's perspective on aging: The shape I'm in

    PubMed Central

    Smith, Jessica; Wright, Jill

    2015-01-01

    Aging is exemplified by progressive, deleterious changes that increase the probability of death. However, while the effects of age are easy to recognize, identification of the processes involved has proved to be much more difficult. Somewhat surprisingly, research using the budding yeast has had a profound impact on our current understanding of the mechanisms involved in aging. Herein, we examine the biological significance and implications surrounding the observation that genetic pathways involved in the modulation of aging and the determination of lifespan in yeast are highly complicated and conserved. PMID:25819684

  17. Septin Filament Formation is Essential in Budding Yeast

    PubMed Central

    McMurray, Michael A.; Bertin, Aurelie; Garcia, Galo; Lam, Lisa; Nogales, Eva; Thorner, Jeremy

    2011-01-01

    SUMMARY Septins are GTP-binding proteins that form ordered, rod-like multimeric complexes and polymerize into filaments, but how such supramolecular structure is related to septin function was unclear. In Saccharomyces cerevisiae, four septins form an apolar hetero-octamer (Cdc11–Cdc12–Cdc3–Cdc10–Cdc10–Cdc3–Cdc12–Cdc11) that associates end-to-end to form filaments. We show that septin filament assembly displays previously unanticipated plasticity. Cells lacking Cdc10 or Cdc11 are able to divide because the now-exposed subunits (Cdc3 or Cdc12, respectively) retain an ability to homodimerize via their so-called G interface, thereby allowing for filament assembly. In such cdc10Δ and cdc11Δ cells, the remaining septins, like wild-type complexes, localize to the cortex at the bud neck and compartmentalize non-septin factors, consistent with a diffusion barrier composed of continuous filaments in intimate contact with the plasma membrane. Conversely, Cdc10 or Cdc11 mutants that cannot self-associate, but “cap” Cdc3 or Cdc12, respectively, prevent filament formation, block cortical localization, and kill cells. PMID:21497764

  18. Epigenetic silencing mechanisms in budding yeast and fruit fly: different paths, same destinations.

    PubMed

    Pirrotta, Vincenzo; Gross, David S

    2005-05-13

    Transcriptional silencing in budding yeast and fruit fly is mediated by fundamentally unrelated proteins that assemble very different chromatin structures. Surprisingly, the repressive mechanisms evolved from these very different materials have similar features, including an epigenetic mode of inheritance and a block to transcription based on interference with the assembly or function of the promoter complex rather than with the binding of gene-specific activators. PMID:15893722

  19. Epigenetic silencing mechanisms in budding yeast and fruit fly: different paths, same destinations.

    PubMed

    Pirrotta, Vincenzo; Gross, David S

    2005-05-13

    Transcriptional silencing in budding yeast and fruit fly is mediated by fundamentally unrelated proteins that assemble very different chromatin structures. Surprisingly, the repressive mechanisms evolved from these very different materials have similar features, including an epigenetic mode of inheritance and a block to transcription based on interference with the assembly or function of the promoter complex rather than with the binding of gene-specific activators.

  20. Quantitative proteomic comparison of stationary/G0 phase cells and tetrads in budding yeast.

    PubMed

    Kumar, Ravinder; Srivastava, Sanjeeva

    2016-01-01

    Most of the microbial cells on earth under natural conditions exist in a dormant condition, commonly known as quiescent state. Quiescent cells exhibit low rates of transcription and translation suggesting that cellular abundance of proteins may be similar in quiescent cells. Therefore, this study aim to compare the proteome of budding yeast cells from two quiescent states viz. stationary phase/G0 and tetrads. Using iTRAQ (isobaric tag for relative and absolute quantitation) based quantitative proteomics we identified 289 proteins, among which around 40 proteins exhibited ±1.5 fold change consistently from the four biological replicates. Proteomics data was validated by western blot and denstiometric analysis of Hsp12 and Spg4. Level of budding yeast 14-3-3 proteins was found to be similar in both the quiescent states, whereas Hsp12 and Spg4 expressed only during stress. FACS (fluorescence-activated cell sorting) analysis showed that budding yeast cells were arrested at G1 stages both in tetrads as well as in stationary phase. We also observed that quiescent states did not express Ime1 (inducer of meiosis). Taken together, our present study demonstrates that the cells in quiescent state may have similar proteome, and accumulation of proteins like Hsp12, Hsp26, and Spg4 may play an important role in retaining viability of the cells during dormancy. PMID:27558777

  1. The Malleable Nature of the Budding Yeast Nuclear Envelope: Flares, Fusion, and Fenestrations.

    PubMed

    Meseroll, Rebecca A; Cohen-Fix, Orna

    2016-11-01

    In eukaryotes, the nuclear envelope (NE) physically separates nuclear components and activities from rest of the cell. The NE also provides rigidity to the nucleus and contributes to chromosome organization. At the same time, the NE is highly dynamic; it must change shape and rearrange its components during development and throughout the cell cycle, and its morphology can be altered in response to mutation and disease. Here we focus on the NE of budding yeast, Saccharomyces cerevisiae, which has several unique features: it remains intact throughout the cell cycle, expands symmetrically during interphase, elongates during mitosis and, expands asymmetrically during mitotic delay. Moreover, its NE is safely breached during mating and when large structures, such as nuclear pore complexes and the spindle pole body, are embedded into its double membrane. The budding yeast NE lacks lamins and yet the nucleus is capable of maintaining a spherical shape throughout interphase. Despite these eccentricities, studies of the budding yeast NE have uncovered interesting, and likely conserved, processes that contribute to NE dynamics. In particular, we discuss the processes that drive and enable NE expansion and the dramatic changes in the NE that lead to extensions and fenestrations. J. Cell. Physiol. 231: 2353-2360, 2016. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  2. Quantitative proteomic comparison of stationary/G0 phase cells and tetrads in budding yeast

    PubMed Central

    Kumar, Ravinder; Srivastava, Sanjeeva

    2016-01-01

    Most of the microbial cells on earth under natural conditions exist in a dormant condition, commonly known as quiescent state. Quiescent cells exhibit low rates of transcription and translation suggesting that cellular abundance of proteins may be similar in quiescent cells. Therefore, this study aim to compare the proteome of budding yeast cells from two quiescent states viz. stationary phase/G0 and tetrads. Using iTRAQ (isobaric tag for relative and absolute quantitation) based quantitative proteomics we identified 289 proteins, among which around 40 proteins exhibited ±1.5 fold change consistently from the four biological replicates. Proteomics data was validated by western blot and denstiometric analysis of Hsp12 and Spg4. Level of budding yeast 14-3-3 proteins was found to be similar in both the quiescent states, whereas Hsp12 and Spg4 expressed only during stress. FACS (fluorescence-activated cell sorting) analysis showed that budding yeast cells were arrested at G1 stages both in tetrads as well as in stationary phase. We also observed that quiescent states did not express Ime1 (inducer of meiosis). Taken together, our present study demonstrates that the cells in quiescent state may have similar proteome, and accumulation of proteins like Hsp12, Hsp26, and Spg4 may play an important role in retaining viability of the cells during dormancy. PMID:27558777

  3. Actin dynamics affect mitochondrial quality control and aging in budding yeast.

    PubMed

    Higuchi, Ryo; Vevea, Jason D; Swayne, Theresa C; Chojnowski, Robert; Hill, Vanessa; Boldogh, Istvan R; Pon, Liza A

    2013-12-01

    Actin cables of budding yeast are bundles of F-actin that extend from the bud tip or neck to the mother cell tip, serve as tracks for bidirectional cargo transport, and undergo continuous movement from buds toward mother cells [1]. This movement, retrograde actin cable flow (RACF), is similar to retrograde actin flow in lamellipodia, growth cones, immunological synapses, dendritic spines, and filopodia [2-5]. In all cases, actin flow is driven by the push of actin polymerization and assembly at the cell cortex, and myosin-driven pulling forces deeper within the cell [6-10]. Therefore, for movement and inheritance from mothers to buds, mitochondria must "swim upstream" against the opposing force of RACF [11]. We find that increasing RACF rates results in increased fitness of mitochondria inherited by buds and that the increase in mitochondrial fitness leads to extended replicative lifespan and increased cellular healthspan. The sirtuin SIR2 is required for normal RACF and mitochondrial fitness, and increasing RACF rates in sir2Δ cells increases mitochondrial fitness and cellular healthspan but does not affect replicative lifespan. These studies support the model that RACF serves as a filter for segregation of fit from less-fit mitochondria during inheritance, which controls cellular lifespan and healthspan. They also support a role for Sir2p in these processes.

  4. Ontogeny of Unstable Chromosomes Generated by Telomere Error in Budding Yeast

    PubMed Central

    Weinert, Ted

    2016-01-01

    DNA replication errors at certain sites in the genome initiate chromosome instability that ultimately leads to stable genomic rearrangements. Where instability begins is often unclear. And, early instability may form unstable chromosome intermediates whose transient nature also hinders mechanistic understanding. We report here a budding yeast model that reveals the genetic ontogeny of genome rearrangements, from initial replication error to unstable chromosome formation to their resolution. Remarkably, the initial error often arises in or near the telomere, and frequently forms unstable chromosomes. Early unstable chromosomes may then resolve to an internal "collection site" where a dicentric forms and resolves to an isochromosome (other outcomes are possible at each step). The initial telomere-proximal unstable chromosome is increased in mutants in telomerase subunits, Tel1, and even Rad9, with no known telomere-specific function. Defects in Tel1 and in Rrm3, a checkpoint protein kinase with a role in telomere maintenance and a DNA helicase, respectively, synergize dramatically to generate unstable chromosomes, further illustrating the consequence of replication error in the telomere. Collectively, our results suggest telomeric replication errors may be a common cause of seemingly unrelated genomic rearrangements located hundreds of kilobases away. PMID:27716774

  5. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    SciTech Connect

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G. ); Liskay, R.M. ); Heffron, F. )

    1991-08-30

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

  6. Force generation by endocytic actin patches in budding yeast.

    PubMed

    Carlsson, Anders E; Bayly, Philip V

    2014-04-15

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value.

  7. Force Generation by Endocytic Actin Patches in Budding Yeast

    PubMed Central

    Carlsson, Anders E.; Bayly, Philip V.

    2014-01-01

    Membrane deformation during endocytosis in yeast is driven by local, templated assembly of a sequence of proteins including polymerized actin and curvature-generating coat proteins such as clathrin. Actin polymerization is required for successful endocytosis, but it is not known by what mechanisms actin polymerization generates the required pulling forces. To address this issue, we develop a simulation method in which the actin network at the protein patch is modeled as an active gel. The deformation of the gel is treated using a finite-element approach. We explore the effects and interplay of three different types of force driving invagination: 1), forces perpendicular to the membrane, generated by differences between actin polymerization rates at the edge of the patch and those at the center; 2), the inherent curvature of the coat-protein layer; and 3), forces parallel to the membrane that buckle the coat protein layer, generated by an actomyosin contractile ring. We find that with optimistic estimates for the stall stress of actin gel growth and the shear modulus of the actin gel, actin polymerization can generate almost enough force to overcome the turgor pressure. In combination with the other mechanisms, actin polymerization can the force over the critical value. PMID:24739159

  8. Comparative 3D genome structure analysis of the fission and the budding yeast.

    PubMed

    Gong, Ke; Tjong, Harianto; Zhou, Xianghong Jasmine; Alber, Frank

    2015-01-01

    We studied the 3D structural organization of the fission yeast genome, which emerges from the tethering of heterochromatic regions in otherwise randomly configured chromosomes represented as flexible polymer chains in an nuclear environment. This model is sufficient to explain in a statistical manner many experimentally determined distinctive features of the fission yeast genome, including chromatin interaction patterns from Hi-C experiments and the co-locations of functionally related and co-expressed genes, such as genes expressed by Pol-III. Our findings demonstrate that some previously described structure-function correlations can be explained as a consequence of random chromatin collisions driven by a few geometric constraints (mainly due to centromere-SPB and telomere-NE tethering) combined with the specific gene locations in the chromosome sequence. We also performed a comparative analysis between the fission and budding yeast genome structures, for which we previously detected a similar organizing principle. However, due to the different chromosome sizes and numbers, substantial differences are observed in the 3D structural genome organization between the two species, most notably in the nuclear locations of orthologous genes, and the extent of nuclear territories for genes and chromosomes. However, despite those differences, remarkably, functional similarities are maintained, which is evident when comparing spatial clustering of functionally related genes in both yeasts. Functionally related genes show a similar spatial clustering behavior in both yeasts, even though their nuclear locations are largely different between the yeast species.

  9. Partial purification of histone H3 proteolytic activity from the budding yeast Saccharomyces cerevisiae.

    PubMed

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-06-01

    The proteolytic clipping of histone tails has recently emerged as a novel form of irreversible post-translational modification (PTM) of histones. Histone clipping has been implicated as a regulatory process leading to the permanent removal of PTMs from histone proteins. However, there is scarcity of literature that describes the identification and characterization of histone-specific proteases. Here, we employed various biochemical methods to report histone H3-specific proteolytic activity from budding yeast. Our results demonstrate that H3 proteolytic activity was associated with sepharose bead matrices and activity was not affected by a variety of stress conditions. We have also identified the existence of an unknown protein that acts as a physiological inhibitor of the H3-clipping activity of yeast H3 protease. Moreover, through protease inhibition assays, we have also characterized yeast H3 protease as a serine protease. Interestingly, unlike glutamate dehydrogenase (GDH), yeast H3 proteolytic activity was not inhibited by Stefin B. Together, our findings suggest the existence of a novel H3 protease in yeast that is different from other reported histone H3 proteases. The presence of histone H3 proteolytic activity, along with the physiological inhibitor in yeast, suggests an interesting molecular mechanism that regulates the activity of histone proteases. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Effect of temperature on replicative aging of the budding yeast Saccharomyces cerevisiae.

    PubMed

    Molon, Mateusz; Zadrag-Tecza, Renata

    2016-04-01

    The use of the budding yeast Saccharomyces cerevisiae in gerontological studies was based on the assumption that the reproduction limit of a single cell (replicative aging) is a consequence of accumulation of a hypothetical universal "senescence factor" within the mother cell. However, some evidence suggests that molecules or structures proposed as the "aging factor", such as rDNA circles, oxidatively damaged proteins (with carbonyl groups) or mitochondria, have little effect on replicative lifespan of yeast cells. Our results also suggest that protein aggregates associated with Hsp104, treated as a marker of yeast aging, do not seem to affect the numeric value of replicative lifespan of yeast. What these results indicate, however, is the need for finding a different way of expressing age and longevity of yeast cells instead of the commonly used number of daughters produced over units of time, as in the case of other organisms. In this paper, we show that the temperature has a stronger influence on the time of life (the total lifespan) than on the reproductive potential of yeast cells.

  11. A Mathematical Model of Mitotic Exit in Budding Yeast: The Role of Polo Kinase

    PubMed Central

    Hancioglu, Baris; Tyson, John J.

    2012-01-01

    Cell cycle progression in eukaryotes is regulated by periodic activation and inactivation of a family of cyclin–dependent kinases (Cdk's). Entry into mitosis requires phosphorylation of many proteins targeted by mitotic Cdk, and exit from mitosis requires proteolysis of mitotic cyclins and dephosphorylation of their targeted proteins. Mitotic exit in budding yeast is known to involve the interplay of mitotic kinases (Cdk and Polo kinases) and phosphatases (Cdc55/PP2A and Cdc14), as well as the action of the anaphase promoting complex (APC) in degrading specific proteins in anaphase and telophase. To understand the intricacies of this mechanism, we propose a mathematical model for the molecular events during mitotic exit in budding yeast. The model captures the dynamics of this network in wild-type yeast cells and 110 mutant strains. The model clarifies the roles of Polo-like kinase (Cdc5) in the Cdc14 early anaphase release pathway and in the G-protein regulated mitotic exit network. PMID:22383977

  12. Regulation of Budding Yeast CENP-A levels Prevents Misincorporation at Promoter Nucleosomes and Transcriptional Defects

    PubMed Central

    Hildebrand, Erica M.; Biggins, Sue

    2016-01-01

    The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-ACse4 is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-ACse4 for degradation. To identify additional mechanisms that prevent CENP-ACse4 misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-ACse4 in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-ACse4 is enriched at promoters that contain histone H2A.ZHtz1 nucleosomes, but that H2A.ZHtz1 is not required for CENP-ACse4 mislocalization. Instead, the INO80 complex, which removes H2A.ZHtz1 from nucleosomes, promotes the ectopic deposition of CENP-ACse4. Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-ACse4. The down-regulated genes are enriched for CENP-ACse4 mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation. PMID:26982580

  13. The plate-washing assay: a simple test for filamentous growth in budding yeast.

    PubMed

    Cullen, Paul J

    2015-02-01

    Filamentous growth is a foraging response that occurs in fungal species. It allows fungal pathogens to invade cells and tissues of a host organism. Budding yeast undergoes filamentous growth and can invade semisolid agar plates, penetrating the agar surface. These cells cannot be removed by rinsing with water and form an invasive scar. The plate-washing assay is an easy first test for filamentous growth and is performed at low cost with minimal reagents. The assay is versatile: It can be used as a teaching tool, is amenable to high-throughput genetic analysis, and is used to evaluate filamentous growth in different fungal species, including pathogens like Candida albicans.

  14. Global reorganization of budding yeast chromosome conformation in different physiological conditions

    PubMed Central

    Tjong, Harianto; Weider, Elodie; Herzog, Mareike; Young, Barry; Brune, Christiane; Müllner, Daniel; Loewen, Christopher; Alber, Frank; Weis, Karsten

    2016-01-01

    The organization of the genome is nonrandom and important for correct function. Specifically, the nuclear envelope plays a critical role in gene regulation. It generally constitutes a repressive environment, but several genes, including the GAL locus in budding yeast, are recruited to the nuclear periphery on activation. Here, we combine imaging and computational modeling to ask how the association of a single gene locus with the nuclear envelope influences the surrounding chromosome architecture. Systematic analysis of an entire yeast chromosome establishes that peripheral recruitment of the GAL locus is part of a large-scale rearrangement that shifts many chromosomal regions closer to the nuclear envelope. This process is likely caused by the presence of several independent anchoring points. To identify novel factors required for peripheral anchoring, we performed a genome-wide screen and demonstrated that the histone acetyltransferase SAGA and the activity of histone deacetylases are needed for this extensive gene recruitment to the nuclear periphery. PMID:26811423

  15. Separable Crossover-Promoting and Crossover-Constraining Aspects of Zip1 Activity during Budding Yeast Meiosis

    PubMed Central

    Voelkel-Meiman, Karen; Johnston, Cassandra; Thappeta, Yashna; Subramanian, Vijayalakshmi V.; Hochwagen, Andreas; MacQueen, Amy J.

    2015-01-01

    Accurate chromosome segregation during meiosis relies on the presence of crossover events distributed among all chromosomes. MutSγ and MutLγ homologs (Msh4/5 and Mlh1/3) facilitate the formation of a prominent group of meiotic crossovers that mature within the context of an elaborate chromosomal structure called the synaptonemal complex (SC). SC proteins are required for intermediate steps in the formation of MutSγ-MutLγ crossovers, but whether the assembled SC structure per se is required for MutSγ-MutLγ-dependent crossover recombination events is unknown. Here we describe an interspecies complementation experiment that reveals that the mature SC is dispensable for the formation of Mlh3-dependent crossovers in budding yeast. Zip1 forms a major structural component of the budding yeast SC, and is also required for MutSγ and MutLγ-dependent crossover formation. Kluyveromyces lactis ZIP1 expressed in place of Saccharomyces cerevisiae ZIP1 in S. cerevisiae cells fails to support SC assembly (synapsis) but promotes wild-type crossover levels in those nuclei that progress to form spores. While stable, full-length SC does not assemble in S. cerevisiae cells expressing K. lactis ZIP1, aggregates of K. lactis Zip1 displayed by S. cerevisiae meiotic nuclei are decorated with SC-associated proteins, and K. lactis Zip1 promotes the SUMOylation of the SC central element protein Ecm11, suggesting that K. lactis Zip1 functionally interfaces with components of the S. cerevisiae synapsis machinery. Moreover, K. lactis Zip1-mediated crossovers rely on S. cerevisiae synapsis initiation proteins Zip3, Zip4, Spo16, as well as the Mlh3 protein, as do the crossovers mediated by S. cerevisiae Zip1. Surprisingly, however, K. lactis Zip1-mediated crossovers are largely Msh4/Msh5 (MutSγ)-independent. This separation-of-function version of Zip1 thus reveals that neither assembled SC nor MutSγ is required for Mlh3-dependent crossover formation per se in budding yeast. Our data

  16. Interaction networks of prion, prionogenic and prion-like proteins in budding yeast, and their role in gene regulation.

    PubMed

    Harbi, Djamel; Harrison, Paul M

    2014-01-01

    Prions are transmissible, propagating alternative states of proteins. Prions in budding yeast propagate heritable phenotypes and can function in large-scale gene regulation, or in some cases occur as diseases of yeast. Other 'prionogenic' proteins are likely prions that have been determined experimentally to form amyloid in vivo, and to have prion-like domains that are able to propagate heritable states. Furthermore, there are over 300 additional 'prion-like' yeast proteins that have similar amino-acid composition to prions (primarily a bias for asparagines and glutamines). Here, we examine the protein functional and interaction networks that involve prion, prionogenic and prion-like proteins. Set against a marked overall preference for N/Q-rich prion-like proteins not to interact with each other, we observe a significant tendency of prion/prionogenic proteins to interact with other, N/Q-rich prion-like proteins. This tendency is mostly due to a small number of networks involving the proteins NUP100p, LSM4p and PUB1p. In general, different data analyses of functional and interaction networks converge to indicate a strong linkage of prionogenic and prion-like proteins, to stress-granule assembly and related biological processes. These results further elucidate how prions may impact gene regulation, and reveal a broader horizon for the functional relevance of N/Q-rich prion-like domains.

  17. Regulation of Mitotic Spindle Disassembly by an Environmental Stress-Sensing Pathway in Budding Yeast

    PubMed Central

    Pigula, Adrianne; Drubin, David G.; Barnes, Georjana

    2014-01-01

    Timely spindle disassembly is essential for coordination of mitotic exit with cytokinesis. In the budding yeast Saccharomyces cerevisiae, the microtubule-associated protein She1 functions in one of at least three parallel pathways that promote spindle disassembly. She1 phosphorylation by the Aurora kinase Ipl1 facilitates a role for She1 in late anaphase, when She1 contributes to microtubule depolymerization and shrinkage of spindle halves. By examining the genetic interactions of known spindle disassembly genes, we identified three genes in the environmental stress-sensing HOG (high-osmolarity glycerol response) pathway, SHO1, PBS2, and HOG1, and found they are necessary for proper localization of She1 to the anaphase spindle and for proper spindle disassembly. HOG pathway mutants exhibited spindle disassembly defects, as well as mislocalization of anillin-related proteins Boi1 and Boi2 from the bud neck. Moreover, Boi2, but not Boi1, plays a role in spindle disassembly that places Boi2 in a pathway with Sho1, Pbs2, and Hog1. Together, our data identify a process by which cells monitor events at the spindle and bud neck and describe a novel role for the HOG pathway in mitotic signaling. PMID:25213170

  18. Endoplasmic Reticulum Dynamics, Inheritance, and Cytoskeletal Interactions in Budding YeastV⃞

    PubMed Central

    Fehrenbacher, K. L.; Davis, D.; Wu, M.; Boldogh, I.; Pon, Liza A.

    2002-01-01

    The endoplasmic reticulum (ER) in Saccharomyces cerevisiae consists of a reticulum underlying the plasma membrane (cortical ER) and ER associated with the nuclear envelope (nuclear ER). We used a Sec63p-green fluorescent protein fusion protein to study motility events associated with inheritance of cortical ER and nuclear ER in living yeast cells. During M phase before nuclear migration, we observed thick, apparently rigid tubular extensions emanating from the nuclear ER that elongate, undergo sweeping motions along the cell cortex, and shorten. Two findings support a role for microtubules in this process. First, extension of tubular structures from the nuclear ER is inhibited by destabilization of microtubules. Second, astral microtubules, structures that undergo similar patterns of extension, cortical surveillance and retraction, colocalize with nuclear ER extensions. During S and G2 phases of the cell cycle, we observed anchorage of the cortical ER at the site of bud emergence and apical bud growth. Thin tubules of the ER that extend from the anchored cortical ER display undulating, apparently random movement and move into the bud as it grows. Finally, we found that cortical ER morphology is sensitive to a filamentous actin–destabilizing drug, latrunculin-A, and to mutations in the actin-encoding ACT1 gene. Our observations support 1) different mechanisms and cytoskeletal mediators for the inheritance of nuclear and cortical ER elements and 2) a mechanism for cortical ER inheritance that is cytoskeleton dependent but relies on anchorage, not directed movement. PMID:11907267

  19. Astral Microtubule Pivoting Promotes Their Search for Cortical Anchor Sites during Mitosis in Budding Yeast

    PubMed Central

    Baumgärtner, Stephan; Tolić, Iva M.

    2014-01-01

    Positioning of the mitotic spindle is crucial for proper cell division. In the budding yeast Saccharomyces cerevisiae, two mechanisms contribute to spindle positioning. In the Kar9 pathway, astral microtubules emanating from the daughter-bound spindle pole body interact via the linker protein Kar9 with the myosin Myo2, which moves the microtubule along the actin cables towards the neck. In the dynein pathway, astral microtubules off-load dynein onto the cortical anchor protein Num1, which is followed by dynein pulling on the spindle. Yet, the mechanism by which microtubules target cortical anchor sites is unknown. Here we quantify the pivoting motion of astral microtubules around the spindle pole bodies, which occurs during spindle translocation towards the neck and through the neck. We show that this pivoting is largely driven by the Kar9 pathway. The microtubules emanating from the daughter-bound spindle pole body pivot faster than those at the mother-bound spindle pole body. The Kar9 pathway reduces the time needed for an astral microtubule inside the daughter cell to start pulling on the spindle. Thus, we propose a new role for microtubule pivoting: By pivoting around the spindle pole body, microtubules explore the space laterally, which helps them search for cortical anchor sites in the context of spindle positioning in budding yeast. PMID:24721997

  20. Budding Yeast Silencing Complexes and Regulation of Sir2 Activity by Protein-Protein Interactions

    PubMed Central

    Tanny, Jason C.; Kirkpatrick, Donald S.; Gerber, Scott A.; Gygi, Steven P.; Moazed, Danesh

    2004-01-01

    Gene silencing in the budding yeast Saccharomyces cerevisiae requires the enzymatic activity of the Sir2 protein, a highly conserved NAD-dependent deacetylase. In order to study the activity of native Sir2, we purified and characterized two budding yeast Sir2 complexes: the Sir2/Sir4 complex, which mediates silencing at mating-type loci and at telomeres, and the RENT complex, which mediates silencing at the ribosomal DNA repeats. Analyses of the protein compositions of these complexes confirmed previously described interactions. We show that the assembly of Sir2 into native silencing complexes does not alter its selectivity for acetylated substrates, nor does it allow the deacetylation of nucleosomal histones. The inability of Sir2 complexes to deacetylate nucleosomes suggests that additional factors influence Sir2 activity in vivo. In contrast, Sir2 complexes show significant enhancement in their affinities for acetylated substrates and their sensitivities to the physiological inhibitor nicotinamide relative to recombinant Sir2. Reconstitution experiments showed that, for the Sir2/Sir4 complex, these differences stem from the physical interaction of Sir2 with Sir4. Finally, we provide evidence that the different nicotinamide sensitivities of Sir2/Sir4 and RENT in vitro could contribute to locus-specific differences in how Sir2 activity is regulated in vivo. PMID:15282295

  1. An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Du, Zhiqiang; Valtierra, Stephanie; Li, Liming

    2014-01-01

    The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI(+)]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI(+)] and [PIN(+)] ([RNQ(+)]) (Genetics, Vol. 197, 685-700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI(+)] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ(+)] or [SWI(+)]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI(+)] prion stabilizes. Our finding provides strong evidence supporting the "cross-seeding" model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.

  2. A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

    PubMed

    Vo, Tommy V; Das, Jishnu; Meyer, Michael J; Cordero, Nicolas A; Akturk, Nurten; Wei, Xiaomu; Fair, Benjamin J; Degatano, Andrew G; Fragoza, Robert; Liu, Lisa G; Matsuyama, Akihisa; Trickey, Michelle; Horibata, Sachi; Grimson, Andrew; Yamano, Hiroyuki; Yoshida, Minoru; Roth, Frederick P; Pleiss, Jeffrey A; Xia, Yu; Yu, Haiyuan

    2016-01-14

    Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution.

  3. A Proteome-wide Fission Yeast Interactome Reveals Network Evolution Principles from Yeasts to Human.

    PubMed

    Vo, Tommy V; Das, Jishnu; Meyer, Michael J; Cordero, Nicolas A; Akturk, Nurten; Wei, Xiaomu; Fair, Benjamin J; Degatano, Andrew G; Fragoza, Robert; Liu, Lisa G; Matsuyama, Akihisa; Trickey, Michelle; Horibata, Sachi; Grimson, Andrew; Yamano, Hiroyuki; Yoshida, Minoru; Roth, Frederick P; Pleiss, Jeffrey A; Xia, Yu; Yu, Haiyuan

    2016-01-14

    Here, we present FissionNet, a proteome-wide binary protein interactome for S. pombe, comprising 2,278 high-quality interactions, of which ∼ 50% were previously not reported in any species. FissionNet unravels previously unreported interactions implicated in processes such as gene silencing and pre-mRNA splicing. We developed a rigorous network comparison framework that accounts for assay sensitivity and specificity, revealing extensive species-specific network rewiring between fission yeast, budding yeast, and human. Surprisingly, although genes are better conserved between the yeasts, S. pombe interactions are significantly better conserved in human than in S. cerevisiae. Our framework also reveals that different modes of gene duplication influence the extent to which paralogous proteins are functionally repurposed. Finally, cross-species interactome mapping demonstrates that coevolution of interacting proteins is remarkably prevalent, a result with important implications for studying human disease in model organisms. Overall, FissionNet is a valuable resource for understanding protein functions and their evolution. PMID:26771498

  4. Electron Tomography Reveals the Steps in Filovirus Budding

    PubMed Central

    Welsch, Sonja; Kolesnikova, Larissa; Krähling, Verena; Riches, James D.; Becker, Stephan; Briggs, John A. G.

    2010-01-01

    The filoviruses, Marburg and Ebola, are non-segmented negative-strand RNA viruses causing severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. The sequence of events that leads to release of filovirus particles from cells is poorly understood. Two contrasting mechanisms have been proposed, one proceeding via a “submarine-like” budding with the helical nucleocapsid emerging parallel to the plasma membrane, and the other via perpendicular “rocket-like” protrusion. Here we have infected cells with Marburg virus under BSL-4 containment conditions, and reconstructed the sequence of steps in the budding process in three dimensions using electron tomography of plastic-embedded cells. We find that highly infectious filamentous particles are released at early stages in infection. Budding proceeds via lateral association of intracellular nucleocapsid along its whole length with the plasma membrane, followed by rapid envelopment initiated at one end of the nucleocapsid, leading to a protruding intermediate. Scission results in local membrane instability at the rear of the virus. After prolonged infection, increased vesiculation of the plasma membrane correlates with changes in shape and infectivity of released viruses. Our observations demonstrate a cellular determinant of virus shape. They reconcile the contrasting models of filovirus budding and allow us to describe the sequence of events taking place during budding and release of Marburg virus. We propose that this represents a general sequence of events also followed by other filamentous and rod-shaped viruses. PMID:20442788

  5. Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

    PubMed

    Trabold, Peter A; Weinberger, Martin; Feng, Li; Burhans, William C

    2005-04-01

    Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA. PMID:15647270

  6. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  7. Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae.

    PubMed

    Ikushiro, Shinichi; Nishikawa, Miyu; Masuyama, Yuuka; Shouji, Tadashi; Fujii, Miharu; Hamada, Masahiro; Nakajima, Noriyuki; Finel, Moshe; Yasuda, Kaori; Kamakura, Masaki; Sakaki, Toshiyuki

    2016-07-01

    Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides. PMID:27241161

  8. Identification of the Molecular Mechanisms for Cell-Fate Selection in Budding Yeast through Mathematical Modeling

    PubMed Central

    Li, Yongkai; Yi, Ming; Zou, Xiufen

    2013-01-01

    The specification and maintenance of cell fates is essential to the development of multicellular organisms. However, the precise molecular mechanisms in cell fate selection are, to our knowledge, poorly understood due to the complexity of multiple interconnected pathways. In this study, model-based quantitative analysis is used to explore how to maintain distinguished cell fates between cell-cycle commitment and mating arrest in budding yeast. We develop a full mathematical model of an interlinked regulatory network based on the available experimental data. By theoretically defining the Start transition point, the model is able to reproduce many experimental observations of the dynamical behaviors in wild-type cells as well as in Ste5-8A and Far1-S87A mutants. Furthermore, we demonstrate that a moderate ratio between Cln1/2→Far1 inhibition and Cln1/2→Ste5 inhibition is required to ensure a successful switch between different cell fates. We also show that the different ratios of the mutual Cln1/2 and Far1 inhibition determine the different cell fates. In addition, based on a new, definition of network entropy, we find that the Start point in wild-type cells coincides with the system’s point of maximum entropy. This result indicates that Start is a transition point in the network entropy. Therefore, we theoretically explain the Start point from a network dynamics standpoint. Moreover, we analyze the biological bistablity of our model through bifurcation analysis. We find that the Cln1/2 and Cln3 production rates and the nonlinearity of SBF regulation on Cln1/2 production are potential determinants for irreversible entry into a new cell fate. Finally, the quantitative computations further reveal that high specificity and fidelity of the cell-cycle and mating pathways can guarantee specific cell-fate selection. These findings show that quantitative analysis and simulations with a mathematical model are useful tools for understanding the molecular mechanisms in

  9. Reinventing Heterochromatin in Budding Yeasts: Sir2 and the Origin Recognition Complex Take Center Stage ▿

    PubMed Central

    Hickman, Meleah A.; Froyd, Cara A.; Rusche, Laura N.

    2011-01-01

    The transcriptional silencing of the cryptic mating-type loci in Saccharomyces cerevisiae is one of the best-studied models of repressive heterochromatin. However, this type of heterochromatin, which is mediated by the Sir proteins, has a distinct molecular composition compared to the more ubiquitous type of heterochromatin found in Schizosaccharomyces pombe, other fungi, animals, and plants and characterized by the presence of HP1 (heterochromatin protein 1). This review discusses how the loss of important heterochromatin proteins, including HP1, in the budding yeast lineage presented an evolutionary opportunity for the development and diversification of alternative varieties of heterochromatin, in which the conserved deacetylase Sir2 and the replication protein Orc1 play key roles. In addition, we highlight how this diversification has been facilitated by gene duplications and has contributed to adaptations in lifestyle. PMID:21764908

  10. Assessment of the Biological Pathways Targeted by Isocyanate Using N-Succinimidyl N-Methylcarbamate in Budding Yeast Saccharomyces cerevisiae

    PubMed Central

    Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S.

    2014-01-01

    Isocyanates, a group of low molecular weight aromatic and aliphatic compounds possesses the functional isocyanate group. They are highly toxic in nature hence; we used N-succinimidyl N-methylcarbamate (NSNM), a surrogate chemical containing a functional isocyanate group to understand the mode of action of this class of compounds. We employed budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by NSNM. Our screening with yeast mutants revealed that it affects chromatin, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair processes. We also show that NSNM acts as an epigenetic modifier as its treatment causes reduction in global histone acetylation and formation of histone adducts. Cells treated with NSNM exhibited increase in mitochondrial membrane potential as well as intracellular ROS levels and the effects were rescued by addition of reduced glutathione to the medium. We also report that deletion of SOD1 and SOD2, the superoxide dismutase in Saccharomyces cerevisiae displayed hypersensitivity to NSNM. Furthermore, NSNM treatment causes rapid depletion of total glutathione and reduced glutathione. We also demonstrated that NSNM induces degradation of Sml1, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. In summary, we define the various biological pathways targeted by isocyanates. PMID:24664350

  11. Assessment of the biological pathways targeted by isocyanate using N-succinimidyl N-methylcarbamate in budding yeast Saccharomyces cerevisiae.

    PubMed

    Azad, Gajendra Kumar; Singh, Vikash; Tomar, Raghuvir S

    2014-01-01

    Isocyanates, a group of low molecular weight aromatic and aliphatic compounds possesses the functional isocyanate group. They are highly toxic in nature hence; we used N-succinimidyl N-methylcarbamate (NSNM), a surrogate chemical containing a functional isocyanate group to understand the mode of action of this class of compounds. We employed budding yeast Saccharomyces cerevisiae as a model organism to study the pathways targeted by NSNM. Our screening with yeast mutants revealed that it affects chromatin, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair processes. We also show that NSNM acts as an epigenetic modifier as its treatment causes reduction in global histone acetylation and formation of histone adducts. Cells treated with NSNM exhibited increase in mitochondrial membrane potential as well as intracellular ROS levels and the effects were rescued by addition of reduced glutathione to the medium. We also report that deletion of SOD1 and SOD2, the superoxide dismutase in Saccharomyces cerevisiae displayed hypersensitivity to NSNM. Furthermore, NSNM treatment causes rapid depletion of total glutathione and reduced glutathione. We also demonstrated that NSNM induces degradation of Sml1, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. In summary, we define the various biological pathways targeted by isocyanates.

  12. Uncoupling of 3'-phosphatase and 5'-kinase functions in budding yeast. Characterization of Saccharomyces cerevisiae DNA 3'-phosphatase (TPP1).

    PubMed

    Vance, J R; Wilson, T E

    2001-05-01

    Polynucleotide kinase is a bifunctional enzyme containing both DNA 3'-phosphatase and 5'-kinase activities seemingly suited to the coupled repair of single-strand nicks in which the phosphate has remained with the 3'-base. We show that the yeast Saccharomyces cerevisiae is able to repair transformed dephosphorylated linear plasmids by non-homologous end joining with considerable efficiency independently of the end-processing polymerase Pol4p. Homology searches and biochemical assays did not reveal a 5'-kinase that would account for this repair, however. Instead, open reading frame YMR156C (here named TPP1) is shown to encode only a polynucleotide kinase-type 3'-phosphatase. Tpp1p bears extensive similarity to the ancient L-2-halo-acid dehalogenase and DDDD phosphohydrolase superfamilies, but is specific for double-stranded DNA. It is present at high levels in cell extracts in a functional form and so does not represent a pseudogene. Moreover, the phosphatase-only nature of this gene is shared by Saccharomyces mikatae YMR156C and Arabidopsis thaliana K15M2.3. Repair of 3'-phosphate and 5'-hydroxyl lesions is thus uncoupled in budding yeast as compared with metazoans. Repair of transformed dephosphorylated plasmids, and 5'-hydroxyl blocking lesions more generally, likely proceeds by a cycle of base removal and resynthesis.

  13. Dilution of the cell cycle inhibitor Whi5 controls budding-yeast cell size.

    PubMed

    Schmoller, Kurt M; Turner, J J; Kõivomägi, M; Skotheim, Jan M

    2015-10-01

    Cell size fundamentally affects all biosynthetic processes by determining the scale of organelles and influencing surface transport. Although extensive studies have identified many mutations affecting cell size, the molecular mechanisms underlying size control have remained elusive. In the budding yeast Saccharomyces cerevisiae, size control occurs in G1 phase before Start, the point of irreversible commitment to cell division. It was previously thought that activity of the G1 cyclin Cln3 increased with cell size to trigger Start by initiating the inhibition of the transcriptional inhibitor Whi5 (refs 6-8). Here we show that although Cln3 concentration does modulate the rate at which cells pass Start, its synthesis increases in proportion to cell size so that its total concentration is nearly constant during pre-Start G1. Rather than increasing Cln3 activity, we identify decreasing Whi5 activity--due to the dilution of Whi5 by cell growth--as a molecular mechanism through which cell size controls proliferation. Whi5 is synthesized in S/G2/M phases of the cell cycle in a largely size-independent manner. This results in smaller daughter cells being born with higher Whi5 concentrations that extend their pre-Start G1 phase. Thus, at its most fundamental level, size control in budding yeast results from the differential scaling of Cln3 and Whi5 synthesis rates with cell size. More generally, our work shows that differential size-dependency of protein synthesis can provide an elegant mechanism to coordinate cellular functions with growth. PMID:26390151

  14. Unconventional genomic architecture in the budding yeast saccharomyces cerevisiae masks the nested antisense gene NAG1.

    PubMed

    Ma, Jun; Dobry, Craig J; Krysan, Damian J; Kumar, Anuj

    2008-08-01

    The genomic architecture of the budding yeast Saccharomyces cerevisiae is typical of other eukaryotes in that genes are spatially organized into discrete and nonoverlapping units. Inherent in this organizational model is the assumption that protein-coding sequences do not overlap completely. Here, we present evidence to the contrary, defining a previously overlooked yeast gene, NAG1 (for nested antisense gene) nested entirely within the coding sequence of the YGR031W open reading frame in an antisense orientation on the opposite strand. NAG1 encodes a 19-kDa protein, detected by Western blotting of hemagglutinin (HA)-tagged Nag1p with anti-HA antibodies and by beta-galactosidase analysis of a NAG1-lacZ fusion. NAG1 is evolutionarily conserved as a unit with YGR031W in bacteria and fungi. Unlike the YGR031WP protein product, however, which localizes to the mitochondria, Nag1p localizes to the cell periphery, exhibiting properties consistent with those of a plasma membrane protein. Phenotypic analysis of a site-directed mutant (nag1-1) disruptive for NAG1 but silent with respect to YGR031W, defines a role for NAG1 in yeast cell wall biogenesis; microarray profiling of nag1-1 indicates decreased expression of genes contributing to cell wall organization, and the nag1-1 mutant is hypersensitive to the cell wall-perturbing agent calcofluor white. Furthermore, production of Nag1p is dependent upon the presence of the cell wall integrity pathway mitogen-activated protein kinase Slt2p and its downstream transcription factor Rlm1p. Thus, NAG1 is important for two reasons. First, it contributes to yeast cell wall biogenesis. Second, its genomic context is novel, raising the possibility that other nested protein-coding genes may exist in eukaryotic genomes.

  15. Single-mRNA counting using fluorescent in situ hybridization in budding yeast

    PubMed Central

    Trcek, Tatjana; Chao, Jeffrey A; Larson, Daniel R; Park, Hye Yoon; Zenklusen, Daniel; Shenoy, Shailesh M; Singer, Robert H

    2014-01-01

    Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d. PMID:22301778

  16. Construction, Verification and Experimental Use of Two Epitope-Tagged Collections of Budding Yeast Strains

    PubMed Central

    Howson, Russell; Huh, Won-Ki; Ghaemmaghami, Sina; Falvo, James V.; Bower, Kiowa; Belle, Archana; Dephoure, Noah; Wykoff, Dennis D.; Weissman, Jonathan S.

    2005-01-01

    A major challenge in the post-genomic era is the development of experimental approaches to monitor the properties of proteins on a proteome-wide level. It would be particularly useful to systematically assay protein subcellular localization, post-translational modifications and protein–protein interactions, both at steady state and in response to environmental stimuli. Development of new reagents and methods will enhance our ability to do so efficiently and systematically. Here we describe the construction of two collections of budding yeast strains that facilitate proteome-wide measurements of protein properties. These collections consist of strains with an epitope tag integrated at the C-terminus of essentially every open reading frame (ORF), one with the tandem affinity purification (TAP) tag, and one with the green fluorescent protein (GFP) tag. We show that in both of these collections we have accurately tagged a high proportion of all ORFs (approximately 75% of the proteome) by confirming expression of the fusion proteins. Furthermore, we demonstrate the use of the TAP collection in performing high-throughput immunoprecipitation experiments. Building on these collections and the methods described in this paper, we hope that the yeast community will expand both the quantity and type of proteome level data available. PMID:18629296

  17. Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

    SciTech Connect

    Choi, Joon-Seok; Lee, Cheol-Koo

    2013-09-13

    Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.

  18. Ndc10 is a platform for inner kinetochore assembly in budding yeast.

    PubMed

    Cho, Uhn-Soo; Harrison, Stephen C

    2012-01-01

    Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1-Ctf13-(Cep3)(2)-(Ndc10)(2) recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 Å resolution of domains I-II (residues 1-402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetochore assembly. PMID:22139014

  19. Ndc10 is a platform for inner kinetochore assembly in budding yeast

    SciTech Connect

    Cho, Uhn-Soo; Harrison, Stephen C.

    2012-01-10

    Kinetochores link centromeric DNA to spindle microtubules and ensure faithful chromosome segregation during mitosis. In point-centromere yeasts, the CBF3 complex Skp1-Ctf13-(Cep3){sub 2}-(Ndc10){sub 2} recognizes a conserved centromeric DNA element through contacts made by Cep3 and Ndc10. We describe here the five-domain organization of Kluyveromyces lactis Ndc10 and the structure at 2.8 {angstrom} resolution of domains I-II (residues 1-402) bound to DNA. The structure resembles tyrosine DNA recombinases, although it lacks both endonuclease and ligase activities. Structural and biochemical data demonstrate that each subunit of the Ndc10 dimer binds a separate fragment of DNA, suggesting that Ndc10 stabilizes a DNA loop at the centromere. We describe in vitro association experiments showing that specific domains of Ndc10 interact with each of the known inner-kinetochore proteins or protein complexes in budding yeast. We propose that Ndc10 provides a central platform for inner-kinetochore assembly.

  20. The Ty1 LTR-retrotransposon of budding yeast, Saccharomyces cerevisiae

    PubMed Central

    Curcio, M. Joan; Lutz, Sheila; Lesage, Pascale

    2015-01-01

    Summary Long-terminal repeat (LTR)-retrotransposons generate a copy of their DNA (cDNA) by reverse transcription of their RNA genome in cytoplasmic nucleocapsids. They are widespread in the eukaryotic kingdom and are the evolutionary progenitors of retroviruses [1]. The Ty1 element of the budding yeast Saccharomyces cerevisiae was the first LTR-retrotransposon demonstrated to mobilize through an RNA intermediate, and not surprisingly, is the best studied. The depth of our knowledge of Ty1 biology stems not only from the predominance of active Ty1 elements in the S. cerevisiae genome but also the ease and breadth of genomic, biochemical and cell biology approaches available to study cellular processes in yeast. This review describes the basic structure of Ty1 and its gene products, the replication cycle, the rapidly expanding compendium of host co-factors known to influence retrotransposition and the nature of Ty1's elaborate symbiosis with its host. Our goal is to illuminate the value of Ty1 as a paradigm to explore the biology of LTR-retrotransposons in multicellular organisms, where the low frequency of retrotransposition events presents a formidable barrier to investigations of retrotransposon biology. PMID:25893143

  1. Computational Analysis of Reciprocal Association of Metabolism and Epigenetics in the Budding Yeast: A Genome-Scale Metabolic Model (GSMM) Approach

    PubMed Central

    Salehzadeh-Yazdi, Ali; Asgari, Yazdan; Saboury, Ali Akbar; Masoudi-Nejad, Ali

    2014-01-01

    Metaboloepigenetics is a newly coined term in biological sciences that investigates the crosstalk between epigenetic modifications and metabolism. The reciprocal relation between biochemical transformations and gene expression regulation has been experimentally demonstrated in cancers and metabolic syndromes. In this study, we explored the metabolism-histone modifications crosstalk by topological analysis and constraint-based modeling approaches in the budding yeast. We constructed nine models through the integration of gene expression data of four mutated histone tails into a genome-scale metabolic model of yeast. Accordingly, we defined the centrality indices of the lowly expressed enzymes in the undirected enzyme-centric network of yeast by CytoHubba plug-in in Cytoscape. To determine the global effects of histone modifications on the yeast metabolism, the growth rate and the range of possible flux values of reactions, we used constraint-based modeling approach. Centrality analysis shows that the lowly expressed enzymes could affect and control the yeast metabolic network. Besides, constraint-based modeling results are in a good agreement with the experimental findings, confirming that the mutations in histone tails lead to non-lethal alterations in the yeast, but have diverse effects on the growth rate and reveal the functional redundancy. PMID:25365344

  2. Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion.

    PubMed Central

    Skibbens, Robert V

    2004-01-01

    From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sister-chromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated. PMID:15020404

  3. The glyoxylate shunt is essential for desiccation tolerance in C. elegans and budding yeast

    PubMed Central

    Erkut, Cihan; Gade, Vamshidhar R; Laxman, Sunil; Kurzchalia, Teymuras V

    2016-01-01

    Many organisms, including species from all kingdoms of life, can survive desiccation by entering a state with no detectable metabolism. To survive, C. elegans dauer larvae and stationary phase S. cerevisiae require elevated amounts of the disaccharide trehalose. We found that dauer larvae and stationary phase yeast switched into a gluconeogenic mode in which metabolism was reoriented toward production of sugars from non-carbohydrate sources. This mode depended on full activity of the glyoxylate shunt (GS), which enables synthesis of trehalose from acetate. The GS was especially critical during preparation of worms for harsh desiccation (preconditioning) and during the entry of yeast into stationary phase. Loss of the GS dramatically decreased desiccation tolerance in both organisms. Our results reveal a novel physiological role for the GS and elucidate a conserved metabolic rewiring that confers desiccation tolerance on organisms as diverse as worm and yeast. DOI: http://dx.doi.org/10.7554/eLife.13614.001 PMID:27090086

  4. A Role for the Budding Yeast Separase, Esp1, in Ty1 Element Retrotransposition

    PubMed Central

    Cheung, Stephanie; Manhas, Savrina; Fang, Nancy; Wang, Kaiqian; Young, Barry; Loewen, Christopher; Mayor, Thibault; Measday, Vivien

    2015-01-01

    Separase/Esp1 is a protease required at the onset of anaphase to cleave cohesin and thereby enable sister chromatid separation. Esp1 also promotes release of the Cdc14 phosphatase from the nucleolus to enable mitotic exit. To uncover other potential roles for separase, we performed two complementary genome-wide genetic interaction screens with a strain carrying the budding yeast esp1-1 separase mutation. We identified 161 genes that when mutated aggravate esp1-1 growth and 44 genes that upon increased dosage are detrimental to esp1-1 viability. In addition to the expected cell cycle and sister chromatid segregation genes that were identified, 24% of the genes identified in the esp1-1 genetic screens have a role in Ty1 element retrotransposition. Retrotransposons, like retroviruses, replicate through reverse transcription of an mRNA intermediate and the resultant cDNA product is integrated into the genome by a conserved transposon or retrovirus encoded integrase protein. We purified Esp1 from yeast and identified an interaction between Esp1 and Ty1 integrase using mass spectrometry that was subsequently confirmed by co-immunoprecipitation analysis. Ty1 transposon mobility and insertion upstream of the SUF16 tRNA gene are both reduced in an esp1-1 strain but increased in cohesin mutant strains. Securin/Pds1, which is required for efficient localization of Esp1 to the nucleus, is also required for efficient Ty1 transposition. We propose that Esp1 serves two roles to mediate Ty1 transposition – one to remove cohesin and the second to target Ty1-IN to chromatin. PMID:25822502

  5. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    PubMed Central

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  6. Sequestration of mRNAs Modulates the Timing of Translation during Meiosis in Budding Yeast

    PubMed Central

    Jin, Liang; Zhang, Kai; Xu, Yifeng; Sternglanz, Rolf

    2015-01-01

    Starvation of diploid cells of the budding yeast Saccharomyces cerevisiae induces them to enter meiosis and differentiate into haploid spores. During meiosis, the precise timing of gene expression is controlled at the level of transcription, and also translation. If cells are returned to rich medium after they have committed to meiosis, the transcript levels of most meiotically upregulated genes decrease rapidly. However, for a subset of transcripts whose translation is delayed until the end of meiosis II, termed protected transcripts, the transcript levels remain stable even after nutrients are reintroduced. The Ime2-Rim4 regulatory circuit controls both the delayed translation and the stability of protected transcripts. These protected mRNAs localize in discrete foci, which are not seen for transcripts of genes with different translational timing and are regulated by Ime2. These results suggest that Ime2 and Rim4 broadly regulate translational delay but that additional factors, such as mRNA localization, modulate this delay to tune the timing of gene expression to developmental transitions during sporulation. PMID:26217015

  7. The budding yeast Msh4 protein functions in chromosome synapsis and the regulation of crossover distribution.

    PubMed Central

    Novak, J E; Ross-Macdonald, P B; Roeder, G S

    2001-01-01

    The budding yeast MSH4 gene encodes a MutS homolog produced specifically in meiotic cells. Msh4 is not required for meiotic mismatch repair or gene conversion, but it is required for wild-type levels of crossing over. Here, we show that a msh4 null mutation substantially decreases crossover interference. With respect to the defect in interference and the level of crossing over, msh4 is similar to the zip1 mutant, which lacks a structural component of the synaptonemal complex (SC). Furthermore, epistasis tests indicate that msh4 and zip1 affect the same subset of meiotic crossovers. In the msh4 mutant, SC formation is delayed compared to wild type, and full synapsis is achieved in only about half of all nuclei. The simultaneous defects in synapsis and interference observed in msh4 (and also zip1 and ndj1/tam1) suggest a role for the SC in mediating interference. The Msh4 protein localizes to discrete foci on meiotic chromosomes and colocalizes with Zip2, a protein involved in the initiation of chromosome synapsis. Both Zip2 and Zip1 are required for the normal localization of Msh4 to chromosomes, raising the possibility that the zip1 and zip2 defects in crossing over are indirect, resulting from the failure to localize Msh4 properly. PMID:11454751

  8. Genes involved in sister chromatid separation and segregation in the budding yeast Saccharomyces cerevisiae.

    PubMed Central

    Biggins, S; Bhalla, N; Chang, A; Smith, D L; Murray, A W

    2001-01-01

    Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair alpha-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor. PMID:11606525

  9. Integrative Analysis of Cell Cycle Control in Budding YeastD⃞

    PubMed Central

    Chen, Katherine C.; Calzone, Laurence; Csikasz-Nagy, Attila; Cross, Frederick R.; Novak, Bela; Tyson, John J.

    2004-01-01

    The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo. PMID:15169868

  10. Polygenic Molecular Architecture Underlying Non-Sexual Cell Aggregation in Budding Yeast

    PubMed Central

    Li, Jiarui; Wang, Lin; Wu, Xiaoping; Fang, Ou; Wang, Luwen; Lu, Chenqi; Yang, Shengjie; Hu, Xiaohua; Luo, Zewei

    2013-01-01

    Cell aggregation in unicellular organisms, induced by either cell non-sexual adhesion to yield flocs and biofilm, or pheromone-driving sexual conjugation is of great significance in cellular stress response, medicine, and brewing industries. Most current literatures have focused on one form of cell aggregation termed flocculation and its major molecular determinants, the flocculation (FLO) family genes. Here, we implemented a map-based approach for dissecting the molecular basis of non-sexual cell aggregation in Saccharomyces cerevisiae. Genome-wide mapping has identified four major quantitative trait loci (QTL) underlying nature variation in the cell aggregation phenotype. High-resolution mapping following up with knockout and allele replacement experiments resolved the QTL into the underlying genes (AMN1, RGA1, FLO1, and FLO8) or even into the causative nucleotide. Genetic variation in the QTL genes can explain up to 46% of phenotypic variation of this trait. Of these genes, AMN1 plays the leading role, differing from the FLO family members, in regulating expression of cell clumping phenotype through inducing cell segregation defect. These findings provide novel insights into the molecular mechanism of how cell aggregation is regulated in budding yeast, and the data will be directly implicated to understand the molecular basis and evolutionary implications of cell aggregation in other fungus species. PMID:23284084

  11. Sequestration of mRNAs Modulates the Timing of Translation during Meiosis in Budding Yeast.

    PubMed

    Jin, Liang; Zhang, Kai; Xu, Yifeng; Sternglanz, Rolf; Neiman, Aaron M

    2015-10-01

    Starvation of diploid cells of the budding yeast Saccharomyces cerevisiae induces them to enter meiosis and differentiate into haploid spores. During meiosis, the precise timing of gene expression is controlled at the level of transcription, and also translation. If cells are returned to rich medium after they have committed to meiosis, the transcript levels of most meiotically upregulated genes decrease rapidly. However, for a subset of transcripts whose translation is delayed until the end of meiosis II, termed protected transcripts, the transcript levels remain stable even after nutrients are reintroduced. The Ime2-Rim4 regulatory circuit controls both the delayed translation and the stability of protected transcripts. These protected mRNAs localize in discrete foci, which are not seen for transcripts of genes with different translational timing and are regulated by Ime2. These results suggest that Ime2 and Rim4 broadly regulate translational delay but that additional factors, such as mRNA localization, modulate this delay to tune the timing of gene expression to developmental transitions during sporulation.

  12. Lipid droplets maintain lipid homeostasis during anaphase for efficient cell separation in budding yeast

    PubMed Central

    Yang, Po-Lin; Hsu, Tzu-Han; Wang, Chao-Wen; Chen, Rey-Huei

    2016-01-01

    The neutral lipids steryl ester and triacylglycerol (TAG) are stored in the membrane-bound organelle lipid droplet (LD) in essentially all eukaryotic cells. It is unclear what physiological conditions require the mobilization or storage of these lipids. Here, we study the budding yeast mutant are1Δ are2Δ dga1Δ lro1Δ, which cannot synthesize the neutral lipids and therefore lacks LDs. This quadruple mutant is delayed at cell separation upon release from mitotic arrest. The cells have abnormal septa, unstable septin assembly during cytokinesis, and prolonged exocytosis at the division site at the end of cytokinesis. Lipidomic analysis shows a marked increase of diacylglycerol (DAG) and phosphatidic acid, the precursors for TAG, in the mutant during mitotic exit. The cytokinesis and separation defects are rescued by adding phospholipid precursors or inhibiting fatty acid synthesis, which both reduce DAG levels. Our results suggest that converting excess lipids to neutral lipids for storage during mitotic exit is important for proper execution of cytokinesis and efficient cell separation. PMID:27307588

  13. Genetic evidence for a role of phospholipase C at the budding yeast kinetochore.

    PubMed

    DeLillo, N; Romero, C; Lin, H; Vancura, A

    2003-05-01

    Chromosome segregation during mitosis requires kinetochores, specialized organelles that mediate chromosome attachment to spindle microtubules. We have shown previously that in budding yeast, Plc1p (phosphoinositide-specific phospholipase C) localizes to centromeric loci, associates with the kinetochore proteins Ndc10p and Cep3p, and affects the function of kinetochores. Deletion of PLC1 results in nocodazole sensitivity, mitotic delay, and a higher frequency of chromosome loss. We report here that despite the nocodazole sensitivity of plc1Delta cells, Plc1p is not required for the spindle checkpoint. However, plc1Delta cells require a functional BUB1/BUB3-dependent spindle checkpoint for viability. PLC1 displays strong genetic interactions with genes encoding components of the inner kinetochore, including NDC10, SKP1, MIF2, CEP1, CEP3, and CTF13. Furthermore, plc1Delta cells display alterations in chromatin structure in the core centromere. Chromatin immunoprecipitation experiments indicate that Plc1p localizes to centromeric loci independently of microtubules, and accumulates at the centromeres during G(2)/M stage of cell cycle. These results are consistent with the view that Plc1p affects kinetochore function, possibly by modulating the structure of centromeric chromatin.

  14. Organelle Size Scaling of the Budding Yeast Vacuole Is Tuned by Membrane Trafficking Rates

    PubMed Central

    Chan, Yee-Hung Mark; Marshall, Wallace F.

    2014-01-01

    Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates. PMID:24806931

  15. Organelle size scaling of the budding yeast vacuole is tuned by membrane trafficking rates.

    PubMed

    Chan, Yee-Hung Mark; Marshall, Wallace F

    2014-05-01

    Organelles serve as biochemical reactors in the cell, and often display characteristic scaling trends with cell size, suggesting mechanisms that coordinate their sizes. In this study, we measure the vacuole-cell size scaling trends in budding yeast using optical microscopy and a novel, to our knowledge, image analysis algorithm. Vacuole volume and surface area both show characteristic scaling trends with respect to cell size that are consistent among different strains. Rapamycin treatment was found to increase vacuole-cell size scaling trends for both volume and surface area. Unexpectedly, these increases did not depend on macroautophagy, as similar increases in vacuole size were observed in the autophagy deficient mutants atg1Δ and atg5Δ. Rather, rapamycin appears to act on vacuole size by inhibiting retrograde membrane trafficking, as the atg18Δ mutant, which is defective in retrograde trafficking, shows similar vacuole size scaling to rapamycin-treated cells and is itself insensitive to rapamycin treatment. Disruption of anterograde membrane trafficking in the apl5Δ mutant leads to complementary changes in vacuole size scaling. These quantitative results lead to a simple model for vacuole size scaling based on proportionality between cell growth rates and vacuole growth rates.

  16. Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1

    PubMed Central

    Kang, Hui; Tsygankov, Denis; Lew, Daniel J.

    2016-01-01

    Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a “bud sensor.” Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence. PMID:27053666

  17. Sensing a bud in the yeast morphogenesis checkpoint: a role for Elm1.

    PubMed

    Kang, Hui; Tsygankov, Denis; Lew, Daniel J

    2016-06-01

    Bud formation by Saccharomyces cerevisiae must be coordinated with the nuclear cycle to enable successful proliferation. Many environmental stresses temporarily disrupt bud formation, and in such circumstances, the morphogenesis checkpoint halts nuclear division until bud formation can resume. Bud emergence is essential for degradation of the mitotic inhibitor, Swe1. Swe1 is localized to the septin cytoskeleton at the bud neck by the Swe1-binding protein Hsl7. Neck localization of Swe1 is required for Swe1 degradation. Although septins form a ring at the presumptive bud site before bud emergence, Hsl7 is not recruited to the septins until after bud emergence, suggesting that septins and/or Hsl7 respond to a "bud sensor." Here we show that recruitment of Hsl7 to the septin ring depends on a combination of two septin-binding kinases: Hsl1 and Elm1. We elucidate which domains of these kinases are needed and show that artificial targeting of those domains suffices to recruit Hsl7 to septin rings even in unbudded cells. Moreover, recruitment of Elm1 is responsive to bud emergence. Our findings suggest that Elm1 plays a key role in sensing bud emergence. PMID:27053666

  18. Reliable cell cycle commitment in budding yeast is ensured by signal integration

    NASA Astrophysics Data System (ADS)

    Tang, Chao

    2014-03-01

    Cells have to make reliable decisions in response to external and/or internal signals that can be noisy and varying. For budding yeast Saccharomyces cerevisiae, cells decide whether and when to commit to cell division at the Start checkpoint. The decision is irreversible and has the physiological significance for coordinating cell growth with cell division. The trigger of the Start, the G1 cyclin Cln3 is a dynamic sensor of the nutrient and cellular conditions with low copy number and rapid turnover time. Here we quantitatively investigate how cells process the information from Cln3 to make the Start decision. By using an inducible Cln3 and monitoring the time cell waits before Start transition (G1 length), we find that G1 length is inversely proportional to Cln3 concentration, which implies that Start is triggered when the integration of Cln3 concentration over time exceeds certain threshold. We identify the Start repressor, Whi5 as the integrator. The instantaneous kinase activity of Cln3-Cdk1 is recorded over time on the phosphorylated Whi5, and the decision is made only when the phosphorylation level of Whi5 reaches a threshold. Furthermore, we find that Whi5 plays an important role for coordinating growth and division - cells modulate Whi5 level in different nutrient conditions to adjust the Start threshold. The strategy of signal integration, which reduces noise and minimizes error and uncertainty, has been found in decision-making behaviors of animals. Our work shows that it is adopted at the cellular level, suggesting a general design principle that may be widely implemented in decision-making and signaling systems.

  19. Phosphorylation and cellular function of the human Rpa2 N-terminus in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Ghospurkar, Padmaja L; Wilson, Timothy M; Liu, Shengqin; Herauf, Anna; Steffes, Jenna; Mueller, Erica N; Oakley, Gregory G; Haring, Stuart J

    2015-02-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms.

  20. Structure of a Bud6/actin complex reveals a novel WH2-like actin monomer recruitment motif

    PubMed Central

    Park, Eunyoung; Graziano, Brian R.; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L.; Eck, Michael J.

    2015-01-01

    SUMMARY In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a “core” domain that binds to the formin and a “flank” domain that binds monomeric actin. Here we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6flank interact with actin; one binds in a groove at the barbed-end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation. PMID:26118535

  1. Structure of a Bud6/Actin Complex Reveals a Novel WH2-like Actin Monomer Recruitment Motif.

    PubMed

    Park, Eunyoung; Graziano, Brian R; Zheng, Wei; Garabedian, Mikael; Goode, Bruce L; Eck, Michael J

    2015-08-01

    In budding yeast, the actin-binding protein Bud6 cooperates with formins Bni1 and Bnr1 to catalyze the assembly of actin filaments. The nucleation-enhancing activity of Bud6 requires both a "core" domain that binds to the formin and a "flank" domain that binds monomeric actin. Here, we describe the structure of the Bud6 flank domain in complex with actin. Two helices in Bud6(flank) interact with actin; one binds in a groove at the barbed end of the actin monomer in a manner closely resembling the helix of WH2 domains, a motif found in many actin nucleation factors. The second helix rises along the face of actin. Mutational analysis verifies the importance of these Bud6-actin contacts for nucleation-enhancing activity. The Bud6 binding site on actin overlaps with that of the formin FH2 domain and is also incompatible with inter-subunit contacts in F-actin, suggesting that Bud6 interacts only transiently with actin monomers during filament nucleation.

  2. Role of endocytosis in localization and maintenance of the spatial markers for bud-site selection in yeast.

    PubMed

    Tuo, Shanshan; Nakashima, Kenichi; Pringle, John R

    2013-01-01

    The yeast Saccharomyces cerevisiae normally selects bud sites (and hence axes of cell polarization) in one of two distinct patterns, the axial pattern of haploid cells and the bipolar pattern of diploid cells. These patterns depend on distinct sets of cortical-marker proteins that transmit positional information through a common signaling pathway based on a Ras-type GTPase. It has been reported previously that various proteins of the endocytic pathway may be involved in determining the bipolar pattern but not the axial pattern. To explore this question systematically, we constructed and analyzed congenic haploid and diploid deletion mutants for 14 genes encoding proteins that are involved in endocytosis. The mutants displayed a wide range of severities in their overall endocytosis defects, as judged by their growth rates and abilities to take up the lipophilic dye FM 4-64. Consistent with the previous reports, none of the mutants displayed a significant defect in axial budding, but they displayed defects in bipolar budding that were roughly correlated with the severities of their overall endocytosis defects. Both the details of the mutant budding patterns and direct examination of GFP-tagged marker proteins suggested that both initial formation and maintenance of the normally persistent bipolar marks depend on endocytosis, as well as polarized exocytosis, in actively growing cells. Interestingly, maintenance of the bipolar marks in non-growing cells did not appear to require normal levels of endocytosis. In some cases, there was a striking lack of correlation between the overall severities of the general-endocytosis defect and the bud-site selection defect, suggesting that various endocytosis proteins may differ in their importance for the uptake of various plasma-membrane targets.

  3. Intersection between the regulators of sister chromatid cohesion establishment and maintenance in budding yeast indicates a multi-step mechanism.

    PubMed

    Noble, Daniel; Kenna, Margaret A; Dix, Melissa; Skibbens, Robert V; Unal, Elçin; Guacci, Vincent

    2006-11-01

    Sister chromatid cohesion is established during S phase and maintained until anaphase. The cohesin complex (Mcd1p/Scc1p, Smc1p, Smc3p Irr1p/Scc3p in budding yeast) serves a structural role as it is required at all times when cohesion exists. Pds5p colocalizes temporally and spatially with cohesin on chromosomes but is thought to serve as a regulator of cohesion maintenance during mitosis. In contrast, Ctf7p/Eco1p is required during S phase for establishment but is not required during mitosis. Here we provide genetic and biochemical evidence that the pathways of cohesion establishment and maintenance are intimately linked. Our results show that mutants in ctf7 and pds5 are synthetically lethal. Moreover, over-expression of either CTF7 or PDS5 exhibits reciprocal suppression of the other mutant's temperature sensitivity. The suppression by CTF7 is specific for pds5 mutants as CTF7 over-expression increases the temperature sensitivity of an mcd1 mutant but has no effect on smc1 or smc3 mutants. Three additional findings provide new insights into the process of cohesion establishment. First, over-expression of ctf7 alleles deficient in acetylase activity exhibit significantly reduced suppression of the pds5 mutant but exacerbated toxicity to the mcd1 mutant. Second, using chromosome spreads and chromatin immuno-precipitation, we find either cohesin complex or Pds5p chromosomal localization is altered in ctf7 mutants. Finally, biochemical analysis reveals that Ctf7p and Pds5p coimmunoprecipitate, which physically links these regulators of cohesion establishment and maintenance. We propose a model whereby Ctf7p and Pds5p cooperate to facilitate efficient establishment by mediating changes in cohesin complex on chromosomes after its deposition. PMID:17102636

  4. Ndj1, a telomere-associated protein, regulates centrosome separation in budding yeast meiosis

    PubMed Central

    Li, Ping; Shao, Yize; Jin, Hui

    2015-01-01

    Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression. PMID:25897084

  5. The long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virus.

    PubMed

    Lista, María José; Voisset, Cécile; Contesse, Marie-Astrid; Friocourt, Gaëlle; Daskalogianni, Chrysoula; Bihel, Frédéric; Fåhraeus, Robin; Blondel, Marc

    2015-09-01

    The Epstein-Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast-based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.

  6. Binding of the Wheat Germ Lectin to Cryptococcus neoformans Suggests an Association of Chitinlike Structures with Yeast Budding and Capsular Glucuronoxylomannan▿ †

    PubMed Central

    Rodrigues, Marcio L.; Alvarez, Mauricio; Fonseca, Fernanda L.; Casadevall, Arturo

    2008-01-01

    The capsule of Cryptococcus neoformans is a complex structure whose assembly requires intermolecular interactions to connect its components into an organized structure. In this study, we demonstrated that the wheat germ agglutinin (WGA), which binds to sialic acids and β-1,4-N-acetylglucosamine (GlcNAc) oligomers, can also bind to cryptococcal capsular structures. Confocal microscopy demonstrated that these structures form round or hooklike projections linking the capsule to the cell wall, as well as capsule-associated structures during yeast budding. Chemical analysis of capsular extracts by gas chromatography coupled to mass spectrometry and high-pH anion-exchange chromatography suggested that the molecules recognized by WGA were firmly associated with the cell wall. Enzymatic treatment, competition assays, and staining with chemically modified WGA revealed that GlcNAc oligomers, but not sialic acids, were the molecules recognized by the lectin. Accordingly, treatment of C. neoformans cells with chitinase released glucuronoxylomannan (GXM) from the cell surface and reduced the capsule size. Chitinase-treated acapsular cells bound soluble GXM in a modified pattern. These results indicate an association of chitin-derived structures with GXM and budding in C. neoformans, which may represent a new mechanism by which the capsular polysaccharide interacts with the cell wall and is rearranged during replication. PMID:18039942

  7. HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern in the yeast Saccharomyces cerevisiae.

    PubMed Central

    Yabe, T; Yamada-Okabe, T; Kasahara, S; Furuichi, Y; Nakajima, T; Ichishima, E; Arisawa, M; Yamada-Okabe, H

    1996-01-01

    We previously isolated the Saccharomyces cerevisiae HKR1 gene that confers on S. cerevisiae cells resistance to HM-1 killer toxin secreted by Hansenula mrakii (S. Kasahara, H. Yamada, T. Mio, Y. Shiratori, C. Miyamoto, T. Yabe, T. Nakajima, E. Ichishima, and Y. Furuichi, J. Bacteriol. 176:1488-1499, 1994). HKR1 encodes a type 1 membrane protein that contains a calcium-binding consensus sequence (EF hand motif) in the cytoplasmic domain. Although the null mutation of HKR1 is lethal, disruption of the 3' part of the coding region, which would result in deletion of the cytoplasmic domain of Hkr1p, did not affect the viability of yeast cells. This partial disruption of HKR1 significantly reduced beta-1,3-glucan synthase activity and the amount of beta-1,3-glucan in the cell wall and altered the axial budding pattern of haploid cells. Neither chitin synthase activity nor chitin content was significantly affected in the cells harboring the partially disrupted HKR1 allele. Immunofluorescence microscopy with an antibody raised against Hkr1p expressed in Escherichia coli revealed that Hkr1p was predominantly localized on the cell surface. The cell surface localization of Hkr1p required the N-terminal signal sequence because the C-terminal half of Hkr1p was detected uniformly in the cells. These results demonstrate that HKR1 encodes a cell surface protein that regulates both cell wall beta-glucan synthesis and budding pattern and suggest that bud site assembly is somehow related to beta-glucan synthesis in S. cerevisiae. PMID:8550469

  8. Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques

    NASA Astrophysics Data System (ADS)

    Bystricky, Kerstin; Heun, Patrick; Gehlen, Lutz; Langowski, Jörg; Gasser, Susan M.

    2004-11-01

    Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of 110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure. higher-order structure | 30-nm fiber | nucleosomes

  9. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1

    PubMed Central

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-01-01

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1–3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4–9 did not influence the cell cycle–regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4–9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1. PMID:27226481

  10. Dual control by Cdk1 phosphorylation of the budding yeast APC/C ubiquitin ligase activator Cdh1.

    PubMed

    Höckner, Sebastian; Neumann-Arnold, Lea; Seufert, Wolfgang

    2016-07-15

    The antagonism between cyclin-dependent kinases (Cdks) and the ubiquitin ligase APC/C-Cdh1 is central to eukaryotic cell cycle control. APC/C-Cdh1 targets cyclin B and other regulatory proteins for degradation, whereas Cdks disable APC/C-Cdh1 through phosphorylation of the Cdh1 activator protein at multiple sites. Budding yeast Cdh1 carries nine Cdk phosphorylation sites in its N-terminal regulatory domain, most or all of which contribute to inhibition. However, the precise role of individual sites has remained unclear. Here, we report that the Cdk phosphorylation sites of yeast Cdh1 are organized into autonomous subgroups and act through separate mechanisms. Cdk sites 1-3 had no direct effect on the APC/C binding of Cdh1 but inactivated a bipartite nuclear localization sequence (NLS) and thereby controlled the partitioning of Cdh1 between cytoplasm and nucleus. In contrast, Cdk sites 4-9 did not influence the cell cycle-regulated localization of Cdh1 but prevented its binding to the APC/C. Cdk sites 4-9 reside near two recently identified APC/C interaction motifs in a pattern conserved with the human Cdh1 orthologue. Thus a Cdk-inhibited NLS goes along with Cdk-inhibited APC/C binding sites in yeast Cdh1 to relay the negative control by Cdk1 phosphorylation of the ubiquitin ligase APC/C-Cdh1.

  11. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast

    PubMed Central

    1994-01-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine- nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

  12. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

    PubMed

    Peterson, J; Zheng, Y; Bender, L; Myers, A; Cerione, R; Bender, A

    1994-12-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

  13. Coupling spindle position with mitotic exit in budding yeast: The multifaceted role of the small GTPase Tem1

    PubMed Central

    Scarfone, Ilaria; Piatti, Simonetta

    2015-01-01

    The budding yeast S. cerevisiae divides asymmetrically and is an excellent model system for asymmetric cell division. As for other asymmetrically dividing cells, proper spindle positioning along the mother-daughter polarity axis is crucial for balanced chromosome segregation. Thus, a surveillance mechanism named Spindle Position Checkpoint (SPOC) inhibits mitotic exit and cytokinesis until the mitotic spindle is properly oriented, thereby preventing the generation of cells with aberrant ploidies. The small GTPase Tem1 is required to trigger a Hippo-like protein kinase cascade, named Mitotic Exit Network (MEN), that is essential for mitotic exit and cytokinesis but also contributes to correct spindle alignment in metaphase. Importantly, Tem1 is the target of the SPOC, which relies on the activity of the GTPase-activating complex (GAP) Bub2-Bfa1 to keep Tem1 in the GDP-bound inactive form. Tem1 forms a hetero-trimeric complex with Bub2-Bfa1 at spindle poles (SPBs) that accumulates asymmetrically on the bud-directed spindle pole during mitosis when the spindle is properly positioned. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. We have recently shown that Tem1 residence at SPBs depends on its nucleotide state and, importantly, asymmetry of the Bub2-Bfa1-Tem1 complex does not promote mitotic exit but rather controls spindle positioning. PMID:26507466

  14. Complete DNA Sequence of Kuraishia capsulata Illustrates Novel Genomic Features among Budding Yeasts (Saccharomycotina)

    PubMed Central

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993T), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ∼13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts. PMID:24317973

  15. Mediated amperometry reveals different modes of yeast responses to sugars.

    PubMed

    Garjonyte, Rasa; Melvydas, Vytautas; Malinauskas, Albertas

    2016-02-01

    Menadione-mediated amperometry at carbon paste electrodes modified with various yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Pichia guilliermondii and Debaryomyces hansenii) was employed to monitor redox activity inside the yeast cells induced by glucose, fructose, sucrose, maltose or galactose. Continuous measurements revealed distinct modes (transient or gradually increasing) of the current development during the first 2 to 3 min after subjection to glucose, fructose and sucrose at electrodes containing S. cerevisiae and non-Saccharomyces strains. Different modes (increasing or decreasing) of the current development after yeast subjection to galactose at electrodes with S. cerevisiae or D. hansenii and at electrodes with C. pulcherrima and P. guilliermondii suggested different mechanisms of galactose assimilation.

  16. Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling.

    PubMed

    Kim, Junwon; Rose, Mark D

    2015-12-01

    Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth

  17. Stable Pseudohyphal Growth in Budding Yeast Induced by Synergism between Septin Defects and Altered MAP-kinase Signaling

    PubMed Central

    Kim, Junwon; Rose, Mark D.

    2015-01-01

    Upon nutrient limitation, budding yeasts like Saccharomyces cerevisiae can be induced to adopt alternate filament-like growth patterns called diploid pseudohyphal or invasive haploid growth. Here, we report a novel constitutive pseudohyphal growth state, sharing some characteristics with classic forms of filamentous growth, but differing in crucial aspects of morphology, growth conditions and genetic regulation. The constitutive pseudohyphal state is observed in fus3 mutants containing various septin assembly defects, which we refer to as sadF growth (septin assembly defect induced filamentation) to distinguish it from classic filamentation pathways. Similar to other filamentous states, sadF cultures comprise aggregated chains of highly elongated cells. Unlike the classic pathways, sadF growth occurs in liquid rich media, requiring neither starvation nor the key pseudohyphal proteins, Flo8p and Flo11p. Moreover sadF growth occurs in haploid strains of S288C genetic background, which normally cannot undergo pseudohyphal growth. The sadF cells undergo highly polarized bud growth during prolonged G2 delays dependent on Swe1p. They contain septin structures distinct from classical pseudo-hyphae and FM4-64 labeling at actively growing tips similar to the Spitzenkörper observed in true hyphal growth. The sadF growth state is induced by synergism between Kss1p-dependent signaling and septin assembly defects; mild disruption of mitotic septins activates Kss1p-dependent gene expression, which exacerbates the septin defects, leading to hyper-activation of Kss1p. Unlike classical pseudo-hyphal growth, sadF signaling requires Ste5, Ste4 and Ste18, the scaffold protein and G-protein β and γ subunits from the pheromone response pathway, respectively. A swe1 mutation largely abolished signaling, breaking the positive feedback that leads to amplification of sadF signaling. Taken together, our findings show that budding yeast can access a stable constitutive pseudohyphal growth

  18. Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe.

    PubMed

    Rostron, Kerry A; Rolph, Carole E; Lawrence, Clare L

    2015-07-01

    Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321-328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses.

  19. alpha-Synuclein budding yeast model: toxicity enhanced by impaired proteasome and oxidative stress.

    PubMed

    Sharma, Nijee; Brandis, Katrina A; Herrera, Sara K; Johnson, Brandon E; Vaidya, Tulaza; Shrestha, Ruja; Debburman, Shubhik K

    2006-01-01

    Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.

  20. Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

    PubMed

    Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J; Klein, Ophir D; Barlow, Linda A

    2014-08-01

    Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation. PMID:24993944

  1. Induction of ectopic taste buds by SHH reveals the competency and plasticity of adult lingual epithelium.

    PubMed

    Castillo, David; Seidel, Kerstin; Salcedo, Ernesto; Ahn, Christina; de Sauvage, Frederic J; Klein, Ophir D; Barlow, Linda A

    2014-08-01

    Taste buds are assemblies of elongated epithelial cells, which are innervated by gustatory nerves that transmit taste information to the brain stem. Taste cells are continuously renewed throughout life via proliferation of epithelial progenitors, but the molecular regulation of this process remains unknown. During embryogenesis, sonic hedgehog (SHH) negatively regulates taste bud patterning, such that inhibition of SHH causes the formation of more and larger taste bud primordia, including in regions of the tongue normally devoid of taste buds. Here, using a Cre-lox system to drive constitutive expression of SHH, we identify the effects of SHH on the lingual epithelium of adult mice. We show that misexpression of SHH transforms lingual epithelial cell fate, such that daughter cells of lingual epithelial progenitors form cell type-replete, onion-shaped taste buds, rather than non-taste, pseudostratified epithelium. These SHH-induced ectopic taste buds are found in regions of the adult tongue previously thought incapable of generating taste organs. The ectopic buds are composed of all taste cell types, including support cells and detectors of sweet, bitter, umami, salt and sour, and recapitulate the molecular differentiation process of endogenous taste buds. In contrast to the well-established nerve dependence of endogenous taste buds, however, ectopic taste buds form independently of both gustatory and somatosensory innervation. As innervation is required for SHH expression by endogenous taste buds, our data suggest that SHH can replace the need for innervation to drive the entire program of taste bud differentiation.

  2. Dietary restriction depends on nutrient composition to extend chronological lifespan in budding yeast Saccharomyces cerevisiae.

    PubMed

    Wu, Ziyun; Liu, Shao Quan; Huang, Dejian

    2013-01-01

    The traditional view on dietary restriction has been challenged with regard to extending lifespan of the fruit fly Drosophila melanogaster. This is because studies have shown that changing the balance of dietary components without reduction of dietary intake can increase lifespan, suggesting that nutrient composition other than dietary restriction play a pivotal role in regulation of longevity. However, this opinion has not been reflected in yeast aging studies. Inspired by this new finding, response surface methodology was applied to evaluate the relationships between nutrients (glucose, amino acids and yeast nitrogen base) and lifespan as well as biomass production in four Saccharomyces cerevisiae strains (wild-type BY4742, sch9Δ, tor1Δ, and sir2Δ mutants) using a high throughput screening assay. Our results indicate that lifespan extension by a typical dietary restriction regime was dependent on the nutrients in media and that nutrient composition was a key determinant for yeast longevity. Four different yeast strains were cultured in various media, which showed similar response surface trends in biomass production and viability at day two but greatly different trends in lifespan. The pH of aging media was dependent on glucose concentration and had no apparent correlation with lifespan under conditions where amino acids and YNB were varied widely, and simply buffering the pH of media could extend lifespan significantly. Furthermore, the results showed that strain sch9Δ was more responsive in nutrient-sensing than the other three strains, suggesting that Sch9 (serine-threonine kinase pathway) was a major nutrient-sensing factor that regulates cell growth, cell size, metabolism, stress resistance and longevity. Overall, our findings support the notion that nutrient composition might be a more effective way than simple dietary restriction to optimize lifespan and biomass production from yeast to other organisms.

  3. Collective effects of SNPs on transgenerational inheritance in Caenorhabditis elegans and budding yeast.

    PubMed

    Zhu, Zuobin; Man, Xian; Xia, Mengying; Huang, Yimin; Yuan, Dejian; Huang, Shi

    2015-07-01

    We studied the collective effects of single nucleotide polymorphisms (SNPs) on transgenerational inheritance in Caenorhabditis elegans recombinant inbred advanced intercross lines (RIAILs) and yeast segregants. We divided the RIAILs and segregants into two groups of high and low minor allele content (MAC). RIAILs with higher MAC needed less generations of benzaldehyde training to gain a stable olfactory imprint and showed a greater change from normal after benzaldehyde training. Yeast segregants with higher MAC showed a more dramatic shortening of the lag phase length after ethanol exposure. The short lag phase as acquired by ethanol training was more dramatically lost after recovery in ethanol free medium for the high MAC group. We also found a preferential association between MAC and traits linked with higher number of additive QTLs. These results suggest a role for the collective effects of SNPs in transgenerational inheritance, and may help explain human variations in disease susceptibility.

  4. The proline metabolism intermediate Δ1-pyrroline-5-carboxylate directly inhibits the mitochondrial respiration in budding yeast.

    PubMed

    Nishimura, Akira; Nasuno, Ryo; Takagi, Hiroshi

    2012-07-30

    The proline metabolism intermediate Δ(1)-pyrroline-5-carboxylate (P5C) induces cell death in animals, plants and yeasts. To elucidate how P5C triggers cell death, we analyzed P5C metabolism, mitochondrial respiration and superoxide anion generation in the yeast Saccharomyces cerevisiae. Gene disruption analysis revealed that P5C-mediated cell death was not due to P5C metabolism. Interestingly, deficiency in mitochondrial respiration suppressed the sensitivity of yeast cells to P5C. In addition, we found that P5C inhibits the mitochondrial respiration and induces a burst of superoxide anions from the mitochondria. We propose that P5C regulates cell death via the inhibition of mitochondrial respiration.

  5. Bck2 Acts through the MADS Box Protein Mcm1 to Activate Cell-Cycle-Regulated Genes in Budding Yeast

    PubMed Central

    Bastajian, Nazareth; Friesen, Helena; Andrews, Brenda J.

    2013-01-01

    The Bck2 protein is a potent genetic regulator of cell-cycle-dependent gene expression in budding yeast. To date, most experiments have focused on assessing a potential role for Bck2 in activation of the G1/S-specific transcription factors SBF (Swi4, Swi6) and MBF (Mbp1, Swi6), yet the mechanism of gene activation by Bck2 has remained obscure. We performed a yeast two-hybrid screen using a truncated version of Bck2 and discovered six novel Bck2-binding partners including Mcm1, an essential protein that binds to and activates M/G1 promoters through Early Cell cycle Box (ECB) elements as well as to G2/M promoters. At M/G1 promoters Mcm1 is inhibited by association with two repressors, Yox1 or Yhp1, and gene activation ensues once repression is relieved by an unknown activating signal. Here, we show that Bck2 interacts physically with Mcm1 to activate genes during G1 phase. We used chromatin immunoprecipitation (ChIP) experiments to show that Bck2 localizes to the promoters of M/G1-specific genes, in a manner dependent on functional ECB elements, as well as to the promoters of G1/S and G2/M genes. The Bck2-Mcm1 interaction requires valine 69 on Mcm1, a residue known to be required for interaction with Yox1. Overexpression of BCK2 decreases Yox1 localization to the early G1-specific CLN3 promoter and rescues the lethality caused by overexpression of YOX1. Our data suggest that Yox1 and Bck2 may compete for access to the Mcm1-ECB scaffold to ensure appropriate activation of the initial suite of genes required for cell cycle commitment. PMID:23675312

  6. Replication dynamics in fission and budding yeasts through DNA polymerase tracking

    PubMed Central

    Vázquez, Enrique

    2015-01-01

    The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof‐read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions. PMID:26293347

  7. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast

    SciTech Connect

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. {sup 1}H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes. - Highlights: • Involvement of yeast TF genes essential for cell growth in lifespan was evaluated. • The essential TF genes, FHL1, RAP1, REB1, and MCM1, regulate replicative lifespan. • Heterozygous deletion of FHL1 changes cellular metabolism related to lifespan.

  8. The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

    PubMed Central

    Heun, Patrick; Laroche, Thierry; Raghuraman, M.K.; Gasser, Susan M.

    2001-01-01

    We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus. PMID:11266454

  9. A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A.

    PubMed

    Hornung, Peter; Troc, Paulina; Malvezzi, Francesca; Maier, Michael; Demianova, Zuzana; Zimniak, Tomasz; Litos, Gabriele; Lampert, Fabienne; Schleiffer, Alexander; Brunner, Matthias; Mechtler, Karl; Herzog, Franz; Marlovits, Thomas C; Westermann, Stefan

    2014-08-18

    Kinetochores are megadalton-sized protein complexes that mediate chromosome-microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1(CENP-U) and Okp1(CENP-Q) form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1-Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers.

  10. A cooperative mechanism drives budding yeast kinetochore assembly downstream of CENP-A

    PubMed Central

    Hornung, Peter; Troc, Paulina; Malvezzi, Francesca; Maier, Michael; Demianova, Zuzana; Zimniak, Tomasz; Litos, Gabriele; Lampert, Fabienne; Schleiffer, Alexander; Brunner, Matthias; Mechtler, Karl; Herzog, Franz; Marlovits, Thomas C.

    2014-01-01

    Kinetochores are megadalton-sized protein complexes that mediate chromosome–microtubule interactions in eukaryotes. How kinetochore assembly is triggered specifically on centromeric chromatin is poorly understood. Here we use biochemical reconstitution experiments alongside genetic and structural analysis to delineate the contributions of centromere-associated proteins to kinetochore assembly in yeast. We show that the conserved kinetochore subunits Ame1CENP-U and Okp1CENP-Q form a DNA-binding complex that associates with the microtubule-binding KMN network via a short Mtw1 recruitment motif in the N terminus of Ame1. Point mutations in the Ame1 motif disrupt kinetochore function by preventing KMN assembly on chromatin. Ame1–Okp1 directly associates with the centromere protein C (CENP-C) homologue Mif2 to form a cooperative binding platform for outer kinetochore assembly. Our results indicate that the key assembly steps, CENP-A recognition and outer kinetochore recruitment, are executed through different yeast constitutive centromere-associated network subunits. This two-step mechanism may protect against inappropriate kinetochore assembly similar to rate-limiting nucleation steps used by cytoskeletal polymers. PMID:25135934

  11. Cadmium induces the activation of cell wall integrity pathway in budding yeast.

    PubMed

    Xiong, Bing; Zhang, Lilin; Xu, Huihui; Yang, Yi; Jiang, Linghuo

    2015-10-01

    MAP kinases are important signaling molecules regulating cell survival, proliferation and differentiation, and can be activated by cadmium stress. In this study, we demonstrate that cadmium induces phosphorylation of the yeast cell wall integrity (CWI) pathway_MAP kinase Slt2, and this cadmium-induced CWI activation is mediated by the cell surface sensor Mid2 through the GEF Rom1, the central regulator Rho1 and Bck1. Nevertheless, cadmium stress does not affect the subcellular localization of Slt2 proteins. In addition, this cadmium-induced CWI activation is independent on the calcium/calcineurin signaling and the high osmolarity glycerol (HOG) signaling pathways in yeast cells. Furthermore, we tested the cadmium sensitivity of 42 paired double-gene deletion mutants between six CWI components and seven components of the HOG pathway. Our results indicate that the CWI pathway is epistatic to the HOG pathway in cadmium sensitivity. However, gene deletion mutations for the Swi4/Swi6 transcription factor complex show synergistic effects with mutations of HOG components in cadmium sensitivity.

  12. Microtubule stability in budding yeast: characterization and dosage suppression of a benomyl-dependent tubulin mutant.

    PubMed Central

    Machin, N A; Lee, J M; Barnes, G

    1995-01-01

    To better understand the dynamic regulation of microtubule structures in yeast, we studied a conditional-lethal beta-tubulin mutation tub2-150. This mutation is unique among the hundreds of tubulin mutations isolated in Saccharomyces cerevisiae in that it appears to cause an increase in the stability of microtubules. We report here that this allele is a mutation of threonine 238 to alanine, and that tub2-150 prevents the spindle from elongating during anaphase, suggesting a nuclear microtubule defect. To identify regulators of microtubule stability and/or anaphase, yeast genes were selected that, when overexpressed, could suppress the tub2-150 temperature-sensitive phenotype. One of these genes, JSN1, encodes a protein of 125 kDa that has limited similarity to a number of proteins of unknown function. Overexpression of the JSN1 gene in a TUB2 strain causes that strain to become more sensitive to benomyl, a microtubule-destabilizing drug. Of a representative group of microtubule mutants, only one other mutation, tub2-404, could be suppressed by JSN1 overexpression, showing that JSN1 is an allele-specific suppressor. As tub2-404 mutants are also defective for spindle elongation, this provides additional support for a role for JSN1 during anaphase. Images PMID:8534919

  13. iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

    2015-08-01

    Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants.

  14. iAID: an improved auxin-inducible degron system for the construction of a 'tight' conditional mutant in the budding yeast Saccharomyces cerevisiae.

    PubMed

    Tanaka, Seiji; Miyazawa-Onami, Mayumi; Iida, Tetsushi; Araki, Hiroyuki

    2015-08-01

    Isolation of a 'tight' conditional mutant of a gene of interest is an effective way of studying the functions of essential genes. Strategies that use ubiquitin-mediated protein degradation to eliminate the product of a gene of interest, such as heat-inducible degron (td) and auxin-inducible degron (AID), are powerful methods for constructing conditional mutants. However, these methods do not work with some genes. Here, we describe an improved AID system (iAID) for isolating tight conditional mutants in the budding yeast Saccharomyces cerevisiae. In this method, transcriptional repression by the 'Tet-OFF' promoter is combined with proteolytic elimination of the target protein by the AID system. To provide examples, we describe the construction of tight mutants of the replication factors Dpb11 and Mcm10, dpb11-iAID, and mcm10-iAID. Because Dpb11 and Mcm10 are required for the initiation of DNA replication, their tight mutants are unable to enter S phase. This is the case for dpb11-iAID and mcm10-iAID cells after the addition of tetracycline and auxin. Both the 'Tet-OFF' promoter and the AID system have been shown to work in model eukaryotes other than budding yeast. Therefore, the iAID system is not only useful in budding yeast, but also can be applied to other model systems to isolate tight conditional mutants. PMID:26081484

  15. DNA resection proteins Sgs1 and Exo1 are required for G1 checkpoint activation in budding yeast

    PubMed Central

    Balogun, Fiyinfolu O.; Truman, Andrew W.; Kron, Stephen J.

    2013-01-01

    Double-strand breaks (DSBs) in budding yeast trigger activation of DNA damage checkpoints, allowing repair to occur. Although resection is necessary for initiating damage-induced cell cycle arrest in G2, no role has been assigned to it in the activation of G1 checkpoint. Here we demonstrate for the first time that the resection proteins Sgs1 and Exo1 are required for efficient G1 checkpoint activation. We find in G1 arrested cells that histone H2A phosphorylation in response to ionizing radiation is independent of Sgs1 and Exo1. In contrast, these proteins are required for damage-induced recruitment of Rfa1 to the DSB sites, phosphorylation of the Rad53 effector kinase, cell cycle arrest and RNR3 expression. Checkpoint activation in G1 requires the catalytic activity of Sgs1, suggesting that it is DNA resection mediated by Sgs1 that stimulates the damage response pathway rather than protein-protein interactions with other DDR proteins. Together, these results implicate DNA resection, which is thought to be minimal in G1, as necessary for activation of the G1 checkpoint. PMID:23835406

  16. Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast.

    PubMed

    Palou, Gloria; Palou, Roger; Zeng, Fanli; Vashisht, Ajay A; Wohlschlegel, James A; Quintana, David G

    2015-09-01

    A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation. PMID:26332045

  17. Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.

    PubMed

    Murata, Kazuyoshi; Esaki, Masatoshi; Ogura, Teru; Arai, Shigeo; Yamamoto, Yuta; Tanaka, Nobuo

    2014-11-01

    Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology.

  18. An in vivo detection system for transient and low-abundant protein interactions and their kinetics in budding yeast.

    PubMed

    Brezovich, Andrea; Schuschnig, Martina; Ammerer, Gustav; Kraft, Claudine

    2015-03-01

    Methylation tracking (M-Track) is a protein-proximity assay in Saccharomyces cerevisiae, allowing the detection of transient protein-protein interactions in living cells. The bait protein is fused to a histone lysine methyl transferase and the prey protein to a methylation acceptor peptide derived from histone 3. Upon interaction, the histone 3 fragment is stably methylated on lysine 9 and can be detected by methylation-specific antibodies. Since methylation marking is irreversible in budding yeast and only takes place in living cells, the occurrence of artifacts during cell lysate preparation is greatly reduced, leading to a more accurate representation of native interactions. So far, this method has been limited to highly abundant or overexpressed proteins. However, many proteins of interest are low-abundant, and overexpression of proteins may interfere with their function, leading to an artificial situation. Here we report the generation of a toolbox including a novel cleavage-enrichment system for the analysis of very low-abundant proteins at their native expression levels. In addition, we developed a system for the parallel analysis of two prey proteins in a single cell, as well as an inducible methylation system. The inducible system allows precise control over the time during which the interaction is detected and can be used to determine interaction kinetics. Furthermore, we generated a set of constructs facilitating the cloning-free genomic tagging of proteins at their endogenous locus by homologous recombination, and their expression from centromeric plasmids.

  19. The budding yeast nuclear envelope adjacent to the nucleolus serves as a membrane sink during mitotic delay.

    PubMed

    Witkin, Keren L; Chong, Yolanda; Shao, Sichen; Webster, Micah T; Lahiri, Sujoy; Walters, Alison D; Lee, Brandon; Koh, Judice L Y; Prinz, William A; Andrews, Brenda J; Cohen-Fix, Orna

    2012-06-19

    The mechanisms that dictate nuclear shape are largely unknown. Here we screened the budding yeast deletion collection for mutants with abnormal nuclear shape. A common phenotype was the appearance of a nuclear extension, particularly in mutants in DNA repair and chromosome segregation genes. Our data suggest that these mutations led to the abnormal nuclear morphology indirectly, by causing a checkpoint-induced cell-cycle delay. Indeed, delaying cells in mitosis by other means also led to the appearance of nuclear extensions, whereas inactivating the DNA damage checkpoint pathway in a DNA repair mutant reduced the fraction of cells with nuclear extensions. Formation of a nuclear extension was specific to a mitotic delay, because cells arrested in S or G2 had round nuclei. Moreover, the nuclear extension always coincided with the nucleolus, while the morphology of the DNA mass remained largely unchanged. Finally, we found that phospholipid synthesis continued unperturbed when cells delayed in mitosis, and inhibiting phospholipid synthesis abolished the formation of nuclear extensions. Our data suggest a mechanism that promotes nuclear envelope expansion during mitosis. When mitotic progression is delayed, cells sequester the added membrane to the nuclear envelope associated with the nucleolus, possibly to avoid disruption of intranuclear organization.

  20. Mek1 Kinase Is Regulated To Suppress Double-Strand Break Repair between Sister Chromatids during Budding Yeast Meiosis▿

    PubMed Central

    Niu, Hengyao; Li, Xue; Job, Emily; Park, Caroline; Moazed, Danesh; Gygi, Steven P.; Hollingsworth, Nancy M.

    2007-01-01

    Mek1 is a meiosis-specific kinase in budding yeast which promotes recombination between homologous chromosomes by suppressing double-strand break (DSB) repair between sister chromatids. Previous work has shown that in the absence of the meiosis-specific recombinase gene, DMC1, cells arrest in prophase due to unrepaired DSBs and that Mek1 kinase activity is required in this situation to prevent repair of the breaks using sister chromatids. This work demonstrates that Mek1 is activated in response to DSBs by autophosphorylation of two conserved threonines, T327 and T331, in the Mek1 activation loop. Using a version of Mek1 that can be conditionally dimerized during meiosis, Mek1 function was shown to be promoted by dimerization, perhaps as a way of enabling autophosphorylation of the activation loop in trans. A putative HOP1-dependent dimerization domain within the C terminus of Mek1 has been identified. Dimerization alone, however, is insufficient for activation, as DSBs and Mek1 recruitment to the meiosis-specific chromosomal core protein Red1 are also necessary. Phosphorylation of S320 in the activation loop inhibits sister chromatid repair specifically in dmc1Δ-arrested cells. Ectopic dimerization of Mek1 bypasses the requirement for S320 phosphorylation, suggesting this phosphorylation is necessary for maintenance of Mek1 dimers during checkpoint-induced arrest. PMID:17526735

  1. Positive feedback promotes mitotic exit via the APC/C-Cdh1-separase-Cdc14 axis in budding yeast.

    PubMed

    Hatano, Yuhki; Naoki, Koike; Suzuki, Asuka; Ushimaru, Takashi

    2016-10-01

    The mitotic inhibitor securin is degraded via the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdc20 after anaphase onset. This triggers activation of the mitotic protease separase and thereby sister chromatid separation. However, only a proportion of securin molecules are degraded at metaphase-anaphase transition and the remaining molecules are still present in anaphase. The roles of securin and separase in late mitosis remain elusive. Here, we show that securin still inhibits separase to repress mitotic exit in anaphase in budding yeast. APC/C-Cdh1-mediated securin degradation at telophase further liberated separase, which promotes Cdc14 release and mitotic exit. Separase executed these events via its proteolytic action and that in the Cdc14 early release (FEAR) network. Cdc14 release further activated APC/C-Cdh1 in the manner of a positive feedback loop. Thus, the positive feedback promotes mitotic exit via the APC/C-Cdh1-separase-Cdc14 axis. This study shows the importance of the two-step degradation mode of securin and the role of separase in mitotic exit.

  2. Transcription factor genes essential for cell proliferation and replicative lifespan in budding yeast.

    PubMed

    Kamei, Yuka; Tai, Akiko; Dakeyama, Shota; Yamamoto, Kaori; Inoue, Yamato; Kishimoto, Yoshifumi; Ohara, Hiroya; Mukai, Yukio

    2015-07-31

    Many of the lifespan-related genes have been identified in eukaryotes ranging from the yeast to human. However, there is limited information available on the longevity genes that are essential for cell proliferation. Here, we investigated whether the essential genes encoding DNA-binding transcription factors modulated the replicative lifespan of Saccharomyces cerevisiae. Heterozygous diploid knockout strains for FHL1, RAP1, REB1, and MCM1 genes showed significantly short lifespan. (1)H-nuclear magnetic resonance analysis indicated a characteristic metabolic profile in the Δfhl1/FHL1 mutant. These results strongly suggest that FHL1 regulates the transcription of lifespan related metabolic genes. Thus, heterozygous knockout strains could be the potential materials for discovering further novel lifespan genes.

  3. Exposure of ELF-EMF and RF-EMF Increase the Rate of Glucose Transport and TCA Cycle in Budding Yeast

    PubMed Central

    Lin, Kang-Wei; Yang, Chuan-Jun; Lian, Hui-Yong; Cai, Peng

    2016-01-01

    In this study, we investigated the transcriptional response to 50 Hz extremely low frequency electromagnetic field (ELF-EMF) and 2.0 GHz radio frequency electromagnetic field (RF-EMF) exposure by Illumina sequencing technology using budding yeast as the model organism. The transcription levels of 28 genes were upregulated and those of four genes were downregulated under ELF-EMF exposure, while the transcription levels of 29 genes were upregulated and those of 24 genes were downregulated under RF-EMF exposure. After validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR), a concordant direction of change both in differential gene expression (DGE) and RT-qPCR was demonstrated for nine genes under ELF-EMF exposure and for 10 genes under RF-EMF exposure. The RT-qPCR results revealed that ELF-EMF and RF-EMF exposure can upregulate the expression of genes involved in glucose transportation and the tricarboxylic acid (TCA) cycle, but not the glycolysis pathway. Energy metabolism is closely related with the cell response to environmental stress including EMF exposure. Our findings may throw light on the mechanism underlying the biological effects of EMF. PMID:27630630

  4. Exposure of ELF-EMF and RF-EMF Increase the Rate of Glucose Transport and TCA Cycle in Budding Yeast.

    PubMed

    Lin, Kang-Wei; Yang, Chuan-Jun; Lian, Hui-Yong; Cai, Peng

    2016-01-01

    In this study, we investigated the transcriptional response to 50 Hz extremely low frequency electromagnetic field (ELF-EMF) and 2.0 GHz radio frequency electromagnetic field (RF-EMF) exposure by Illumina sequencing technology using budding yeast as the model organism. The transcription levels of 28 genes were upregulated and those of four genes were downregulated under ELF-EMF exposure, while the transcription levels of 29 genes were upregulated and those of 24 genes were downregulated under RF-EMF exposure. After validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR), a concordant direction of change both in differential gene expression (DGE) and RT-qPCR was demonstrated for nine genes under ELF-EMF exposure and for 10 genes under RF-EMF exposure. The RT-qPCR results revealed that ELF-EMF and RF-EMF exposure can upregulate the expression of genes involved in glucose transportation and the tricarboxylic acid (TCA) cycle, but not the glycolysis pathway. Energy metabolism is closely related with the cell response to environmental stress including EMF exposure. Our findings may throw light on the mechanism underlying the biological effects of EMF. PMID:27630630

  5. Exposure of ELF-EMF and RF-EMF Increase the Rate of Glucose Transport and TCA Cycle in Budding Yeast

    PubMed Central

    Lin, Kang-Wei; Yang, Chuan-Jun; Lian, Hui-Yong; Cai, Peng

    2016-01-01

    In this study, we investigated the transcriptional response to 50 Hz extremely low frequency electromagnetic field (ELF-EMF) and 2.0 GHz radio frequency electromagnetic field (RF-EMF) exposure by Illumina sequencing technology using budding yeast as the model organism. The transcription levels of 28 genes were upregulated and those of four genes were downregulated under ELF-EMF exposure, while the transcription levels of 29 genes were upregulated and those of 24 genes were downregulated under RF-EMF exposure. After validation by reverse transcription quantitative polymerase chain reaction (RT-qPCR), a concordant direction of change both in differential gene expression (DGE) and RT-qPCR was demonstrated for nine genes under ELF-EMF exposure and for 10 genes under RF-EMF exposure. The RT-qPCR results revealed that ELF-EMF and RF-EMF exposure can upregulate the expression of genes involved in glucose transportation and the tricarboxylic acid (TCA) cycle, but not the glycolysis pathway. Energy metabolism is closely related with the cell response to environmental stress including EMF exposure. Our findings may throw light on the mechanism underlying the biological effects of EMF.

  6. In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p.

    PubMed

    Smith, Carlas; Lari, Azra; Derrer, Carina Patrizia; Ouwehand, Anette; Rossouw, Ammeret; Huisman, Maximiliaan; Dange, Thomas; Hopman, Mark; Joseph, Aviva; Zenklusen, Daniel; Weis, Karsten; Grunwald, David; Montpetit, Ben

    2015-12-21

    Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼ 200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport.

  7. In vivo single-particle imaging of nuclear mRNA export in budding yeast demonstrates an essential role for Mex67p

    PubMed Central

    Smith, Carlas; Lari, Azra; Derrer, Carina Patrizia; Ouwehand, Anette; Rossouw, Ammeret; Huisman, Maximiliaan; Dange, Thomas; Hopman, Mark; Joseph, Aviva; Zenklusen, Daniel

    2015-01-01

    Many messenger RNA export proteins have been identified; yet the spatial and temporal activities of these proteins and how they determine directionality of messenger ribonucleoprotein (mRNP) complex export from the nucleus remain largely undefined. Here, the bacteriophage PP7 RNA-labeling system was used in Saccharomyces cerevisiae to follow single-particle mRNP export events with high spatial precision and temporal resolution. These data reveal that mRNP export, consisting of nuclear docking, transport, and cytoplasmic release from a nuclear pore complex (NPC), is fast (∼200 ms) and that upon arrival in the cytoplasm, mRNPs are frequently confined near the nuclear envelope. Mex67p functions as the principal mRNP export receptor in budding yeast. In a mex67-5 mutant, delayed cytoplasmic release from NPCs and retrograde transport of mRNPs was observed. This proves an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport. PMID:26694837

  8. Mediated amperometry reveals different modes of yeast responses to sugars.

    PubMed

    Garjonyte, Rasa; Melvydas, Vytautas; Malinauskas, Albertas

    2016-02-01

    Menadione-mediated amperometry at carbon paste electrodes modified with various yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Pichia guilliermondii and Debaryomyces hansenii) was employed to monitor redox activity inside the yeast cells induced by glucose, fructose, sucrose, maltose or galactose. Continuous measurements revealed distinct modes (transient or gradually increasing) of the current development during the first 2 to 3 min after subjection to glucose, fructose and sucrose at electrodes containing S. cerevisiae and non-Saccharomyces strains. Different modes (increasing or decreasing) of the current development after yeast subjection to galactose at electrodes with S. cerevisiae or D. hansenii and at electrodes with C. pulcherrima and P. guilliermondii suggested different mechanisms of galactose assimilation. PMID:26523505

  9. Natural variation in non-coding regions underlying phenotypic diversity in budding yeast.

    PubMed

    Salinas, Francisco; de Boer, Carl G; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo, Luis F; Martínez, Claudio; Cubillos, Francisco A

    2016-01-01

    Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype. PMID:26898953

  10. Natural variation in non-coding regions underlying phenotypic diversity in budding yeast

    PubMed Central

    Salinas, Francisco; de Boer, Carl G.; Abarca, Valentina; García, Verónica; Cuevas, Mara; Araos, Sebastian; Larrondo, Luis F.; Martínez, Claudio; Cubillos, Francisco A.

    2016-01-01

    Linkage mapping studies in model organisms have typically focused their efforts in polymorphisms within coding regions, ignoring those within regulatory regions that may contribute to gene expression variation. In this context, differences in transcript abundance are frequently proposed as a source of phenotypic diversity between individuals, however, until now, little molecular evidence has been provided. Here, we examined Allele Specific Expression (ASE) in six F1 hybrids from Saccharomyces cerevisiae derived from crosses between representative strains of the four main lineages described in yeast. ASE varied between crosses with levels ranging between 28% and 60%. Part of the variation in expression levels could be explained by differences in transcription factors binding to polymorphic cis-regulations and to differences in trans-activation depending on the allelic form of the TF. Analysis on highly expressed alleles on each background suggested ASN1 as a candidate transcript underlying nitrogen consumption differences between two strains. Further promoter allele swap analysis under fermentation conditions confirmed that coding and non-coding regions explained aspartic and glutamic acid consumption differences, likely due to a polymorphism affecting Uga3 binding. Together, we provide a new catalogue of variants to bridge the gap between genotype and phenotype. PMID:26898953

  11. A series of conditional shuttle vectors for targeted genomic integration in budding yeast.

    PubMed

    Chou, Chia-Ching; Patel, Michael T; Gartenberg, Marc R

    2015-05-01

    The capacity of Saccharomyces cerevisiae to repair exposed DNA ends by homologous recombination has long been used by experimentalists to assemble plasmids from DNA fragments in vivo. While this approach works well for engineering extrachromosomal vectors, it is not well suited to the generation, recovery and reuse of integrative vectors. Here, we describe the creation of a series of conditional centromeric shuttle vectors, termed pXR vectors, that can be used for both plasmid assembly in vivo and targeted genomic integration. The defining feature of pXR vectors is that the DNA segment bearing the centromere and origin of replication, termed CEN/ARS, is flanked by a pair of loxP sites. Passaging the vectors through bacteria that express Cre recombinase reduces the loxP-CEN/ARS-loxP module to a single loxP site, thereby eliminating the ability to replicate autonomously in yeast. Each vector also contains a selectable marker gene, as well as a fragment of the HO locus, which permits targeted integration at a neutral genomic site. The pXR vectors provide a convenient and robust method to assemble DNAs for targeted genomic modifications.

  12. Cytosolic chaperones mediate quality control of higher-order septin assembly in budding yeast

    PubMed Central

    Johnson, Courtney R.; Weems, Andrew D.; Brewer, Jennifer M.; Thorner, Jeremy; McMurray, Michael A.

    2015-01-01

    Septin hetero-oligomers polymerize into cytoskeletal filaments with essential functions in many eukaryotic cell types. Mutations within the oligomerization interface that encompasses the GTP-binding pocket of a septin (its “G interface”) cause thermoinstability of yeast septin hetero-oligomer assembly, and human disease. When coexpressed with its wild-type counterpart, a G interface mutant is excluded from septin filaments, even at moderate temperatures. We show that this quality control mechanism is specific to G interface mutants, operates during de novo septin hetero-oligomer assembly, and requires specific cytosolic chaperones. Chaperone overexpression lowers the temperature permissive for proliferation of cells expressing a G interface mutant as the sole source of a given septin. Mutations that perturb the septin G interface retard release from these chaperones, imposing a kinetic delay on the availability of nascent septin molecules for higher-order assembly. Un­expectedly, the disaggregase Hsp104 contributes to this delay in a manner that does not require its “unfoldase” activity, indicating a latent “holdase” activity toward mutant septins. These findings provide new roles for chaperone-mediated kinetic partitioning of non-native proteins and may help explain the etiology of septin-linked human diseases. PMID:25673805

  13. Sumoylation of the BLM ortholog, Sgs1, promotes telomere–telomere recombination in budding yeast

    PubMed Central

    Lu, Chia-Yin; Tsai, Cheng-Hui; Brill, Steven J.; Teng, Shu-Chun

    2010-01-01

    BLM and WRN are members of the RecQ family of DNA helicases, and in humans their loss is associated with syndromes characterized by genome instability and cancer predisposition. As the only RecQ DNA helicase in the yeast Saccharomyces cerevisiae, Sgs1 is known to safeguard genome integrity through its role in DNA recombination. Interestingly, WRN, BLM and Sgs1 are all known to be modified by the small ubiquitin-related modifier (SUMO), although the significance of this posttranslational modification remains elusive. Here, we demonstrate that Sgs1 is specifically sumoylated under the stress of DNA double strand breaks. The major SUMO attachment site in Sgs1 is lysine 621, which lies between the Top3 binding domain and the DNA helicase domain. Surprisingly, sumoylation of K621 was found to be uniquely required for Sgs1’s role in telomere–telomere recombination. In contrast, sumoylation was dispensable for Sgs1’s roles in DNA damage tolerance, supppression of direct repeat and rDNA recombination, and promotion of top3Δ slow growth. Our results demonstrate that although modification by SUMO is a conserved feature of RecQ family DNA helicases, the major sites of modification are located on different domains of the protein in different organisms. We suggest that sumoylation of different domains of RecQ DNA helicases from different organisms contributes to conserved roles in regulating telomeric recombination. PMID:19906698

  14. Organelle Size Scaling of the Budding Yeast Vacuole by Relative Growth and Inheritance.

    PubMed

    Chan, Yee-Hung M; Reyes, Lorena; Sohail, Saba M; Tran, Nancy K; Marshall, Wallace F

    2016-05-01

    It has long been noted that larger animals have larger organs compared to smaller animals of the same species, a phenomenon termed scaling [1]. Julian Huxley proposed an appealingly simple model of "relative growth"-in which an organ and the whole body grow with their own intrinsic rates [2]-that was invoked to explain scaling in organs from fiddler crab claws to human brains. Because organ size is regulated by complex, unpredictable pathways [3], it remains unclear whether scaling requires feedback mechanisms to regulate organ growth in response to organ or body size. The molecular pathways governing organelle biogenesis are simpler than organogenesis, and therefore organelle size scaling in the cell provides a more tractable case for testing Huxley's model. We ask the question: is it possible for organelle size scaling to arise if organelle growth is independent of organelle or cell size? Using the yeast vacuole as a model, we tested whether mutants defective in vacuole inheritance, vac8Δ and vac17Δ, tune vacuole biogenesis in response to perturbations in vacuole size. In vac8Δ/vac17Δ, vacuole scaling increases with the replicative age of the cell. Furthermore, vac8Δ/vac17Δ cells continued generating vacuole at roughly constant rates even when they had significantly larger vacuoles compared to wild-type. With support from computational modeling, these results suggest there is no feedback between vacuole biogenesis rates and vacuole or cell size. Rather, size scaling is determined by the relative growth rates of the vacuole and the cell, thus representing a cellular version of Huxley's model.

  15. Regulation of Cdc42 polarization by the Rsr1 GTPase and Rga1, a Cdc42 GTPase-activating protein, in budding yeast

    PubMed Central

    Lee, Mid Eum; Lo, Wing-Cheong; Miller, Kristi E.; Chou, Ching-Shan; Park, Hay-Oak

    2015-01-01

    ABSTRACT Cdc42 plays a central role in establishing polarity in yeast and animals, yet how polarization of Cdc42 is achieved in response to spatial cues is poorly understood. Using live-cell imaging, we found distinct dynamics of Cdc42 polarization in haploid budding yeast in correlation with two temporal steps of the G1 phase. The position at which the Cdc42–GTP cluster develops changes rapidly around the division site during the first step but becomes stabilized in the second step, suggesting that an axis of polarized growth is determined in mid G1. Cdc42 polarization in the first step and its proper positioning depend on Rsr1 and its GTPase-activating protein (GAP) Bud2. Interestingly, Rga1, a Cdc42 GAP, exhibits transient localization to a site near the bud neck and to the division site during cytokinesis and G1, and this temporal change of Rga1 distribution is necessary for determination of a proper growth site. Mathematical modeling suggests that a proper axis of Cdc42 polarization in haploid cells might be established through a biphasic mechanism involving sequential positive feedback and transient negative feedback. PMID:25908844

  16. A septin from the filamentous fungus A. nidulans induces atypical pseudohyphae in the budding yeast S. cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Septins were first discovered in Saccharomyces cerevisiae where they form a scaffold that organizes the bud site and are a component of the morphogenesis checkpoint that coordinates budding with mitosis. Five of the seven S. cerevisiae septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) colocalize as a rin...

  17. The Rim15-Endosulfine-PP2ACdc55 Signalling Module Regulates Entry into Gametogenesis and Quiescence via Distinct Mechanisms in Budding Yeast

    PubMed Central

    Sarkar, Sourav; Dalgaard, Jacob Z.; Millar, Jonathan B. A.; Arumugam, Prakash

    2014-01-01

    Quiescence and gametogenesis represent two distinct survival strategies in response to nutrient starvation in budding yeast. Precisely how environmental signals are sensed by yeast cells to trigger quiescence and gametogenesis is not fully understood. A conserved signalling module consisting of Greatwall kinase, Endosulfine and Protein Phosphatase PP2ACdc55 proteins regulates entry into mitosis in Xenopus egg extracts and meiotic maturation in flies. We report here that an analogous signalling module consisting of the serine-threonine kinase Rim15, the Endosulfines Igo1 and Igo2 and the Protein Phosphatase PP2ACdc55, regulates entry into both quiescence and gametogenesis in budding yeast. PP2ACdc55 inhibits entry into gametogenesis and quiescence. Rim15 promotes entry into gametogenesis and quiescence by converting Igo1 into an inhibitor of PP2ACdc55 by phosphorylating at a conserved serine residue. Moreover, we show that the Rim15-Endosulfine-PP2ACdc55 pathway regulates entry into quiescence and gametogenesis by distinct mechanisms. In addition, we show that Igo1 and Igo2 are required for pre-meiotic autophagy but the lack of pre-meiotic autophagy is insufficient to explain the sporulation defect of igo1Δ igo2Δ cells. We propose that the Rim15-Endosulfine-PP2ACdc55 signalling module triggers entry into quiescence and gametogenesis by regulating dephosphorylation of distinct substrates. PMID:24968058

  18. Modelling of Yeast Mating Reveals Robustness Strategies for Cell-Cell Interactions.

    PubMed

    Chen, Weitao; Nie, Qing; Yi, Tau-Mu; Chou, Ching-Shan

    2016-07-01

    Mating of budding yeast cells is a model system for studying cell-cell interactions. Haploid yeast cells secrete mating pheromones that are sensed by the partner which responds by growing a mating projection toward the source. The two projections meet and fuse to form the diploid. Successful mating relies on precise coordination of dynamic extracellular signals, signaling pathways, and cell shape changes in a noisy background. It remains elusive how cells mate accurately and efficiently in a natural multi-cell environment. Here we present the first stochastic model of multiple mating cells whose morphologies are driven by pheromone gradients and intracellular signals. Our novel computational framework encompassed a moving boundary method for modeling both a-cells and α-cells and their cell shape changes, the extracellular diffusion of mating pheromones dynamically coupled with cell polarization, and both external and internal noise. Quantification of mating efficiency was developed and tested for different model parameters. Computer simulations revealed important robustness strategies for mating in the presence of noise. These strategies included the polarized secretion of pheromone, the presence of the α-factor protease Bar1, and the regulation of sensing sensitivity; all were consistent with data in the literature. In addition, we investigated mating discrimination, the ability of an a-cell to distinguish between α-cells either making or not making α-factor, and mating competition, in which multiple a-cells compete to mate with one α-cell. Our simulations were consistent with previous experimental results. Moreover, we performed a combination of simulations and experiments to estimate the diffusion rate of the pheromone a-factor. In summary, we constructed a framework for simulating yeast mating with multiple cells in a noisy environment, and used this framework to reproduce mating behaviors and to identify strategies for robust cell-cell interactions. PMID

  19. Modelling of Yeast Mating Reveals Robustness Strategies for Cell-Cell Interactions

    PubMed Central

    Chen, Weitao; Nie, Qing; Yi, Tau-Mu; Chou, Ching-Shan

    2016-01-01

    Mating of budding yeast cells is a model system for studying cell-cell interactions. Haploid yeast cells secrete mating pheromones that are sensed by the partner which responds by growing a mating projection toward the source. The two projections meet and fuse to form the diploid. Successful mating relies on precise coordination of dynamic extracellular signals, signaling pathways, and cell shape changes in a noisy background. It remains elusive how cells mate accurately and efficiently in a natural multi-cell environment. Here we present the first stochastic model of multiple mating cells whose morphologies are driven by pheromone gradients and intracellular signals. Our novel computational framework encompassed a moving boundary method for modeling both a-cells and α-cells and their cell shape changes, the extracellular diffusion of mating pheromones dynamically coupled with cell polarization, and both external and internal noise. Quantification of mating efficiency was developed and tested for different model parameters. Computer simulations revealed important robustness strategies for mating in the presence of noise. These strategies included the polarized secretion of pheromone, the presence of the α-factor protease Bar1, and the regulation of sensing sensitivity; all were consistent with data in the literature. In addition, we investigated mating discrimination, the ability of an a-cell to distinguish between α-cells either making or not making α-factor, and mating competition, in which multiple a-cells compete to mate with one α-cell. Our simulations were consistent with previous experimental results. Moreover, we performed a combination of simulations and experiments to estimate the diffusion rate of the pheromone a-factor. In summary, we constructed a framework for simulating yeast mating with multiple cells in a noisy environment, and used this framework to reproduce mating behaviors and to identify strategies for robust cell-cell interactions. PMID

  20. The Ddc1-Mec3-Rad17 Sliding Clamp Regulates Histone-Histone Chaperone Interactions and DNA Replication-coupled Nucleosome Assembly in Budding Yeast*

    PubMed Central

    Burgess, Rebecca J.; Han, Junhong; Zhang, Zhiguo

    2014-01-01

    The maintenance of genome integrity is regulated in part by chromatin structure and factors involved in the DNA damage response pathway. Nucleosome assembly is a highly regulated process that restores chromatin structure after DNA replication, DNA repair, and gene transcription. During S phase the histone chaperones Asf1, CAF-1, and Rtt106 coordinate to deposit newly synthesized histones H3-H4 onto replicated DNA in budding yeast. Here we describe synthetic genetic interactions between RTT106 and the DDC1-MEC3-RAD17 (9-1-1) complex, a sliding clamp functioning in the S phase DNA damage and replication checkpoint response, upon treatment with DNA damaging agents. The DNA damage sensitivity of rad17Δ rtt106Δ cells depends on the function of Rtt106 in nucleosome assembly. Epistasis analysis reveals that 9-1-1 complex components interact with multiple DNA replication-coupled nucleosome assembly factors, including Rtt106, CAF-1, and lysine residues of H3-H4. Furthermore, rad17Δ cells exhibit defects in the deposition of newly synthesized H3-H4 onto replicated DNA. Finally, deletion of RAD17 results in increased association of Asf1 with checkpoint kinase Rad53, which may lead to the observed reduction in Asf1-H3 interaction in rad17Δ mutant cells. In addition, we observed that the interaction between histone H3-H4 with histone chaperone CAF-1 or Rtt106 increases in cells lacking Rad17. These results support the idea that the 9-1-1 checkpoint protein regulates DNA replication-coupled nucleosome assembly in part through regulating histone-histone chaperone interactions. PMID:24573675

  1. Budding yeast protein extraction and purification for the study of function, interactions, and post-translational modifications.

    PubMed

    Szymanski, Eva Paige; Kerscher, Oliver

    2013-01-01

    Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and

  2. A Visual Screen of Protein Localization during Sporulation Identifies New Components of Prospore Membrane-Associated Complexes in Budding Yeast

    PubMed Central

    Lam, Chien; Santore, Ethan; Lavoie, Elizabeth; Needleman, Leor; Fiacco, Nicholas; Kim, Carey

    2014-01-01

    During ascospore formation in Saccharomyces cerevisiae, the secretory pathway is reorganized to create new intracellular compartments, termed prospore membranes. Prospore membranes engulf the nuclei produced by the meiotic divisions, giving rise to individual spores. The shape and growth of prospore membranes are constrained by cytoskeletal structures, such as septin proteins, that associate with the membranes. Green fluorescent protein (GFP) fusions to various proteins that associate with septins at the bud neck during vegetative growth as well as to proteins encoded by genes that are transcriptionally induced during sporulation were examined for their cellular localization during prospore membrane growth. We report localizations for over 100 different GFP fusions, including over 30 proteins localized to the prospore membrane compartment. In particular, the screen identified IRC10 as a new component of the leading-edge protein complex (LEP), a ring structure localized to the lip of the prospore membrane. Localization of Irc10 to the leading edge is dependent on SSP1, but not ADY3. Loss of IRC10 caused no obvious phenotype, but an ady3 irc10 mutant was completely defective in sporulation and displayed prospore membrane morphologies similar to those of an ssp1 strain. These results reveal the architecture of the LEP and provide insight into the evolution of this membrane-organizing complex. PMID:24390141

  3. A role for the rap GTPase YlRsr1 in cellular morphogenesis and the involvement of YlRsr1 and the ras GTPase YlRas2 in bud site selection in the dimorphic yeast Yarrowia lipolytica.

    PubMed

    Li, Yun-Qing; Li, Min; Zhao, Xiao-Feng; Gao, Xiang-Dong

    2014-05-01

    Yarrowia lipolytica is a dimorphic yeast species that can grow in the ovoid yeast form or in the elongated pseudohyphal or hyphal form depending on the growth conditions. Here, we show that the Rap GTPase Rsr1 of Y. lipolytica (YlRsr1) plays an important role in cellular morphogenesis in this microorganism. Cells deleted for YlRSR1 exhibited impaired polarized growth during yeast-form growth. Pseudohyphal and hyphal development were also abnormal. YlRsr1 is also important for cell growth, since the deletion of YlRSR1 in cells lacking the Ras GTPase YlRas2 caused lethality. Y. lipolytica cells bud in a bipolar pattern in which the cells produce the new buds at the two poles. YlRsr1 plays a prominent role in this bud site selection process. YlRsr1's function in bud site selection absolutely requires the cycling of YlRsr1 between the GTP- and GDP-bound states but its function in cellular morphogenesis does not, suggesting that the two processes are differentially regulated. Interestingly, the Ras GTPase YlRas2 is also involved in the control of bud site selection, as Ylras2Δ cells were severely impaired in bipolar bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2 are important for YlRas2's function in bud site selection. However, they are not essential for this process, suggesting that the mechanism by which YlRas2 acts is different from that of YlRsr1. Our results suggest that YlRsr1 is regulated by the GTPase-activating protein (GAP) YlBud2 and partially by YlCdc25, the potential guanine nucleotide exchange factor (GEF) for YlRas2.

  4. Structural and functional studies of Bud23-Trm112 reveal 18S rRNA N7-G1575 methylation occurs on late 40S precursor ribosomes.

    PubMed

    Létoquart, Juliette; Huvelle, Emmeline; Wacheul, Ludivine; Bourgeois, Gabrielle; Zorbas, Christiane; Graille, Marc; Heurgué-Hamard, Valérie; Lafontaine, Denis L J

    2014-12-23

    The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N(7)-methylguanosine (m(7)G) introduced at position 1575 on 18S rRNA by Bud23-Trm112 is at a ridge forming a steric block between P- and E-site tRNAs. Here we report atomic resolution structures of Bud23-Trm112 in the apo and S-adenosyl-L-methionine (SAM)-bound forms. Bud23 and Trm112 interact through formation of a β-zipper involving main-chain atoms, burying an important hydrophobic surface and stabilizing the complex. The structures revealed that the coactivator Trm112 undergoes an induced fit to accommodate its methyltransferase (MTase) partner. We report important structural similarity between the active sites of Bud23 and Coffea canephora xanthosine MTase, leading us to propose and validate experimentally a model for G1575 coordination. We identify Bud23 residues important for Bud23-Trm112 complex formation and recruitment to pre-ribosomes. We report that though Bud23-Trm112 binds precursor ribosomes at an early nucleolar stage, m(7)G methylation occurs at a late step of small subunit biogenesis, implying specifically delayed catalytic activation. Finally, we show that Bud23-Trm112 interacts directly with the box C/D snoRNA U3-associated DEAH RNA helicase Dhr1 supposedly involved in central pseudoknot formation; this suggests that Bud23-Trm112 might also contribute to controlling formation of this irreversible and dramatic structural reorganization essential to overall folding of small subunit rRNA. Our study contributes important new elements to our understanding of key molecular aspects of human ribosomopathy syndromes associated with WBSCR22 (human Bud23) malfunction.

  5. RIM15 antagonistic pleiotropy is responsible for differences in fermentation and stress response kinetics in budding yeast.

    PubMed

    Kessi-Pérez, Eduardo I; Araos, Sebastián; García, Verónica; Salinas, Francisco; Abarca, Valentina; Larrondo, Luis F; Martínez, Claudio; Cubillos, Francisco A

    2016-05-01

    Different natural yeast populations have faced dissimilar selective pressures due to the heterogeneous fermentation substrates available around the world; this increases the genetic and phenotypic diversity in Saccharomyces cerevisiae In this context, we expect prominent differences between isolates when exposed to a particular condition, such as wine or sake musts. To better comprehend the mechanisms underlying niche adaptation between two S. cerevisiae isolates obtained from wine and sake fermentation processes, we evaluated fermentative and fungicide resistance phenotypes and identify the molecular origin of such adaptive variation. Multiple regions were associated with fermentation rate under different nitrogen conditions and fungicide resistance, with a single QTL co-localizing in all traits. Analysis around this region identified RIM15 as the causative locus driving fungicide sensitivity, together with efficient nitrogen utilization and glycerol production in the wine strain. A null RIM15 variant confers a greater fermentation rate through the utilization of available glucose instead of its storage. However, this variant has a detrimental effect on fungicide resistance since complex sugars are not synthesized and transported into the membrane. Together, our results reveal the antagonist pleiotropic nature of a RIM15 null variant, positively affecting a series of fermentation related phenotypes, but apparently detrimental in the wild. PMID:26945894

  6. Integrative phenomics reveals insight into the structure of phenotypic diversity in budding yeast

    PubMed Central

    Skelly, Daniel A.; Merrihew, Gennifer E.; Riffle, Michael; Connelly, Caitlin F.; Kerr, Emily O.; Johansson, Marnie; Jaschob, Daniel; Graczyk, Beth; Shulman, Nicholas J.; Wakefield, Jon; Cooper, Sara J.; Fields, Stanley; Noble, William S.; Muller, Eric G.D.; Davis, Trisha N.; Dunham, Maitreya J.; MacCoss, Michael J.; Akey, Joshua M.

    2013-01-01

    To better understand the quantitative characteristics and structure of phenotypic diversity, we measured over 14,000 transcript, protein, metabolite, and morphological traits in 22 genetically diverse strains of Saccharomyces cerevisiae. More than 50% of all measured traits varied significantly across strains [false discovery rate (FDR) = 5%]. The structure of phenotypic correlations is complex, with 85% of all traits significantly correlated with at least one other phenotype (median = 6, maximum = 328). We show how high-dimensional molecular phenomics data sets can be leveraged to accurately predict phenotypic variation between strains, often with greater precision than afforded by DNA sequence information alone. These results provide new insights into the spectrum and structure of phenotypic diversity and the characteristics influencing the ability to accurately predict phenotypes. PMID:23720455

  7. Human SEC13Rp functions in yeast and is located on transport vesicles budding from the endoplasmic reticulum

    PubMed Central

    1995-01-01

    In the yeast Saccharomyces cerevisiae, Sec13p is required for intracellular protein transport from the ER to the Golgi apparatus, and has also been identified as a component of the COPII vesicle coat structure. Recently, a human cDNA encoding a protein 53% identical to yeast Sec13p has been isolated. In this report, we apply the genetic assays of complementation and synthetic lethality to demonstrate the conservation of function between this human protein, designated SEC13Rp, and yeast Sec13p. We show that two reciprocal human/yeast fusion constructs, encoding the NH2-terminal half of one protein and the COOH-terminal half of the other, can each complement the secretion defect of a sec13-1 mutant at 36 degrees C. The chimera encoding the NH2-terminal half of the yeast protein and the COOH-terminal half of the human protein is also able to complement a SEC13 deletion. Overexpression of either the entire human SEC13Rp protein or the chimera encoding the NH2-terminal half of the human protein and the COOH-terminal half of the yeast protein inhibits the growth of a sec13- 1 mutant at 24 degrees C; this growth inhibition is not seen in a wild- type strain nor in other sec mutants, suggesting that the NH2-terminal half of SEC13Rp may compete with Sec13-1p for a common target. We show by immunoelectronmicroscopy of mammalian cells that SEC13Rp (like the putative mammalian homologues of the COPII subunits Sar1p and Sec23p) resides in the region of the transitional ER. We also show that the distribution of SEC13Rp is not affected by brefeldin A treatment. This report presents the first demonstration of a putative mammalian COPII component functioning in yeast, and highlights a potentially useful approach for the study of conserved mammalian proteins in a genetically tractable system. PMID:7876304

  8. Ca(2+) homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca(2+) storage.

    PubMed

    D'hooge, Petra; Coun, Catherina; Van Eyck, Vincent; Faes, Liesbeth; Ghillebert, Ruben; Mariën, Lore; Winderickx, Joris; Callewaert, Geert

    2015-08-01

    Yeast has proven to be a powerful tool to elucidate the molecular aspects of several biological processes in higher eukaryotes. As in mammalian cells, yeast intracellular Ca(2+) signalling is crucial for a myriad of biological processes. Yeast cells also bear homologs of the major components of the Ca(2+) signalling toolkit in mammalian cells, including channels, co-transporters and pumps. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar Ca(2+) transporters, combined with manipulations to estimate intracellular Ca(2+) storage, we evaluated the contribution of individual transport systems to intracellular Ca(2+) homeostasis. Yeast strains lacking Pmr1 and/or Cod1, two ion pumps implicated in ER/Golgi Ca(2+) homeostasis, displayed a fragmented vacuolar phenotype and showed increased vacuolar Ca(2+) uptake and Ca(2+) influx across the plasma membrane. In the pmr1Δ strain, these effects were insensitive to calcineurin activity, independent of Cch1/Mid1 Ca(2+) channels and Pmc1 but required Vcx1. By contrast, in the cod1Δ strain increased vacuolar Ca(2+) uptake was not affected by Vcx1 deletion but was largely dependent on Pmc1 activity. Our analysis further corroborates the distinct roles of Vcx1 and Pmc1 in vacuolar Ca(2+) uptake and point to the existence of not-yet identified Ca(2+) influx pathways.

  9. Asymmetry of the Budding Yeast Tem1 GTPase at Spindle Poles Is Required for Spindle Positioning But Not for Mitotic Exit

    PubMed Central

    Scarfone, Ilaria; Venturetti, Marianna; Hotz, Manuel; Lengefeld, Jette; Barral, Yves; Piatti, Simonetta

    2015-01-01

    The asymmetrically dividing yeast S. cerevisiae assembles a bipolar spindle well after establishing the future site of cell division (i.e., the bud neck) and the division axis (i.e., the mother-bud axis). A surveillance mechanism called spindle position checkpoint (SPOC) delays mitotic exit and cytokinesis until the spindle is properly positioned relative to the mother-bud axis, thereby ensuring the correct ploidy of the progeny. SPOC relies on the heterodimeric GTPase-activating protein Bub2/Bfa1 that inhibits the small GTPase Tem1, in turn essential for activating the mitotic exit network (MEN) kinase cascade and cytokinesis. The Bub2/Bfa1 GAP and the Tem1 GTPase form a complex at spindle poles that undergoes a remarkable asymmetry during mitosis when the spindle is properly positioned, with the complex accumulating on the bud-directed old spindle pole. In contrast, the complex remains symmetrically localized on both poles of misaligned spindles. The mechanism driving asymmetry of Bub2/Bfa1/Tem1 in mitosis is unclear. Furthermore, whether asymmetry is involved in timely mitotic exit is controversial. We investigated the mechanism by which the GAP Bub2/Bfa1 controls GTP hydrolysis on Tem1 and generated a series of mutants leading to constitutive Tem1 activation. These mutants are SPOC-defective and invariably lead to symmetrical localization of Bub2/Bfa1/Tem1 at spindle poles, indicating that GTP hydrolysis is essential for asymmetry. Constitutive tethering of Bub2 or Bfa1 to both spindle poles impairs SPOC response but does not impair mitotic exit. Rather, it facilitates mitotic exit of MEN mutants, likely by increasing the residence time of Tem1 at spindle poles where it gets active. Surprisingly, all mutant or chimeric proteins leading to symmetrical localization of Bub2/Bfa1/Tem1 lead to increased symmetry at spindle poles of the Kar9 protein that mediates spindle positioning and cause spindle misalignment. Thus, asymmetry of the Bub2/Bfa1/Tem1 complex is

  10. Timed Deletion of Twist1 in the Limb Bud Reveals Age-Specific Impacts on Autopod and Zeugopod Patterning

    PubMed Central

    Loebel, David A. F.; Hor, Angelyn C. C.; Bildsoe, Heidi K.; Tam, Patrick P. L.

    2014-01-01

    Twist1 encodes a transcription factor that plays a vital role in limb development. We have used a tamoxifen-inducible Cre transgene, Ubc-CreERT2, to generate time-specific deletions of Twist1 by inducing Cre activity in mouse embryos at different ages from embryonic (E) day 9.5 onwards. A novel forelimb phenotype of supernumerary pre-axial digits and enlargement or partial duplication of the distal radius was observed when Cre activity was induced at E9.5. Gene expression analysis revealed significant upregulation of Hoxd10, Hoxd11 and Grem1 in the anterior half of the forelimb bud at E11.5. There is also localized upregulation of Ptch1, Hand2 and Hoxd13 at the site of ectopic digit formation, indicating a posterior molecular identity for the supernumerary digits. The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other Twist1 conditional knockout mutants. This outcome may be attributed to the deferment of Twist1 ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud. PMID:24893291

  11. Deletion of a Cys-His motif from the Alpharetrovirus nucleocapsid domain reveals late domain mutant-like budding defects

    SciTech Connect

    Lee, Eun-Gyung; Linial, Maxine L. . E-mail: mlinial@fhcrc.org

    2006-03-30

    The Rous sarcoma virus (RSV) Gag polyprotein is the only protein required for virus assembly and release. We previously found that deletion of either one of the two Cys-His (CH) motifs in the RSV nucleocapsid (NC) protein did not abrogate Gag-Gag interactions, RNA binding, or packaging but greatly reduced virus production (E-G. Lee, A. Alidina et al., J. Virol. 77: 2010-2020, 2003). In this report, we have further investigated the effects of mutations in the CH motifs on virus assembly and release. Precise deletion of either CH motif, without affecting surrounding basic residues, reduced virus production by approximately 10-fold, similar to levels seen for late (L) domain mutants. Strikingly, transmission electron microscopy revealed that virions of both {delta}CH1 and {delta}CH2 mutants were assembled normally at the plasma membrane but were arrested in budding. Virus particles remained tethered to the membrane or to each other, reminiscent of L domain mutants, although the release defect appears to be independent of the L domain functions. Therefore, two CH motifs are likely to be required for budding independent of a requirement for either Gag-Gag interactions or RNA packaging.

  12. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation

    SciTech Connect

    Ninomiya-Tsuji, Jun ); Nomoto, Satoshi; Matsumoto, Kunihiro ); Yasuda, Hideyo ); Reed, S.I. )

    1991-10-15

    The authors have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34{sup CDC2} kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34{sup CDC2} kinase and more significantly is homologous to Xenopus Eg1 kinase, suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34{sup CDC2} kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G{sub 1} or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages.

  13. PRIMED: PRIMEr database for deleting and tagging all fission and budding yeast genes developed using the open-source genome retrieval script (GRS).

    PubMed

    Cummings, Michael T; Joh, Richard I; Motamedi, Mo

    2015-01-01

    The fission (Schizosaccharomyces pombe) and budding (Saccharomyces cerevisiae) yeasts have served as excellent models for many seminal discoveries in eukaryotic biology. In these organisms, genes are deleted or tagged easily by transforming cells with PCR-generated DNA inserts, flanked by short (50-100 bp) regions of gene homology. These PCR reactions use especially designed long primers, which, in addition to the priming sites, carry homology for gene targeting. Primer design follows a fixed method but is tedious and time-consuming especially when done for a large number of genes. To automate this process, we developed the Python-based Genome Retrieval Script (GRS), an easily customizable open-source script for genome analysis. Using GRS, we created PRIMED, the complete PRIMEr D atabase for deleting and C-terminal tagging genes in the main S. pombe and five of the most commonly used S. cerevisiae strains. Because of the importance of noncoding RNAs (ncRNAs) in many biological processes, we also included the deletion primer set for these features in each genome. PRIMED are accurate and comprehensive and are provided as downloadable Excel files, removing the need for future primer design, especially for large-scale functional analyses. Furthermore, the open-source GRS can be used broadly to retrieve genome information from custom or other annotated genomes, thus providing a suitable platform for building other genomic tools by the yeast or other research communities.

  14. Replication and active demethylation represent partially overlapping mechanisms for erasure of H3K4me3 in budding yeast.

    PubMed

    Radman-Livaja, Marta; Liu, Chih Long; Friedman, Nir; Schreiber, Stuart L; Rando, Oliver J

    2010-02-01

    Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as "long-lived" and that is involved in memory during cell state inheritance in metazoans. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, nearly complete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast. PMID:20140185

  15. Peroxiredoxin-mediated redox regulation of the nuclear localization of Yap1, a transcription factor in budding yeast.

    PubMed

    Okazaki, Shoko; Naganuma, Akira; Kuge, Shusuke

    2005-01-01

    A redox reaction involving cysteine thiol-disulfide exchange is crucial for the intracellular monitoring of oxidation status. The yeast transcription factor Yap1 is activated by formation of a disulfide bond, which inhibits nuclear export in response to peroxide stress, with resultant enhancement of the nuclear localization of Yap1. A glutathione peroxidase-like protein, Gpx3, which has peroxiredoxin activity, is required for formation of the disulfide bond in Yap1. We show here that the requirement for Gpx3 in the regulation of Yap1 is strain-specific. Thus, Tsa1, a ubiquitous thioredoxin peroxidase, is required for the activation of Yap1 in yeast strain Y700, which is derived from W303. The strain-specific utilization of different peroxiredoxins appears to be determined by Ybp1, a Yap1-binding protein. The Ybp1 of Y700 has a nonsense mutation, and a wild-type YBP1 gene can restore the Gpx3-dependent activation of Yap1. These results suggest that Tsa1, a ubiquitous peroxiredoxin, has the potential for transducing redox signals to a particular sensor protein. PMID:15706081

  16. PP2ACdc55’s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function

    PubMed Central

    Kerr, Gary W.; Wong, Jin Huei; Arumugam, Prakash

    2016-01-01

    PP2ACdc55 is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2ACdc55 activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2ACdc55 prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2ACdc55’s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function. PMID:27455870

  17. PP2A(Cdc55)'s role in reductional chromosome segregation during achiasmate meiosis in budding yeast is independent of its FEAR function.

    PubMed

    Kerr, Gary W; Wong, Jin Huei; Arumugam, Prakash

    2016-07-26

    PP2A(Cdc55) is a highly conserved serine-threonine protein phosphatase that is involved in diverse cellular processes. In budding yeast, meiotic cells lacking PP2A(Cdc55) activity undergo a premature exit from meiosis I which results in a failure to form bipolar spindles and divide nuclei. This defect is largely due to its role in negatively regulating the Cdc Fourteen Early Anaphase Release (FEAR) pathway. PP2A(Cdc55) prevents nucleolar release of the Cdk (Cyclin-dependent kinase)-antagonising phosphatase Cdc14 by counteracting phosphorylation of the nucleolar protein Net1 by Cdk. CDC55 was identified in a genetic screen for monopolins performed by isolating suppressors of spo11Δ spo12Δ lethality suggesting that Cdc55 might have a role in meiotic chromosome segregation. We investigated this possibility by isolating cdc55 alleles that suppress spo11Δ spo12Δ lethality and show that this suppression is independent of PP2A(Cdc55)'s FEAR function. Although the suppressor mutations in cdc55 affect reductional chromosome segregation in the absence of recombination, they have no effect on chromosome segregation during wild type meiosis. We suggest that Cdc55 is required for reductional chromosome segregation during achiasmate meiosis and this is independent of its FEAR function.

  18. Mps1 and Ipl1/Aurora B Act Sequentially to Correctly Orient Chromosomes on the Meiotic Spindle of Budding Yeast

    PubMed Central

    Meyer, Régis E.; Kim, Seoyoung; Obeso, David; Straight, Paul D.; Winey, Mark; Dawson, Dean S.

    2013-01-01

    The conserved kinases Mps1 and Ipl1/Aurora B are critical for enabling chromosomes to attach to microtubules such that partner chromosomes will be segregated correctly from each other, but the precise roles of these kinases have been unclear. Here, imaging of live yeast cells was performed to elucidate the stages of chromosome-microtubule interactions, and their regulation by Ipl1 and Mps1, through meiosis I. Ipl1 was found to release kinetochore-microtubule (kMT) associations following meiotic entry, liberating chromosomes to begin homologous pairing. Surprisingly, most chromosome pairs were found to begin their spindle interactions with incorrect kMT attachments. Ipl1 released these improper connections while Mps1 triggered the formation of new force-generating microtubule attachments. This microtubule release and reattachment cycle can prevent catastrophic chromosome segregation errors in meiosis. PMID:23371552

  19. Osmolarity hypersensitivity of hog1 deleted mutants is suppressed by mutation in KSS1 in budding yeast Saccharomyces cerevisiae.

    PubMed

    Lee, Soo-Jung; Park, Shi-Young; Na, Jong-Gil; Kim, Yung-Jin

    2002-03-19

    An osmosensing mechanism of Saccharomyces cerevisiae involves a mitogen-activated protein kinase (MAPK) cascade (HOG pathway). This study aimed to investigate the response of the yeast to osmotic stress. A mutant strain, in which the HOG1 gene was disrupted by TRP1, was constructed. A spontaneous mutant, named YJY45, which suppresses the osmosensitive growth phenotype of the hog1 deletion mutant, was selected and showed a secondary phenotype of temperature sensitivity on YPD containing 0.5 M NaCl at 37 degrees C. Our data indicate that the spontaneous mutation in YJY45 mutant was mapped in KSS1, which is one of the MAPK family. The mutation in KSS1 suppresses the osmolarity-hypersensitive phenotype of the hog1 deletion mutation and restores GPD1 induction. PMID:12007647

  20. Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

    PubMed

    Minakawa, Tomoko; Ueda, Keiichi; Tanaka, Miyuu; Tanaka, Natsuki; Kuwamura, Mitsuru; Izawa, Takeshi; Konno, Toshihiro; Yamate, Jyoji; Itano, Eiko Nakagawa; Sano, Ayako; Wada, Shinpei

    2016-08-01

    Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

  1. Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

    PubMed

    Minakawa, Tomoko; Ueda, Keiichi; Tanaka, Miyuu; Tanaka, Natsuki; Kuwamura, Mitsuru; Izawa, Takeshi; Konno, Toshihiro; Yamate, Jyoji; Itano, Eiko Nakagawa; Sano, Ayako; Wada, Shinpei

    2016-08-01

    Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin. PMID:26883513

  2. Membrane bioreactor wastewater treatment plants reveal diverse yeast and protist communities of potential significance in biofouling.

    PubMed

    Liébana, Raquel; Arregui, Lucía; Belda, Ignacio; Gamella, Luis; Santos, Antonio; Marquina, Domingo; Serrano, Susana

    2015-01-01

    The yeast community was studied in a municipal full-scale membrane bioreactor wastewater treatment plant (MBR-WWTP). The unexpectedly high diversity of yeasts indicated that the activated sludge formed a suitable environment for them to proliferate, with cellular concentrations of 2.2 ± 0.8 × 10(3) CFU ml(-1). Sixteen species of seven genera were present in the biological reactor, with Ascomycetes being the most prevalent group (93%). Most isolates were able to grow in a synthetic wastewater medium, adhere to polyethylene surfaces, and develop biofilms of variable complexity. The relationship between yeast populations and the protists in the MBR-WWTP was also studied, revealing that some protist species preyed on and ingested yeasts. These results suggest that yeast populations may play a role in the food web of a WWTP and, to some extent, contribute to membrane biofouling in MBR systems. PMID:25588128

  3. Membrane bioreactor wastewater treatment plants reveal diverse yeast and protist communities of potential significance in biofouling.

    PubMed

    Liébana, Raquel; Arregui, Lucía; Belda, Ignacio; Gamella, Luis; Santos, Antonio; Marquina, Domingo; Serrano, Susana

    2015-01-01

    The yeast community was studied in a municipal full-scale membrane bioreactor wastewater treatment plant (MBR-WWTP). The unexpectedly high diversity of yeasts indicated that the activated sludge formed a suitable environment for them to proliferate, with cellular concentrations of 2.2 ± 0.8 × 10(3) CFU ml(-1). Sixteen species of seven genera were present in the biological reactor, with Ascomycetes being the most prevalent group (93%). Most isolates were able to grow in a synthetic wastewater medium, adhere to polyethylene surfaces, and develop biofilms of variable complexity. The relationship between yeast populations and the protists in the MBR-WWTP was also studied, revealing that some protist species preyed on and ingested yeasts. These results suggest that yeast populations may play a role in the food web of a WWTP and, to some extent, contribute to membrane biofouling in MBR systems.

  4. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts.

    PubMed

    Mehlgarten, Constance; Krijger, Jorrit-Jan; Lemnian, Ioana; Gohr, André; Kasper, Lydia; Diesing, Anne-Kathrin; Grosse, Ivo; Breunig, Karin D

    2015-01-01

    Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to

  5. Divergent Evolution of the Transcriptional Network Controlled by Snf1-Interacting Protein Sip4 in Budding Yeasts

    PubMed Central

    Mehlgarten, Constance; Krijger, Jorrit-Jan; Lemnian, Ioana; Gohr, André; Kasper, Lydia; Diesing, Anne-Kathrin; Grosse, Ivo; Breunig, Karin D.

    2015-01-01

    Cellular responses to starvation are of ancient origin since nutrient limitation has always been a common challenge to the stability of living systems. Hence, signaling molecules involved in sensing or transducing information about limiting metabolites are highly conserved, whereas transcription factors and the genes they regulate have diverged. In eukaryotes the AMP-activated protein kinase (AMPK) functions as a central regulator of cellular energy homeostasis. The yeast AMPK ortholog SNF1 controls the transcriptional network that counteracts carbon starvation conditions by regulating a set of transcription factors. Among those Cat8 and Sip4 have overlapping DNA-binding specificity for so-called carbon source responsive elements and induce target genes upon SNF1 activation. To analyze the evolution of the Cat8-Sip4 controlled transcriptional network we have compared the response to carbon limitation of Saccharomyces cerevisiae to that of Kluyveromyces lactis. In high glucose, S. cerevisiae displays tumor cell-like aerobic fermentation and repression of respiration (Crabtree-positive) while K. lactis has a respiratory-fermentative life-style, respiration being regulated by oxygen availability (Crabtree-negative), which is typical for many yeasts and for differentiated higher cells. We demonstrate divergent evolution of the Cat8-Sip4 network and present evidence that a role of Sip4 in controlling anabolic metabolism has been lost in the Saccharomyces lineage. We find that in K. lactis, but not in S. cerevisiae, the Sip4 protein plays an essential role in C2 carbon assimilation including induction of the glyoxylate cycle and the carnitine shuttle genes. Induction of KlSIP4 gene expression by KlCat8 is essential under these growth conditions and a primary function of KlCat8. Both KlCat8 and KlSip4 are involved in the regulation of lactose metabolism in K. lactis. In chromatin-immunoprecipitation experiments we demonstrate binding of both, KlSip4 and KlCat8, to

  6. Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Kron, S J; Styles, C A; Fink, G R

    1994-01-01

    Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and daughters bud asynchronously. Mothers bud immediately but daughters grow in G1 until they achieve a critical cell size. By contrast, PH cells divide symmetrically, restricting mitosis until the bud grows to the size of the mother. Thus, mother and daughter bud synchronously in the next cycle, without a G1 delay before Start. YF and PH cells also exhibit distinct bud-site selection patterns. YF cells are bipolar, producing their second and subsequent buds at either pole. PH cells are unipolar, producing their second and subsequent buds only from the end opposite the junction with their mother. We propose that in PH cells a G2 cell-size checkpoint delays mitosis until bud size reaches that of the mother cell. We conclude that yeast and PH forms are distinct cell types each with a unique cell cycle, budding pattern, and cell shape. Images PMID:7841518

  7. Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

    PubMed

    Nishimoto, Takuto; Furuta, Masakazu; Kataoka, Michihiko; Kishida, Masao

    2015-03-01

    Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation.

  8. Evidence for inositol triphosphate as a second messenger for glucose-induced calcium signalling in budding yeast.

    PubMed

    Tisi, Renata; Belotti, Fiorella; Wera, Stefaan; Winderickx, Joris; Thevelein, Johan M; Martegani, Enzo

    2004-02-01

    The Saccharomyces cerevisiae phospholipase C Plc1 is involved in cytosolic transient glucose-induced calcium increase, which also requires the Gpr1/Gpa2 receptor/G protein complex and glucose hexokinases. Differing from mammalian cells, this increase in cytosolic calcium concentration is mainly due to an influx from the external medium. No inositol triphosphate receptor homologue has been identified in the S. cerevisiae genome; and, therefore, the transduction mechanism from Plc1 activation to calcium flux generation still has to be identified. Inositol triphosphate (IP(3)) in yeast is rapidly transformed into IP(4) and IP(5) by a dual kinase, Arg82. Then another kinase, Ipk1, phosphorylates the IP(5) into IP(6). In mutant cells that do not express either of these kinases, the glucose-induced calcium signal was not only detectable but was even wider than in the wild-type strain. IP(3) accumulation upon glucose addition was completely absent in the plc1Delta strain and was amplified both by deletion of either ARG82 or IPK1 genes and by overexpression of PLC1. These results taken together suggest that Plc1p activation by glucose, leading to cleavage of PIP(2) and generation of IP(3), seems to be sufficient for raising the calcium level in the cytosol. This is the first indication for a physiological role of IP(3) signalling in S. cerevisiae. Many aspects about the signal transduction mechanism and the final effectors require further study.

  9. Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

    PubMed

    Nishimoto, Takuto; Furuta, Masakazu; Kataoka, Michihiko; Kishida, Masao

    2015-03-01

    Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation. PMID:25416226

  10. Control of Polarized Growth by the Rho Family GTPase Rho4 in Budding Yeast: Requirement of the N-Terminal Extension of Rho4 and Regulation by the Rho GTPase-Activating Protein Bem2

    PubMed Central

    Gong, Ting; Liao, Yuan; He, Fei; Yang, Yang; Yang, Dan-Dan; Chen, Xiang-Dong

    2013-01-01

    In the budding yeast Saccharomyces cerevisiae, Rho4 GTPase partially plays a redundant role with Rho3 in the control of polarized growth, as deletion of RHO4 and RHO3 together, but not RHO4 alone, caused lethality and a loss of cell polarity at 30°C. Here, we show that overexpression of the constitutively active rho4Q131L mutant in an rdi1Δ strain caused a severe growth defect and generated large, round, unbudded cells, suggesting that an excess of Rho4 activity could block bud emergence. We also generated four temperature-sensitive rho4-Ts alleles in a rho3Δ rho4Δ strain. These mutants showed growth and morphological defects at 37°C. Interestingly, two rho4-Ts alleles contain mutations that cause amino acid substitutions in the N-terminal region of Rho4. Rho4 possesses a long N-terminal extension that is unique among the six Rho GTPases in the budding yeast but is common in Rho4 homologs in other yeasts and filamentous fungi. We show that the N-terminal extension plays an important role in Rho4 function since rho3Δ rho4Δ61 cells expressing truncated Rho4 lacking amino acids (aa) 1 to 61 exhibited morphological defects at 24°C and a growth defect at 37°C. Furthermore, we show that Rho4 interacts with Bem2, a Rho GTPase-activating protein (RhoGAP) for Cdc42 and Rho1, by yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and glutathione S-transferase (GST) pulldown assays. Bem2 specifically interacts with the GTP-bound form of Rho4, and the interaction is mediated by its RhoGAP domain. Overexpression of BEM2 aggravates the defects of rho3Δ rho4 mutants. These results suggest that Bem2 might be a novel GAP for Rho4. PMID:23264647

  11. Rapid and quantitative imaging of excitation polarized fluorescence reveals ordered septin dynamics in live yeast.

    PubMed

    DeMay, Bradley S; Noda, Naoki; Gladfelter, Amy S; Oldenbourg, Rudolf

    2011-08-17

    We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of polarized fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of fluorescence tagging with the structural specificity of polarized light analysis.

  12. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals "aging factors" and mechanism of lifespan asymmetry.

    PubMed

    Yang, Jing; McCormick, Mark A; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K; Li, Hao

    2015-09-22

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials ("aging factors") through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms.

  13. Systematic analysis of asymmetric partitioning of yeast proteome between mother and daughter cells reveals “aging factors” and mechanism of lifespan asymmetry

    PubMed Central

    Yang, Jing; McCormick, Mark A.; Zheng, Jiashun; Xie, Zhengwei; Tsuchiya, Mitsuhiro; Tsuchiyama, Scott; El-Samad, Hana; Ouyang, Qi; Kaeberlein, Matt; Kennedy, Brian K.; Li, Hao

    2015-01-01

    Budding yeast divides asymmetrically, giving rise to a mother cell that progressively ages and a daughter cell with full lifespan. It is generally assumed that mother cells retain damaged, lifespan limiting materials (“aging factors”) through asymmetric division. However, the identity of these aging factors and the mechanisms through which they limit lifespan remain poorly understood. Using a flow cytometry-based, high-throughput approach, we quantified the asymmetric partitioning of the yeast proteome between mother and daughter cells during cell division, discovering 74 mother-enriched and 60 daughter-enriched proteins. While daughter-enriched proteins are biased toward those needed for bud construction and genome maintenance, mother-enriched proteins are biased towards those localized in the plasma membrane and vacuole. Deletion of 23 of the 74 mother-enriched proteins leads to lifespan extension, a fraction that is about six times that of the genes picked randomly from the genome. Among these lifespan-extending genes, three are involved in endosomal sorting/endosome to vacuole transport, and three are nitrogen source transporters. Tracking the dynamic expression of specific mother-enriched proteins revealed that their concentration steadily increases in the mother cells as they age, but is kept relatively low in the daughter cells via asymmetric distribution. Our results suggest that some mother-enriched proteins may increase to a concentration that becomes deleterious and lifespan-limiting in aged cells, possibly by upsetting homeostasis or leading to aberrant signaling. Our study provides a comprehensive resource for analyzing asymmetric cell division and aging in yeast, which should also be valuable for understanding similar phenomena in other organisms. PMID:26351681

  14. Papulacandin B resistance in budding and fission yeasts: isolation and characterization of a gene involved in (1,3)beta-D-glucan synthesis in Saccharomyces cerevisiae.

    PubMed Central

    Castro, C; Ribas, J C; Valdivieso, M H; Varona, R; del Rey, F; Duran, A

    1995-01-01

    Papulacandin B, an antifungal agent that interferes with the synthesis of yeast cell wall (1,3)beta-D-glucan, was used to isolate resistant mutants in Schizosaccharomyces pombe and Saccharomyces cerevisiae. The resistance to papulacandin B always segregated as a recessive character that defines a single complementation group in both yeasts (pbr1+ and PBR1, respectively). Determination of several kinetic parameters of (1,3)beta-D-glucan synthase activity revealed no differences between S. pombe wild-type and pbr1 mutant strains except in the 50% inhibitory concentration for papulacandin B of the synthases (about a 50-fold increase in mutant activity). Inactivation of the synthase activity of both yeasts after in vivo treatment with the antifungal agent showed that mutant synthases were more resistant than the corresponding wild-type ones. Detergent dissociation of the S. pombe synthase into soluble and particulate fractions and subsequent reconstitution indicated that the resistance character of pbr1 mutants resides in the particulate fraction of the enzyme. Cloning and sequencing of PBR1 from S. cerevisiae revealed a gene identical to others recently reported (FKS1, ETG1, CWH53, and CND1). Its disruption leads to reduced levels of both (1,3)beta-D-glucan synthase activity and the alkali-insoluble cell wall fraction. Transformants containing the PBR1 gene reverse the defect in (1,3)beta-D-glucan synthase. It is concluded that Pbr1p is probably part of the (1,3)beta-D-glucan synthase complex. PMID:7592316

  15. Bud-Neck Scaffolding as a Possible Driving Force in ESCRT-Induced Membrane Budding

    PubMed Central

    Mercker, Moritz; Marciniak-Czochra, Anna

    2015-01-01

    Membrane budding is essential for processes such as protein sorting and transport. Recent experimental results with ESCRT proteins reveal a novel budding mechanism, with proteins emerging in bud necks but separated from the entire bud surface. Using an elastic model, we show that ESCRT protein shapes are sufficient to spontaneously create experimentally observed structures, with protein-membrane interactions leading to protein scaffolds in bud-neck regions. Furthermore, the model reproduces experimentally observed budding directions and bud sizes. Finally, our results reveal that membrane-mediated sorting has the capability of creating structures more complicated than previously assumed. PMID:25692588

  16. Selective autophagy in budding yeast

    PubMed Central

    Suzuki, Kuninori

    2013-01-01

    Autophagy is a bulk degradation system, widely conserved in eukaryotes. Upon starvation, autophagosomes enclose a portion of the cytoplasm and ultimately fuse with the vacuole. The contents of autophagosomes are degraded in the vacuole, and recycled to maintain the intracellular amino-acid pool required for protein synthesis and survival under starvation conditions. Previously, autophagy was thought to be an essentially nonselective pathway, but recent evidence suggests that autophagosomes carry selected cargoes. These studies have identified two categories of selective autophagy – one highly selective and dependent on autophagy-related 11 (Atg11); another, less selective, that is, independent of Atg11. The former, selective category comprises the Cvt pathway, mitophagy, pexophagy and piecemeal microautophagy of the nucleus; acetaldehyde dehydrogenase 6 degradation and ribophagy belong to the latter, less selective category. In this review, I focus on the mechanisms and the physiological roles of these selective types of autophagy. PMID:22705847

  17. Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics.

    PubMed

    Boone, Cory H T; Grove, Ryan A; Adamcova, Dana; Braga, Camila P; Adamec, Jiri

    2016-07-01

    Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death. PMID:27193513

  18. Genome-wide Fitness Profiles Reveal a Requirement for Autophagy During Yeast Fermentation

    PubMed Central

    Piggott, Nina; Cook, Michael A.; Tyers, Mike; Measday, Vivien

    2011-01-01

    The ability of cells to respond to environmental changes and adapt their metabolism enables cell survival under stressful conditions. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is particularly well adapted to the harsh conditions of anaerobic wine fermentation. However, S. cerevisiae gene function has not been previously systematically interrogated under conditions of industrial fermentation. We performed a genome-wide study of essential and nonessential S. cerevisiae gene requirements during grape juice fermentation to identify deletion strains that are either depleted or enriched within the viable fermentative population. Genes that function in autophagy and ubiquitin-proteasome degradation are required for optimal survival during fermentation, whereas genes that function in ribosome assembly and peroxisome biogenesis impair fitness during fermentation. We also uncover fermentation phenotypes for 139 uncharacterized genes with no previously known cellular function. We demonstrate that autophagy is induced early in wine fermentation in a nitrogen-replete environment, suggesting that autophagy may be triggered by other forms of stress that arise during fermentation. These results provide insights into the complex fermentation process and suggest possible means for improvement of industrial fermentation strains. PMID:22384346

  19. Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast

    PubMed Central

    Vaga, Stefania; Bernardo-Faura, Marti; Cokelaer, Thomas; Maiolica, Alessio; Barnes, Christopher A; Gillet, Ludovic C; Hegemann, Björn; van Drogen, Frank; Sharifian, Hoda; Klipp, Edda; Peter, Matthias; Saez-Rodriguez, Julio; Aebersold, Ruedi

    2014-01-01

    Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways. PMID:25492886

  20. Xbp1 Directs Global Repression of Budding Yeast Transcription during the Transition to Quiescence and Is Important for the Longevity and Reversibility of the Quiescent State

    PubMed Central

    Miles, Shawna; Li, Lihong; Davison, Jerry; Breeden, Linda L.

    2013-01-01

    Pure populations of quiescent yeast can be obtained from stationary phase cultures that have ceased proliferation after exhausting glucose and other carbon sources from their environment. They are uniformly arrested in the G1 phase of the cell cycle, and display very high thermo-tolerance and longevity. We find that G1 arrest is initiated before all the glucose has been scavenged from the media. Maintaining G1 arrest requires transcriptional repression of the G1 cyclin, CLN3, by Xbp1. Xbp1 is induced as glucose is depleted and it is among the most abundant transcripts in quiescent cells. Xbp1 binds and represses CLN3 transcription and in the absence of Xbp1, or with extra copies of CLN3, cells undergo ectopic divisions and produce very small cells. The Rad53-mediated replication stress checkpoint reinforces the arrest and becomes essential when Cln3 is overproduced. The XBP1 transcript also undergoes metabolic oscillations under glucose limitation and we identified many additional transcripts that oscillate out of phase with XBP1 and have Xbp1 binding sites in their promoters. Further global analysis revealed that Xbp1 represses 15% of all yeast genes as they enter the quiescent state and over 500 of these transcripts contain Xbp1 binding sites in their promoters. Xbp1-repressed transcripts are highly enriched for genes involved in the regulation of cell growth, cell division and metabolism. Failure to repress some or all of these targets leads xbp1 cells to enter a permanent arrest or senescence with a shortened lifespan. PMID:24204289

  1. High-resolution analysis of four efficient yeast replication origins reveals new insights into the ORC and putative MCM binding elements.

    PubMed

    Chang, Fujung; May, Caitlin D; Hoggard, Timothy; Miller, Jeremy; Fox, Catherine A; Weinreich, Michael

    2011-08-01

    In budding yeast, the eukaryotic initiator protein ORC (origin recognition complex) binds to a bipartite sequence consisting of an 11 bp ACS element and an adjacent B1 element. However, the genome contains many more matches to this consensus than actually bind ORC or function as origins in vivo. Although ORC-dependent loading of the replicative MCM helicase at origins is enhanced by a distal B2 element, less is known about this element. Here, we analyzed four highly active origins (ARS309, ARS319, ARS606 and ARS607) by linker scanning mutagenesis and found that sequences adjacent to the ACS contributed substantially to origin activity and ORC binding. Using the sequences of four additional B2 elements we generated a B2 multiple sequence alignment and identified a shared, degenerate 8 bp sequence that was enriched within 228 known origins. In addition, our high-resolution analysis revealed that not all origins exist within nucleosome free regions: a class of Sir2-regulated origins has a stably positioned nucleosome overlapping or near B2. This study illustrates the conserved yet flexible nature of yeast origin architecture to promote ORC binding and origin activity, and helps explain why a strong match to the ORC binding site is insufficient to identify origins within the genome.

  2. A Geometrically-Constrained Mathematical Model of Mammary Gland Ductal Elongation Reveals Novel Cellular Dynamics within the Terminal End Bud

    PubMed Central

    Chauviere, Arnaud; Landua, John; Sreekumar, Amulya; Cristini, Vittorio; Rosen, Jeffrey; Lewis, Michael T.

    2016-01-01

    Mathematics is often used to model biological systems. In mammary gland development, mathematical modeling has been limited to acinar and branching morphogenesis and breast cancer, without reference to normal duct formation. We present a model of ductal elongation that exploits the geometrically-constrained shape of the terminal end bud (TEB), the growing tip of the duct, and incorporates morphometrics, region-specific proliferation and apoptosis rates. Iterative model refinement and behavior analysis, compared with biological data, indicated that the traditional metric of nipple to the ductal front distance, or percent fat pad filled to evaluate ductal elongation rate can be misleading, as it disregards branching events that can reduce its magnitude. Further, model driven investigations of the fates of specific TEB cell types confirmed migration of cap cells into the body cell layer, but showed their subsequent preferential elimination by apoptosis, thus minimizing their contribution to the luminal lineage and the mature duct. PMID:27115287

  3. A Geometrically-Constrained Mathematical Model of Mammary Gland Ductal Elongation Reveals Novel Cellular Dynamics within the Terminal End Bud.

    PubMed

    Paine, Ingrid; Chauviere, Arnaud; Landua, John; Sreekumar, Amulya; Cristini, Vittorio; Rosen, Jeffrey; Lewis, Michael T

    2016-04-01

    Mathematics is often used to model biological systems. In mammary gland development, mathematical modeling has been limited to acinar and branching morphogenesis and breast cancer, without reference to normal duct formation. We present a model of ductal elongation that exploits the geometrically-constrained shape of the terminal end bud (TEB), the growing tip of the duct, and incorporates morphometrics, region-specific proliferation and apoptosis rates. Iterative model refinement and behavior analysis, compared with biological data, indicated that the traditional metric of nipple to the ductal front distance, or percent fat pad filled to evaluate ductal elongation rate can be misleading, as it disregards branching events that can reduce its magnitude. Further, model driven investigations of the fates of specific TEB cell types confirmed migration of cap cells into the body cell layer, but showed their subsequent preferential elimination by apoptosis, thus minimizing their contribution to the luminal lineage and the mature duct. PMID:27115287

  4. DiOC6 staining reveals organelle structure and dynamics in living yeast cells.

    PubMed

    Koning, A J; Lum, P Y; Williams, J M; Wright, R

    1993-01-01

    When present at low concentrations, the fluorescent lipophilic dye, DiOC6, stains mitochondria in living yeast cells [Pringle et al.: Methods in Cell Biol. 31:357-435, 1989; Weisman et al.: Proc. Natl. Acad. Sci. U.S.A. 87:1076-1080, 1990]. However, we found that the nuclear envelope and endoplasmic reticulum were specifically stained if the dye concentration was increased or if certain respiratory-deficient yeast strains were examined. The quality of nuclear envelope staining with DiOC6 was sufficiently sensitive to reveal alterations in the nuclear envelope known as karmellae. These membranes were previously apparent only by electron microscopy. At the high dye concentrations required to stain the nuclear envelope, wild-type cells could no longer grow on non-fermentable carbon sources. In spite of this effect on mitochondrial function, the presence of high dye concentration did not adversely affect cell viability or general growth characteristics when strains were grown under standard conditions on glucose. Consequently, time-lapse confocal microscopy was used to examine organelle dynamics in living yeast cells stained with DiOC6. These in vivo observations correlated very well with previous electron microscopic studies, including analyses of mitochondria, karmellae, and mitosis. For example, cycles of mitochondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time-lapse studies of living DiOC6-stained cells. This technique also revealed new aspects of nuclear disposition and interactions with other organelles. For example, the nucleus and vacuole appeared to form a structurally coupled unit that could undergo coordinated movements. Furthermore, unlike the general view that nuclear movements occur only in association with division, the nucleus/vacuole underwent dramatic migrations around the cell periphery as cells exited from stationary phase. In addition to the large migrations or

  5. Accounting for experimental noise reveals that mRNA levels, amplified by post-transcriptional processes, largely determine steady-state protein levels in yeast.

    PubMed

    Csárdi, Gábor; Franks, Alexander; Choi, David S; Airoldi, Edoardo M; Drummond, D Allan

    2015-05-01

    Cells respond to their environment by modulating protein levels through mRNA transcription and post-transcriptional control. Modest observed correlations between global steady-state mRNA and protein measurements have been interpreted as evidence that mRNA levels determine roughly 40% of the variation in protein levels, indicating dominant post-transcriptional effects. However, the techniques underlying these conclusions, such as correlation and regression, yield biased results when data are noisy, missing systematically, and collinear---properties of mRNA and protein measurements---which motivated us to revisit this subject. Noise-robust analyses of 24 studies of budding yeast reveal that mRNA levels explain more than 85% of the variation in steady-state protein levels. Protein levels are not proportional to mRNA levels, but rise much more rapidly. Regulation of translation suffices to explain this nonlinear effect, revealing post-transcriptional amplification of, rather than competition with, transcriptional signals. These results substantially revise widely credited models of protein-level regulation, and introduce multiple noise-aware approaches essential for proper analysis of many biological phenomena.

  6. Accounting for Experimental Noise Reveals That mRNA Levels, Amplified by Post-Transcriptional Processes, Largely Determine Steady-State Protein Levels in Yeast

    PubMed Central

    Csárdi, Gábor; Franks, Alexander; Choi, David S.; Airoldi, Edoardo M.; Drummond, D. Allan

    2015-01-01

    Cells respond to their environment by modulating protein levels through mRNA transcription and post-transcriptional control. Modest observed correlations between global steady-state mRNA and protein measurements have been interpreted as evidence that mRNA levels determine roughly 40% of the variation in protein levels, indicating dominant post-transcriptional effects. However, the techniques underlying these conclusions, such as correlation and regression, yield biased results when data are noisy, missing systematically, and collinear---properties of mRNA and protein measurements---which motivated us to revisit this subject. Noise-robust analyses of 24 studies of budding yeast reveal that mRNA levels explain more than 85% of the variation in steady-state protein levels. Protein levels are not proportional to mRNA levels, but rise much more rapidly. Regulation of translation suffices to explain this nonlinear effect, revealing post-transcriptional amplification of, rather than competition with, transcriptional signals. These results substantially revise widely credited models of protein-level regulation, and introduce multiple noise-aware approaches essential for proper analysis of many biological phenomena. PMID:25950722

  7. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells

    PubMed Central

    Carlile, Thomas M.; Rojas-Duran, Maria F.; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M.; Gilbert, Wendy V.

    2014-01-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs1, enhances the function of transfer RNA and ribosomal RNA by stabilizing RNA structure2–8. mRNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function – it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding center9,10. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological was unclear. Here we present a comprehensive analysis of pseudouridylation in yeast and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as 100 novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1–4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease11–13. PMID:25192136

  8. From the Cover: Toward a protein-protein interaction map of the budding yeast: A comprehensive system to examine two-hybrid interactions in all possible combinations between the yeast proteins

    NASA Astrophysics Data System (ADS)

    Ito, Takashi; Tashiro, Kosuke; Muta, Shigeru; Ozawa, Ritsuko; Chiba, Tomoko; Nishizawa, Mayumi; Yamamoto, Kiyoshi; Kuhara, Satoru; Sakaki, Yoshiyuki

    2000-02-01

    Protein-protein interactions play pivotal roles in various aspects of the structural and functional organization of the cell, and their complete description is indispensable to thorough understanding of the cell. As an approach toward this goal, here we report a comprehensive system to examine two-hybrid interactions in all of the possible combinations between proteins of Saccharomyces cerevisiae. We cloned all of the yeast ORFs individually as a DNA-binding domain fusion ("bait") in a MATa strain and as an activation domain fusion ("prey") in a MATα strain, and subsequently divided them into pools, each containing 96 clones. These bait and prey clone pools were systematically mated with each other, and the transformants were subjected to strict selection for the activation of three reporter genes followed by sequence tagging. Our initial examination of ≈4 × 106 different combinations, constituting ≈10% of the total to be tested, has revealed 183 independent two-hybrid interactions, more than half of which are entirely novel. Notably, the obtained binary data allow us to extract more complex interaction networks, including the one that may explain a currently unsolved mechanism for the connection between distinct steps of vesicular transport. The approach described here thus will provide many leads for integration of various cellular functions and serve as a major driving force in the completion of the protein-protein interaction map.

  9. Bulk Segregant Analysis Reveals the Genetic Basis of a Natural Trait Variation in Fission Yeast

    PubMed Central

    Hu, Wen; Suo, Fang; Du, Li-Lin

    2015-01-01

    Although the fission yeast Schizosaccharomyces pombe is a well-established model organism, studies of natural trait variations in this species remain limited. To assess the feasibility of segregant-pool-based mapping of phenotype-causing genes in natural strains of fission yeast, we investigated the cause of a maltose utilization defect (Mal-) of the S. pombe strain CBS5557 (originally known as Schizosaccharomyces malidevorans). Analyzing the genome sequence of CBS5557 revealed 955 nonconservative missense substitutions, and 61 potential loss-of-function variants including 47 frameshift indels, 13 early stop codons, and 1 splice site mutation. As a side benefit, our analysis confirmed 146 sequence errors in the reference genome and improved annotations of 27 genes. We applied bulk segregant analysis to map the causal locus of the Mal- phenotype. Through sequencing the segregant pools derived from a cross between CBS5557 and the laboratory strain, we located the locus to within a 2.23-Mb chromosome I inversion found in most S. pombe isolates including CBS5557. To map genes within the inversion region that occupies 18% of the genome, we created a laboratory strain containing the same inversion. Analyzing segregants from a cross between CBS5557 and the inversion-containing laboratory strain narrowed down the locus to a 200-kb interval and led us to identify agl1, which suffers a 5-bp deletion in CBS5557, as the causal gene. Interestingly, loss of agl1 through a 34-kb deletion underlies the Mal- phenotype of another S. pombe strain CGMCC2.1628. This work adapts and validates the bulk segregant analysis method for uncovering trait-gene relationship in natural fission yeast strains. PMID:26615217

  10. Population genomic analysis reveals highly conserved mitochondrial genomes in the yeast species Lachancea thermotolerans.

    PubMed

    Freel, Kelle C; Friedrich, Anne; Hou, Jing; Schacherer, Joseph

    2014-10-01

    The increasing availability of mitochondrial (mt) sequence data from various yeasts provides a tool to study genomic evolution within and between different species. While the genomes from a range of lineages are available, there is a lack of information concerning intraspecific mtDNA diversity. Here, we analyzed the mt genomes of 50 strains from Lachancea thermotolerans, a protoploid yeast species that has been isolated from several locations (Europe, Asia, Australia, South Africa, and North / South America) and ecological sources (fruit, tree exudate, plant material, and grape and agave fermentations). Protein-coding genes from the mtDNA were used to construct a phylogeny, which reflected a similar, yet less resolved topology than the phylogenetic tree of 50 nuclear genes. In comparison to its sister species Lachancea kluyveri, L. thermotolerans has a smaller mt genome. This is due to shorter intergenic regions and fewer introns, of which the latter are only found in COX1. We revealed that L. kluyveri and L. thermotolerans share similar levels of intraspecific divergence concerning the nuclear genomes. However, L. thermotolerans has a more highly conserved mt genome with the coding regions characterized by low rates of nonsynonymous substitution. Thus, in the mt genomes of L. thermotolerans, stronger purifying selection and lower mutation rates potentially shape genome diversity in contract to what was found for L. kluyveri, demonstrating that the factors driving mt genome evolution are different even between closely related species. PMID:25212859

  11. Expression profiling reveals an unexpected growth-stimulating effect of surplus iron on the yeast Saccharomyces cerevisiae.

    PubMed

    Du, Yang; Cheng, Wang; Li, Wei-Fang

    2012-08-01

    Iron homeostasis plays a crucial role in growth and division of cells in all kingdoms of life. Although yeast iron metabolism has been extensively studied, little is known about the molecular mechanism of response to surplus iron. In this study, expression profiling of Saccharomyces cerevisiae in the presence of surplus iron revealed a dual effect at 1 and 4 h. A cluster of stress-responsive genes was upregulated via activation of the stress-resistance transcription factor Msn4, which indicated the stress effect of surplus iron on yeast metabolism. Genes involved in aerobic metabolism and several anabolic pathways are also upregulated in iron-surplus conditions, which could significantly accelerate yeast growth. This dual effect suggested that surplus iron might participate in a more complex metabolic network, in addition to serving as a stress inducer. These findings contribute to our understanding of the global response of yeast to the fluctuating availability of iron in the environment.

  12. Comprehensive polyadenylation site maps in yeast and human reveal pervasive alternative polyadenylation.

    PubMed

    Ozsolak, Fatih; Kapranov, Philipp; Foissac, Sylvain; Kim, Sang Woo; Fishilevich, Elane; Monaghan, A Paula; John, Bino; Milos, Patrice M

    2010-12-10

    The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated poly(A)(+) RNAs. We observed differences in sequence composition surrounding canonical and noncanonical human polyadenylation sites, suggesting novel noncoding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription. PMID:21145465

  13. Physiological differences between bud breaking and flowering after dormancy completion revealed by DAM and FT/TFL1 expression in Japanese pear (Pyrus pyrifolia).

    PubMed

    Ito, Akiko; Saito, Takanori; Sakamoto, Daisuke; Sugiura, Toshihiko; Bai, Songling; Moriguchi, Takaya

    2016-01-01

    The regulatory mechanisms underlying bud breaking (scale leaf elongation) and flowering in the lateral flower buds of Japanese pear (Pyrus pyrifolia Nakai 'Kosui') are unknown. To more fully characterize these processes, we treated pear trees with different amounts of chilling initiated at different times. Chilling for ∼900 h at 6 °C always induced bud breaking (scale elongation in ≥70% lateral flower bud) when provided between October and February, whereas chilling provided earlier (between October and December) was less effective on flowering (floret growth and development) than later chilling and the flowering rate increased with longer chilling durations. During chilling, the expression of pear DAMs (PpMADS13-1, 13-2 and 13-3) in lateral flower buds decreased as chilling accumulated irrespective of the timing of chilling. In addition, pear TFL1 (PpTFL1-1a) in the lateral flower buds was expressed at higher levels when the time interval for chilling was earlier. On the other hand, during forcing at 15 °C after chilling, the expression pattern of all three PpMADS13 genes was similar among the treatments, and the expression levels seemed lower in the treatment where scale leaves of the lateral flower bud elongated faster, whereas pear FT (PpFT2a) was expressed at higher levels in the buds whose flower clusters elongated more vigorously during forcing. From these results, we infer that flowering time may be mediated via the balance of flowering-related genes FT and TFL1, whereas bud breaking may be regulated via the DAM genes in Japanese pear.

  14. Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.

    PubMed

    Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H; Murray, Johanne M; Carr, Antony M; Elledge, Stephen J; Rhind, Nicholas

    2016-06-28

    The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes.

  15. Cryotomography of Budding Influenza A Virus Reveals Filaments with Diverse Morphologies that Mostly Do Not Bear a Genome at Their Distal End

    PubMed Central

    Vijayakrishnan, Swetha; Loney, Colin; Jackson, David; Suphamungmee, Worawit; Rixon, Frazer J.; Bhella, David

    2013-01-01

    Influenza viruses exhibit striking variations in particle morphology between strains. Clinical isolates of influenza A virus have been shown to produce long filamentous particles while laboratory-adapted strains are predominantly spherical. However, the role of the filamentous phenotype in the influenza virus infectious cycle remains undetermined. We used cryo-electron tomography to conduct the first three-dimensional study of filamentous virus ultrastructure in particles budding from infected cells. Filaments were often longer than 10 microns and sometimes had bulbous heads at their leading ends, some of which contained tubules we attribute to M1 while none had recognisable ribonucleoprotein (RNP) and hence genome segments. Long filaments that did not have bulbs were infrequently seen to bear an ordered complement of RNPs at their distal ends. Imaging of purified virus also revealed diverse filament morphologies; short rods (bacilliform virions) and longer filaments. Bacilliform virions contained an ordered complement of RNPs while longer filamentous particles were narrower and mostly appeared to lack this feature, but often contained fibrillar material along their entire length. The important ultrastructural differences between these diverse classes of particles raise the possibility of distinct morphogenetic pathways and functions during the infectious process. PMID:23754946

  16. Molecular Phylogeny of Sequenced Saccharomycetes Reveals Polyphyly of the Alternative Yeast Codon Usage

    PubMed Central

    Mühlhausen, Stefanie; Kollmar, Martin

    2014-01-01

    The universal genetic code defines the translation of nucleotide triplets, called codons, into amino acids. In many Saccharomycetes a unique alteration of this code affects the translation of the CUG codon, which is normally translated as leucine. Most of the species encoding CUG alternatively as serine belong to the Candida genus and were grouped into a so-called CTG clade. However, the “Candida genus” is not a monophyletic group and several Candida species are known to use the standard CUG translation. The codon identity could have been changed in a single branch, the ancestor of the Candida, or to several branches independently leading to a polyphyletic alternative yeast codon usage (AYCU). In order to resolve the monophyly or polyphyly of the AYCU, we performed a phylogenomics analysis of 26 motor and cytoskeletal proteins from 60 sequenced yeast species. By investigating the CUG codon positions with respect to sequence conservation at the respective alignment positions, we were able to unambiguously assign the standard code or AYCU. Quantitative analysis of the highly conserved leucine and serine alignment positions showed that 61.1% and 17% of the CUG codons coding for leucine and serine, respectively, are at highly conserved positions, whereas only 0.6% and 2.3% of the CUG codons, respectively, are at positions conserved in the respective other amino acid. Plotting the codon usage onto the phylogenetic tree revealed the polyphyly of the AYCU with Pachysolen tannophilus and the CTG clade branching independently within a time span of 30–100 Ma. PMID:25646540

  17. Structure-function analysis of yeast piD261/Bud32, an atypical protein kinase essential for normal cell life.

    PubMed

    Facchin, Sonia; Lopreiato, Raffaele; Stocchetto, Silvia; Arrigoni, Giorgio; Cesaro, Luca; Marin, Oriano; Carignani, Giovanna; Pinna, Lorenzo A

    2002-06-01

    The Saccharomyces cerevisiae YGR262c/BUD32 gene, whose disruption causes a severe pleiotropic phenotype, encodes a 261-residue putative protein kinase, piD261, whose structural homologues have been identified in a variety of organisms, including humans, and whose function is unknown. We have demonstrated previously that piD261, expressed in Escherichia coli as a recombinant protein, is a Ser/Thr kinase, as judged by its ability to autophosphorylate and to phosphorylate casein. Here we describe a mutational analysis showing that, despite low sequence similarity, the invariant residues representing the signature of protein kinases are conserved in piD261 and in its structural homologues, but are embedded in an altered context, suggestive of unique mechanistic properties. Especially noteworthy are: (i) three unique inserts of unknown function within the N-terminal lobe, (ii) the lack of a lysyl residue which in all other Ser/Thr kinases participates in the catalytic event by interacting with the transferred ATP gamma-phosphate, and which in piD261 is replaced by a threonine, and (iii) an exceedingly short activation loop including two serines, Ser-187 and Ser-189, whose autophosphorylation accounts for the appearance of an upshifted band upon SDS/PAGE. A mutant in which these serines are replaced by alanines was devoid of the upshifted band and displayed reduced catalytic activity. This would include piD261 in the category of protein kinases activated by phosphorylation, although it lacks the RD (Arg-Asp) motif which is typical of these enzymes.

  18. The crystal structure of yeast CCT reveals intrinsic asymmetry of eukaryotic cytosolic chaperonins

    PubMed Central

    Dekker, Carien; Roe, S Mark; McCormack, Elizabeth A; Beuron, Fabienne; Pearl, Laurence H; Willison, Keith R

    2011-01-01

    The cytosolic chaperonin CCT is a 1-MDa protein-folding machine essential for eukaryotic life. The CCT interactome shows involvement in folding and assembly of a small range of proteins linked to essential cellular processes such as cytoskeleton assembly and cell-cycle regulation. CCT has a classic chaperonin architecture, with two heterogeneous 8-membered rings stacked back-to-back, enclosing a folding cavity. However, the mechanism by which CCT assists folding is distinct from other chaperonins, with no hydrophobic wall lining a potential Anfinsen cage, and a sequential rather than concerted ATP hydrolysis mechanism. We have solved the crystal structure of yeast CCT in complex with actin at 3.8 Å resolution, revealing the subunit organisation and the location of discrete patches of co-evolving ‘signature residues' that mediate specific interactions between CCT and its substrates. The intrinsic asymmetry is revealed by the structural individuality of the CCT subunits, which display unique configurations, substrate binding properties, ATP-binding heterogeneity and subunit–subunit interactions. The location of the evolutionarily conserved N-terminus of Cct5 on the outside of the barrel, confirmed by mutational studies, is unique to eukaryotic cytosolic chaperonins. PMID:21701561

  19. Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells.

    PubMed

    Carlile, Thomas M; Rojas-Duran, Maria F; Zinshteyn, Boris; Shin, Hakyung; Bartoli, Kristen M; Gilbert, Wendy V

    2014-11-01

    Post-transcriptional modification of RNA nucleosides occurs in all living organisms. Pseudouridine, the most abundant modified nucleoside in non-coding RNAs, enhances the function of transfer RNA and ribosomal RNA by stabilizing the RNA structure. Messenger RNAs were not known to contain pseudouridine, but artificial pseudouridylation dramatically affects mRNA function--it changes the genetic code by facilitating non-canonical base pairing in the ribosome decoding centre. However, without evidence of naturally occurring mRNA pseudouridylation, its physiological relevance was unclear. Here we present a comprehensive analysis of pseudouridylation in Saccharomyces cerevisiae and human RNAs using Pseudo-seq, a genome-wide, single-nucleotide-resolution method for pseudouridine identification. Pseudo-seq accurately identifies known modification sites as well as many novel sites in non-coding RNAs, and reveals hundreds of pseudouridylated sites in mRNAs. Genetic analysis allowed us to assign most of the new modification sites to one of seven conserved pseudouridine synthases, Pus1-4, 6, 7 and 9. Notably, the majority of pseudouridines in mRNA are regulated in response to environmental signals, such as nutrient deprivation in yeast and serum starvation in human cells. These results suggest a mechanism for the rapid and regulated rewiring of the genetic code through inducible mRNA modifications. Our findings reveal unanticipated roles for pseudouridylation and provide a resource for identifying the targets of pseudouridine synthases implicated in human disease.

  20. Draft Genome Sequence of the Deep-Sea Basidiomycetous Yeast Cryptococcus sp. Strain Mo29 Reveals Its Biotechnological Potential

    PubMed Central

    Rédou, Vanessa; Kumar, Abhishek; Hainaut, Matthieu; Henrissat, Bernard; Record, Eric; Barbier, Georges

    2016-01-01

    Cryptococcus sp. strain Mo29 was isolated from the Rainbow hydrothermal site on the Mid-Atlantic Ridge. Here, we present the draft genome sequence of this basidiomycetous yeast strain, which has highlighted its biotechnological potential as revealed by the presence of genes involved in the synthesis of secondary metabolites and biotechnologically important enzymes. PMID:27389259

  1. Digital gene expression analysis of male and female bud transition in Metasequoia reveals high activity of MADS-box transcription factors and hormone-mediated sugar pathways

    PubMed Central

    Zhao, Ying; Liang, Haiying; Li, Lan; Tang, Sha; Han, Xiao; Wang, Congpeng; Xia, Xinli; Yin, Weilun

    2015-01-01

    Metasequoia glyptostroboides is a famous redwood tree of ecological and economic importance, and requires more than 20 years of juvenile-to-adult transition before producing female and male cones. Previously, we induced reproductive buds using a hormone solution in juvenile Metasequoia trees as young as 5-to-7 years old. In the current study, hormone-treated shoots found in female and male buds were used to identify candidate genes involved in reproductive bud transition in Metasequoia. Samples from hormone-treated cone reproductive shoots and naturally occurring non-cone setting shoots were analyzed using 24 digital gene expression (DGE) tag profiles using Illumina, generating a total of 69,520 putative transcripts. Next, 32 differentially and specifically expressed transcripts were determined using quantitative real-time polymerase chain reaction, including the upregulation of MADS-box transcription factors involved in male bud transition and flowering time control proteins involved in female bud transition. These differentially expressed transcripts were associated with 243 KEGG pathways. Among the significantly changed pathways, sugar pathways were mediated by hormone signals during the vegetative-to-reproductive phase transition, including glycolysis/gluconeogenesis and sucrose and starch metabolism pathways. Key enzymes were identified in these pathways, including alcohol dehydrogenase (NAD) and glutathione dehydrogenase for the glycolysis/gluconeogenesis pathway, and glucanphosphorylase for sucrose and starch metabolism pathways. Our results increase our understanding of the reproductive bud transition in gymnosperms. In addition, these studies on hormone-mediated sugar pathways increase our understanding of the relationship between sugar and hormone signaling during female and male bud initiation in Metasequoia. PMID:26157452

  2. Analysis of dormant bud (Banjhi) specific transcriptome of tea (Camellia sinensis (L.) O. Kuntze) from cDNA library revealed dormancy-related genes.

    PubMed

    Thirugnanasambantham, Krishnaraj; Prabu, Gajjeraman; Palanisamy, Senthilkumar; Chandrabose, Suresh Ramraj Subhas; Mandal, Abul Kalam Azad

    2013-02-01

    Bud dormancy is of ecological and economical interest due to its impact on tea (Camellia sinensis (L.) O. Kuntze) plant growth and yield. Growth regulation associated with dormancy is an essential element in plant's life cycle that leads to changes in expression of large number of genes. In order to identify and provide a picture of the transcriptome profile, cDNA library was constructed from dormant bud (banjhi) of tea. Sequence and gene ontology analysis of 3,500 clones, in many cases, enabled their functional categorization concerning the bud growth. Based on the cDNA library data, the putative role of identified genes from tea is discussed in relation to growth and dormancy, which includes morphogenesis, cellular differentiation, tropism, cell cycle, signaling, and various metabolic pathways. There was a higher representation of unknown processes such as unknown molecular functions (65.80 %), unknown biological processes (62.46 %), and unknown cellular components (67.42 %). However, these unknown transcripts represented a novel component of transcripts in tea plant bud growth and/or dormancy development. The identified transcripts and expressed sequence tags provides a valuable public resource and preliminary insights into the molecular mechanisms of bud dormancy regulation. Further, the findings will be the target of future expression experiments, particularly for further identification of dormancy-related genes in this species.

  3. A Conserved Non-Canonical Docking Mechanism Regulates the Binding of Dual Specificity Phosphatases to Cell Integrity Mitogen-Activated Protein Kinases (MAPKs) in Budding and Fission Yeasts

    PubMed Central

    Sacristán-Reviriego, Almudena; Madrid, Marisa; Cansado, José; Martín, Humberto; Molina, María

    2014-01-01

    Dual-specificity MAPK phosphatases (MKPs) are essential for the negative regulation of MAPK pathways. Similar to other MAPK-interacting proteins, most MKPs bind MAPKs through specific docking domains known as D-motifs. However, we found that the Saccharomyces cerevisiae MKP Msg5 binds the MAPK Slt2 within the cell wall integrity (CWI) pathway through a distinct motif (IYT). Here, we demonstrate that the IYT motif mediates binding of the Msg5 paralogue Sdp1 to Slt2 as well as of the MKP Pmp1 to its CWI MAPK counterpart Pmk1 in the evolutionarily distant yeast Schizosaccharomyces pombe. As a consequence, removal of the IYT site in Msg5, Sdp1 and Pmp1 reduces MAPK trapping caused by the overexpression of catalytically inactive versions of these phosphatases. Accordingly, an intact IYT site is necessary for inactive Sdp1 to prevent nuclear accumulation of Slt2. We also show that both Ile and Tyr but not Thr are essential for the functionality of the IYT motif. These results provide mechanistic insight into MKP-MAPK interplay and stress the relevance of this conserved non-canonical docking site in the regulation of the CWI pathway in fungi. PMID:24465549

  4. Budding yeast Rad50, Mre11, Xrs2, and Hdf1, but not Rad52, are involved in the formation of deletions on a dicentric plasmid.

    PubMed

    Tsukamoto, Y; Kato, J; Ikeda, H

    1997-08-01

    We have previously shown that the RAD50, RAD52, MRE11, XRS2, and HDF1 genes of Saccharomyces cervisiae are involved in the formation of deletions by illegitimate recombination on a monocentric plasmid. In this study, we investigated the effects of mutations of these genes on formation of deletions of a dicentric plasmid, in which DNA double-strand breaks are expected to occur frequently because the two centromeres are pulled to opposite poles in mitosis. We transformed yeast cells with a dicentric plasmid, and after incubation for a few division cycles, cells carrying deleted plasmids were detected using negative selection markers. Deletions occurred at a higher frequency than on the monocentric plasmid and there were short regions of homology at the recombination junctions as observed on the monocentric plasmid. In rad50, mre11, xrs2, and hdf1 mutants, the frequency of occurrence of deletions was reduced by about 50-fold, while in the rad52 mutant, it was comparable to that in the wild-type strain. The end-joining functions of Rad50, Mre11, Xrs2, and Hdf1, suggest that these proteins play important roles in the joining of DNA ends produced on the dicentric plasmid during mitosis. PMID:9294039

  5. A population study of killer viruses reveals different evolutionary histories of two closely related Saccharomyces sensu stricto yeasts.

    PubMed

    Chang, Shang-Lin; Leu, Jun-Yi; Chang, Tien-Hsien

    2015-08-01

    Microbes have evolved ways of interference competition to gain advantage over their ecological competitors. The use of secreted killer toxins by yeast cells through acquiring double-stranded RNA viruses is one such prominent example. Although the killer behaviour has been well studied in laboratory yeast strains, our knowledge regarding how killer viruses are spread and maintained in nature and how yeast cells co-evolve with viruses remains limited. We investigated these issues using a panel of 81 yeast populations belonging to three Saccharomyces sensu stricto species isolated from diverse ecological niches and geographic locations. We found that killer strains are rare among all three species. In contrast, killer toxin resistance is widespread in Saccharomyces paradoxus populations, but not in Saccharomyces cerevisiae or Saccharomyces eubayanus populations. Genetic analyses revealed that toxin resistance in S. paradoxus is often caused by dominant alleles that have independently evolved in different populations. Molecular typing identified one M28 and two types of M1 killer viruses in those killer strains. We further showed that killer viruses of the same type could lead to distinct killer phenotypes under different host backgrounds, suggesting co-evolution between the viruses and hosts in different populations. Taken together, our data suggest that killer viruses vary in their evolutionary histories even within closely related yeast species.

  6. Structure of the Yeast DEAD Box Protein Mss116p Reveals Two Wedges that Crimp RNA

    SciTech Connect

    Del Campo, Mark; Lambowitz, Alan M.

    2010-01-12

    The yeast DEAD box protein Mss116p is a general RNA chaperone that functions in mitochondrial group I and II intron splicing, translational activation, and RNA end processing. Here we determined high-resolution X-ray crystal structures of Mss116p complexed with an RNA oligonucleotide and ATP analogs AMP-PNP, ADP-BeF{sub 3}, or ADP-AlF{sub 4}{sup -}. The structures show the entire helicase core acting together with a functionally important C-terminal extension. In all structures, the helicase core is in a closed conformation with a wedge {alpha} helix bending RNA 3' of the central bound nucleotides, as in previous DEAD box protein structures. Notably, Mss116p's C-terminal extension also bends RNA 5' of the central nucleotides, resulting in RNA crimping. Despite reported functional differences, we observe few structural changes in ternary complexes with different ATP analogs. The structures constrain models of DEAD box protein function and reveal a strand separation mechanism in which a protein uses two wedges to act as a molecular crimper.

  7. Mutations at the Subunit Interface of Yeast Proliferating Cell Nuclear Antigen Reveal a Versatile Regulatory Domain

    PubMed Central

    Halmai, Miklos; Frittmann, Orsolya; Szabo, Zoltan; Daraba, Andreea; Gali, Vamsi K.; Balint, Eva; Unk, Ildiko

    2016-01-01

    Proliferating cell nuclear antigen (PCNA) plays a key role in many cellular processes and due to that it interacts with a plethora of proteins. The main interacting surfaces of Saccharomyces cerevisiae PCNA have been mapped to the interdomain connecting loop and to the carboxy-terminal domain. Here we report that the subunit interface of yeast PCNA also has regulatory roles in the function of several DNA damage response pathways. Using site-directed mutagenesis we engineered mutations at both sides of the interface and investigated the effect of these alleles on DNA damage response. Genetic experiments with strains bearing the mutant alleles revealed that mutagenic translesion synthesis, nucleotide excision repair, and homologous recombination are all regulated through residues at the subunit interface. Moreover, genetic characterization of one of our mutants identifies a new sub-branch of nucleotide excision repair. Based on these results we conclude that residues at the subunit boundary of PCNA are not only important for the formation of the trimer structure of PCNA, but they constitute a regulatory protein domain that mediates different DNA damage response pathways, as well. PMID:27537501

  8. Structure of the Yeast Polarity Protein Sro7 Reveals a SNARE Regulatory Mechanism

    SciTech Connect

    Hattendorf, D.A.; Andreeva, A.; Gangar, A.; Brennwald, P.J.; Weis, W.I.; /Stanford U., Med. School /North Carolina U.

    2007-07-09

    Polarized exocytosis requires coordination between the actin cytoskeleton and the exocytic machinery responsible for fusion of secretory vesicles at specific sites on the plasma membrane. Fusion requires formation of a complex between a vesicle-bound R-SNARE and plasma membrane Qa, Qb and Qc SNARE proteins. Proteins in the lethal giant larvae protein family, including lethal giant larvae and tomosyn in metazoans and Sro7 in yeast, interact with Q-SNAREs and are emerging as key regulators of polarized exocytosis. The crystal structure of Sro7 reveals two seven-bladed WD40 {beta}-propellers followed by a 60-residue-long 'tail', which is bound to the surface of the amino-terminal propeller. Deletion of the Sro7 tail enables binding to the Qbc SNARE region of Sec9 and this interaction inhibits SNARE complex assembly. The N-terminal domain of Sec9 provides a second, high-affinity Sro7 interaction that is unaffected by the tail. The results suggest that Sro7 acts as an allosteric regulator of exocytosis through interactions with factors that control the tail. Sequence alignments indicate that lethal giant larvae and tomosyn have a two-{beta}-propeller fold similar to that of Sro7, but only tomosyn appears to retain the regulatory tail.

  9. Msa1 and Msa2 Modulate G1-Specific Transcription to Promote G1 Arrest and the Transition to Quiescence in Budding Yeast

    PubMed Central

    Miles, Shawna; Croxford, Matthew W.; Abeysinghe, Amali P.; Breeden, Linda L.

    2016-01-01

    Yeast that naturally exhaust their glucose source can enter a quiescent state that is characterized by reduced cell size, and high cell density, stress tolerance and longevity. The transition to quiescence involves highly asymmetric cell divisions, dramatic reprogramming of transcription and global changes in chromatin structure and chromosome topology. Cells enter quiescence from G1 and we find that there is a positive correlation between the length of G1 and the yield of quiescent cells. The Swi4 and Swi6 transcription factors, which form the SBF transcription complex and promote the G1 to S transition in cycling cells, are also critical for the transition to quiescence. Swi6 forms a second complex with Mbp1 (MBF), which is not required for quiescence. These are the functional analogues of the E2F complexes of higher eukaryotes. Loss of the RB analogue, Whi5, and the related protein Srl3/Whi7, delays G1 arrest, but it also delays recovery from quiescence. Two MBF- and SBF-Associated proteins have been identified that have little effect on SBF or MBF activity in cycling cells. We show that these two related proteins, Msa1 and Msa2, are specifically required for the transition to quiescence. Like the E2F complexes that are quiescence-specific, Msa1 and Msa2 are required to repress the transcription of many SBF target genes, including SWI4, the CLN2 cyclin and histones, specifically after glucose is exhausted from the media. They also activate transcription of many MBF target genes. msa1msa2 cells fail to G1 arrest and rapidly lose viability upon glucose exhaustion. msa1msa2 mutants that survive this transition are very large, but they attain the same thermo-tolerance and longevity of wild type quiescent cells. This indicates that Msa1 and Msa2 are required for successful transition to quiescence, but not for the maintenance of that state. PMID:27272642

  10. Msa1 and Msa2 Modulate G1-Specific Transcription to Promote G1 Arrest and the Transition to Quiescence in Budding Yeast.

    PubMed

    Miles, Shawna; Croxford, Matthew W; Abeysinghe, Amali P; Breeden, Linda L

    2016-06-01

    Yeast that naturally exhaust their glucose source can enter a quiescent state that is characterized by reduced cell size, and high cell density, stress tolerance and longevity. The transition to quiescence involves highly asymmetric cell divisions, dramatic reprogramming of transcription and global changes in chromatin structure and chromosome topology. Cells enter quiescence from G1 and we find that there is a positive correlation between the length of G1 and the yield of quiescent cells. The Swi4 and Swi6 transcription factors, which form the SBF transcription complex and promote the G1 to S transition in cycling cells, are also critical for the transition to quiescence. Swi6 forms a second complex with Mbp1 (MBF), which is not required for quiescence. These are the functional analogues of the E2F complexes of higher eukaryotes. Loss of the RB analogue, Whi5, and the related protein Srl3/Whi7, delays G1 arrest, but it also delays recovery from quiescence. Two MBF- and SBF-Associated proteins have been identified that have little effect on SBF or MBF activity in cycling cells. We show that these two related proteins, Msa1 and Msa2, are specifically required for the transition to quiescence. Like the E2F complexes that are quiescence-specific, Msa1 and Msa2 are required to repress the transcription of many SBF target genes, including SWI4, the CLN2 cyclin and histones, specifically after glucose is exhausted from the media. They also activate transcription of many MBF target genes. msa1msa2 cells fail to G1 arrest and rapidly lose viability upon glucose exhaustion. msa1msa2 mutants that survive this transition are very large, but they attain the same thermo-tolerance and longevity of wild type quiescent cells. This indicates that Msa1 and Msa2 are required for successful transition to quiescence, but not for the maintenance of that state. PMID:27272642

  11. Shuffling of discrete tRNASer regions reveals differently utilized identity elements in yeast and methanogenic archaea.

    PubMed

    Gruic-Sovulj, Ita; Jaric, Jelena; Dulic, Morana; Cindric, Mario; Weygand-Durasevic, Ivana

    2006-08-01

    Seryl-tRNA synthetases (SerRSs) from methanogenic archaea possess distinct evolutionary origin and show minimal sequence similarity with counterparts from bacteria, eukaryotes and other archaea. Here we show that SerRS from yeast Saccharomyces cerevisiae and archaeon Methanococcus maripaludis (ScSerRS and MmSerRS, respectively) display significantly different ability to serylate heterologous tRNA(Ser). Recognition in yeast was shown to be more stringent than in archaeon. While cross-aminoacylation of M. maripaludis tRNA(Ser) (MmtRNA(Ser)) by yeast SerRS barely occurs, yeast tRNA(Ser) (SctRNA(Ser)) was shown to be a good substrate for heterologous MmSerRS. To investigate the contribution of different tRNA regions for the recognition by yeast and archaeal SerRS, chimeric tRNAs bearing separated domains of SctRNA(Ser) in MmtRNA(Ser) framework were produced by in vitro transcription and subjected to kinetic and gel mobility shift analysis with both enzymes. Generally, the recognition in M. maripaludis seems to be relatively relaxed toward tertiary elements of tRNA(Ser) structure and relies on the direct recognition of identity nucleotides. On the other hand, expression of tRNA(Ser) identity elements in yeast seems to be more sensitive toward surrounding sequence context. In both systems variable arm of tRNA was recognized as a major identity region with a strong influence on SerRS:tRNA binding. Acceptor domain of SctRNA(Ser) was also shown to be important for serylation in yeast. We propose that cognate interactions between N-terminal domain of yeast SerRS and variable region of SctRNA(Ser) place the acceptor stem into the enzyme's active site and lead to increased affinity toward serine and efficient serylation of tRNA. The same effect was not observed in M. maripaludis. Unlike its yeast counterpart, MmSerRS forms only one type of covalent complex with MmtRNA(Ser), regardless of the tRNA/SerRS molar ratio. Stoichiometry of the complex, one tRNA per dimeric SerRS, was

  12. Screening a yeast library of temperature-sensitive mutants reveals a role for actin in tombusvirus RNA recombination.

    PubMed

    Prasanth, K Reddisiva; Kovalev, Nikolay; de Castro Martín, Isabel Fernández; Baker, Jannine; Nagy, Peter D

    2016-02-01

    Genetic recombination in RNA viruses drives the evolutionary arms race with host's antiviral strategies and recombination also facilitates adaptation of viruses to new hosts. In this paper, the authors used tombusvirus and a temperature-sensitive (ts) mutant library of yeast to identify 40 host proteins affecting viral recombination in yeast model host. Subsequent detailed analysis with two identified actin-related proteins, Act1p and Arp3p, has revealed that the wt actin network helps TBSV to maintain low level viral recombination. Pharmacological inhibition of actin in plant protoplasts confirmed the role of the actin network in tombusvirus recombination. An in vitro approach revealed the altered activity of the tombusvirus replicase in the presence of mutated Act1p. The authors show more efficient recruitment of a cellular DEAD-box helicase, which enhances tombusvirus recombination, into the membrane-bound replicase in Act1p mutant yeast. Overall, this work shows that the actin network affects tombusvirus recombination in yeast and plant cells.

  13. The Gcn2 Regulator Yih1 Interacts with the Cyclin Dependent Kinase Cdc28 and Promotes Cell Cycle Progression through G2/M in Budding Yeast

    PubMed Central

    Silva, Richard C.; Dautel, Martina; Di Genova, Bruno M.; Amberg, David C.; Castilho, Beatriz A.; Sattlegger, Evelyn

    2015-01-01

    The Saccharomyces cerevisiae protein Yih1, when overexpressed, inhibits the eIF2 alpha kinase Gcn2 by competing for Gcn1 binding. However, deletion of YIH1 has no detectable effect on Gcn2 activity, suggesting that Yih1 is not a general inhibitor of Gcn2, and has no phenotypic defect identified so far. Thus, its physiological role is largely unknown. Here, we show that Yih1 is involved in the cell cycle. Yeast lacking Yih1 displays morphological patterns and DNA content indicative of a delay in the G2/M phases of the cell cycle, and this phenotype is independent of Gcn1 and Gcn2. Accordingly, the levels of phosphorylated eIF2α, which show a cell cycle-dependent fluctuation, are not altered in cells devoid of Yih1. We present several lines of evidence indicating that Yih1 is in a complex with Cdc28. Yih1 pulls down endogenous Cdc28 in vivo and this interaction is enhanced when Cdc28 is active, suggesting that Yih1 modulates the function of Cdc28 in specific stages of the cell cycle. We also demonstrate, by Bimolecular Fluorescence Complementation, that endogenous Yih1 and Cdc28 interact with each other, confirming Yih1 as a bona fide Cdc28 binding partner. Amino acid substitutions within helix H2 of the RWD domain of Yih1 enhance Yih1-Cdc28 association. Overexpression of this mutant, but not of wild type Yih1, leads to a phenotype similar to that of YIH1 deletion, supporting the view that Yih1 is involved through Cdc28 in the regulation of the cell cycle. We further show that IMPACT, the mammalian homologue of Yih1, interacts with CDK1, the mammalian counterpart of Cdc28, indicating that the involvement with the cell cycle is conserved. Together, these data provide insights into the cellular function of Yih1/IMPACT, and provide the basis for future studies on the role of this protein in the cell cycle. PMID:26176233

  14. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

    PubMed

    Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A; Boddy, Michael N

    2015-09-25

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function.

  15. Floral Initiation in Response to Planting Date Reveals the Key Role of Floral Meristem Differentiation Prior to Budding in Canola (Brassica napus L.)

    PubMed Central

    Zhang, Yaofeng; Zhang, Dongqing; Yu, Huasheng; Lin, Baogang; Fu, Ying; Hua, Shuijin

    2016-01-01

    In Brassica napus, floral development is a decisive factor in silique formation, and it is influenced by many cultivation practices including planting date. However, the effect of planting date on floral initiation in canola is poorly understood at present. A field experiment was conducted using a split plot design, in which three planting dates (early, 15 September, middle, 1 October, and late, 15 October) served as main plot and five varieties differing in maturity (1358, J22, Zhongshuang 11, Zheshuang 8, and Zheyou 50) employed as subplot. The purpose of this study was to shed light on the process of floral meristem (FM) differentiation, the influence of planting date on growth period (GP) and floral initiation, and silique formation. The main stages of FM developments can be divided into four stages: first, the transition from shoot apical meristem to FM; second, flower initiation; third, gynoecium and androecium differentiation; and fourth, bud formation. Our results showed that all genotypes had increased GPs from sowing to FM differentiation as planting date was delayed while the GPs from FM differentiation to budding varied year by year except the very early variety, 1358. Based on the number of flowers present at the different reproductive stages, the flowers produced from FM differentiation to budding closely approximated the final silique even though the FM differentiated continuously after budding and peaked generally at the middle flowering stage. The ratio of siliques to maximum flower number ranged from 48 to 80%. These results suggest that (1) the period from FM differentiation to budding is vital for effective flower and silique formation although there was no significant correlation between the length of the period and effective flowers and siliques, and (2) the increased number of flowers from budding were generally ineffective. Therefore, maximizing flower numbers prior to budding will improve silique numbers, and reducing FM degeneration should

  16. Floral Initiation in Response to Planting Date Reveals the Key Role of Floral Meristem Differentiation Prior to Budding in Canola (Brassica napus L.).

    PubMed

    Zhang, Yaofeng; Zhang, Dongqing; Yu, Huasheng; Lin, Baogang; Fu, Ying; Hua, Shuijin

    2016-01-01

    In Brassica napus, floral development is a decisive factor in silique formation, and it is influenced by many cultivation practices including planting date. However, the effect of planting date on floral initiation in canola is poorly understood at present. A field experiment was conducted using a split plot design, in which three planting dates (early, 15 September, middle, 1 October, and late, 15 October) served as main plot and five varieties differing in maturity (1358, J22, Zhongshuang 11, Zheshuang 8, and Zheyou 50) employed as subplot. The purpose of this study was to shed light on the process of floral meristem (FM) differentiation, the influence of planting date on growth period (GP) and floral initiation, and silique formation. The main stages of FM developments can be divided into four stages: first, the transition from shoot apical meristem to FM; second, flower initiation; third, gynoecium and androecium differentiation; and fourth, bud formation. Our results showed that all genotypes had increased GPs from sowing to FM differentiation as planting date was delayed while the GPs from FM differentiation to budding varied year by year except the very early variety, 1358. Based on the number of flowers present at the different reproductive stages, the flowers produced from FM differentiation to budding closely approximated the final silique even though the FM differentiated continuously after budding and peaked generally at the middle flowering stage. The ratio of siliques to maximum flower number ranged from 48 to 80%. These results suggest that (1) the period from FM differentiation to budding is vital for effective flower and silique formation although there was no significant correlation between the length of the period and effective flowers and siliques, and (2) the increased number of flowers from budding were generally ineffective. Therefore, maximizing flower numbers prior to budding will improve silique numbers, and reducing FM degeneration should

  17. Floral Initiation in Response to Planting Date Reveals the Key Role of Floral Meristem Differentiation Prior to Budding in Canola (Brassica napus L.).

    PubMed

    Zhang, Yaofeng; Zhang, Dongqing; Yu, Huasheng; Lin, Baogang; Fu, Ying; Hua, Shuijin

    2016-01-01

    In Brassica napus, floral development is a decisive factor in silique formation, and it is influenced by many cultivation practices including planting date. However, the effect of planting date on floral initiation in canola is poorly understood at present. A field experiment was conducted using a split plot design, in which three planting dates (early, 15 September, middle, 1 October, and late, 15 October) served as main plot and five varieties differing in maturity (1358, J22, Zhongshuang 11, Zheshuang 8, and Zheyou 50) employed as subplot. The purpose of this study was to shed light on the process of floral meristem (FM) differentiation, the influence of planting date on growth period (GP) and floral initiation, and silique formation. The main stages of FM developments can be divided into four stages: first, the transition from shoot apical meristem to FM; second, flower initiation; third, gynoecium and androecium differentiation; and fourth, bud formation. Our results showed that all genotypes had increased GPs from sowing to FM differentiation as planting date was delayed while the GPs from FM differentiation to budding varied year by year except the very early variety, 1358. Based on the number of flowers present at the different reproductive stages, the flowers produced from FM differentiation to budding closely approximated the final silique even though the FM differentiated continuously after budding and peaked generally at the middle flowering stage. The ratio of siliques to maximum flower number ranged from 48 to 80%. These results suggest that (1) the period from FM differentiation to budding is vital for effective flower and silique formation although there was no significant correlation between the length of the period and effective flowers and siliques, and (2) the increased number of flowers from budding were generally ineffective. Therefore, maximizing flower numbers prior to budding will improve silique numbers, and reducing FM degeneration should

  18. Floral Initiation in Response to Planting Date Reveals the Key Role of Floral Meristem Differentiation Prior to Budding in Canola (Brassica napus L.)

    PubMed Central

    Zhang, Yaofeng; Zhang, Dongqing; Yu, Huasheng; Lin, Baogang; Fu, Ying; Hua, Shuijin

    2016-01-01

    In Brassica napus, floral development is a decisive factor in silique formation, and it is influenced by many cultivation practices including planting date. However, the effect of planting date on floral initiation in canola is poorly understood at present. A field experiment was conducted using a split plot design, in which three planting dates (early, 15 September, middle, 1 October, and late, 15 October) served as main plot and five varieties differing in maturity (1358, J22, Zhongshuang 11, Zheshuang 8, and Zheyou 50) employed as subplot. The purpose of this study was to shed light on the process of floral meristem (FM) differentiation, the influence of planting date on growth period (GP) and floral initiation, and silique formation. The main stages of FM developments can be divided into four stages: first, the transition from shoot apical meristem to FM; second, flower initiation; third, gynoecium and androecium differentiation; and fourth, bud formation. Our results showed that all genotypes had increased GPs from sowing to FM differentiation as planting date was delayed while the GPs from FM differentiation to budding varied year by year except the very early variety, 1358. Based on the number of flowers present at the different reproductive stages, the flowers produced from FM differentiation to budding closely approximated the final silique even though the FM differentiated continuously after budding and peaked generally at the middle flowering stage. The ratio of siliques to maximum flower number ranged from 48 to 80%. These results suggest that (1) the period from FM differentiation to budding is vital for effective flower and silique formation although there was no significant correlation between the length of the period and effective flowers and siliques, and (2) the increased number of flowers from budding were generally ineffective. Therefore, maximizing flower numbers prior to budding will improve silique numbers, and reducing FM degeneration should

  19. Natural Variation in the Yeast Glucose-Signaling Network Reveals a New Role for the Mig3p Transcription Factor

    PubMed Central

    Lewis, Jeffrey A.; Gasch, Audrey P.

    2012-01-01

    The Crabtree effect, in which fermentative metabolism is preferred at the expense of respiration, is a hallmark of budding yeast’s glucose response and a model for the Warburg effect in human tumors. While the glucose-responsive transcriptional repressors Mig1p and Mig2p play well-characterized roles in the Crabtree effect, little function for the related Mig3p transcription factor has been uncovered, despite numerous investigations of laboratory yeast strains. Here we studied a wild isolate of Saccharomyces cerevisiae to uncover a critical role for Mig3p that has been lost in S288c-derived laboratory strains. We found that Mig3p affects the expression of hundreds of glucose-responsive genes in the oak strain YPS163, both during growth under standard conditions and upon ethanol treatment. Our results suggest that Mig3p may act as a multifunctional activator/repressor that plays separate roles under standard vs. stress conditions and that this function has been largely lost in the lab strains. Population analysis suggests that the lab strain and several wild strains harbor mutations that diminish Mig3p function. Thus, by expanding our attention to multiple genetic backgrounds, we have uncovered an important missing link in a key metabolic response. PMID:23275883

  20. Whole-Transcriptome Analysis of Differentially Expressed Genes in the Vegetative Buds, Floral Buds and Buds of Chrysanthemum morifolium

    PubMed Central

    Liu, Hua; Sun, Ming; Du, Dongliang; Pan, Huitang; Cheng, Tangren; Wang, Jia; Zhang, Qixiang

    2015-01-01

    Background Chrysanthemum morifolium is an important floral crop that is cultivated worldwide. However, due to a lack of genomic resources, very little information is available concerning the molecular mechanisms of flower development in chrysanthemum. Results The transcriptomes of chrysanthemum vegetative buds, floral buds and buds were sequenced using Illumina paired-end sequencing technology. A total of 15.4 Gb of reads were assembled into 91,367 unigenes with an average length of 739 bp. A total of 43,137 unigenes showed similarity to known proteins in the Swissprot or NCBI non-redundant protein databases. Additionally, 25,424, 24,321 and 13,704 unigenes were assigned to 56 gene ontology (GO) categories, 25 EuKaryotic Orthologous Groups (KOG) categories, and 285 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. A total of 1,876 differentially expressed genes (DEGs) (1,516 up-regulated, 360 down-regulated) were identified between vegetative buds and floral buds, and 3,300 DEGs (1,277 up-regulated, 1,706 down-regulated) were identified between floral buds and buds. Many genes encoding important transcription factors (e.g., AP2, MYB, MYC, WRKY, NAC and CRT) as well as proteins involved in carbohydrate metabolism, protein kinase activity, plant hormone signal transduction, and the defense responses, among others, were considerably up-regulated in floral buds. Genes involved in the photoperiod pathway and flower organ determination were also identified. These genes represent important candidate genes for molecular cloning and functional analysis to study flowering regulation in chrysanthemum. Conclusion This comparative transcriptome analysis revealed significant differences in gene expression and signaling pathway components between the vegetative buds, floral buds and buds of Chrysanthemum morifolium. A wide range of genes was implicated in regulating the phase transition from vegetative to reproductive growth. These results should aid

  1. A Genomewide Screen Reveals a Role of Mitochondria in Anaerobic Uptake of Sterols in Yeast

    PubMed Central

    Reiner, Sonja; Micolod, Delphine; Zellnig, Günther; Schneiter, Roger

    2006-01-01

    The mechanisms that govern intracellular transport of sterols in eukaryotic cells are not well understood. Saccharomyces cerevisiae is a facultative anaerobic organism that becomes auxotroph for sterols and unsaturated fatty acids in the absence of oxygen. To identify pathways that are required for uptake and transport of sterols, we performed a systematic screen of the yeast deletion mutant collection for genes that are required for growth under anaerobic conditions. Of the ∼4800 nonessential genes represented in the deletion collection, 37 were essential for growth under anaerobic conditions. These affect a wide range of cellular functions, including biosynthetic pathways for certain amino acids and cofactors, reprogramming of transcription and translation, mitochondrial function and biogenesis, and membrane trafficking. Thirty-three of these mutants failed to grow on lipid-supplemented media when combined with a mutation in HEM1, which mimics anaerobic conditions in the presence of oxygen. Uptake assays with radio- and fluorescently labeled cholesterol revealed that 17 of the 33 mutants strongly affect uptake and/or esterification of exogenously supplied cholesterol. Examination of the subcellular distribution of sterols in these uptake mutants by cell fractionation and fluorescence microscopy indicates that some of the mutants block incorporation of cholesterol into the plasma membrane, a presumably early step in sterol uptake. Unexpectedly, the largest class of uptake mutants is affected in mitochondrial functions, and many of the uptake mutants show electron-dense mitochondrial inclusions. These results indicate that a hitherto uncharacterized mitochondrial function is required for sterol uptake and/or transport under anaerobic conditions and are discussed in light of the fact that mitochondrial import of cholesterol is required for steroidogenesis in vertebrate cells. PMID:16251356

  2. Plasmids with E2 epitope tags: tagging modules for N- and C-terminal PCR-based gene targeting in both budding and fission yeast, and inducible expression vectors for fission yeast.

    PubMed

    Tamm, Tiina

    2009-01-01

    A single-step PCR-based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10-amino acids (epitope E2a: SSTSSDFRDR)- and 12 amino acids (epitope E2b: GVSSTSSDFRDR)-long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C-terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM-series plasmid. The N-terminal E2a and E2b tagging modules were based on pOM-series plasmid. The pOM-series plasmids were selected for this study because of their use of the Cre-loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N-terminal or C-terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti-E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2-tagged strains. PMID:19180640

  3. Nucleocytoplasmic Distribution of Budding Yeast Protein Kinase A Regulatory Subunit Bcy1 Requires Zds1 and Is Regulated by Yak1-Dependent Phosphorylation of Its Targeting Domain

    PubMed Central

    Griffioen, Gerard; Branduardi, Paola; Ballarini, Annalisa; Anghileri, Paola; Norbeck, Joakim; Baroni, Maurizio D.; Ruis, Helmut

    2001-01-01

    In Saccharomyces cerevisiae the subcellular distribution of Bcy1 is carbon source dependent. In glucose-grown cells, Bcy1 is almost exclusively nuclear, while it appears more evenly distributed between nucleus and cytoplasm in carbon source-derepressed cells. Here we show that phosphorylation of its N-terminal domain directs Bcy1 to the cytoplasm. Biochemical fractionation revealed that the cytoplasmic fraction contains mostly phosphorylated Bcy1, whereas unmodified Bcy1 is predominantly present in the nuclear fraction. Site-directed mutagenesis of two clusters (I and II) of serines near the N terminus to alanine resulted in an enhanced nuclear accumulation of Bcy1 in ethanol-grown cells. In contrast, substitutions to Asp led to a dramatic increase of cytoplasmic localization in glucose-grown cells. Bcy1 modification was found to be dependent on Yak1 kinase and, consequently, in ethanol-grown yak1 cells the Bcy1 remained nuclear. A two-hybrid screen aimed to isolate genes encoding proteins that interact with the Bcy1 N-terminal domain identified Zds1. In ethanol-grown zds1 cells, cytoplasmic localization of Bcy1 was largely absent, while overexpression of ZDS1 led to increased cytoplasmic Bcy1 localization. Zds1 does not regulate Bcy1 modification since this was found to be unaffected in zds1 cells. However, in zds1 cells cluster II-mediated, but not cluster I-mediated, cytoplasmic localization of Bcy1 was found to be absent. Altogether, these results suggest that Zds1-mediated cytoplasmic localization of Bcy1 is regulated by carbon source-dependent phosphorylation of cluster II serines, while cluster I acts in a Zds1-independent manner. PMID:11134339

  4. Active Trans-Plasma Membrane Water Cycling in Yeast Is Revealed by NMR

    PubMed Central

    Zhang, Yajie; Poirier-Quinot, Marie; Springer, Charles S.; Balschi, James A.

    2011-01-01

    Plasma membrane water transport is a crucial cellular phenomenon. Net water movement in response to an osmotic gradient changes cell volume. Steady-state exchange of water molecules, with no net flux or volume change, occurs by passive diffusion through the phospholipid bilayer and passage through membrane proteins. The hypothesis is tested that plasma membrane water exchange also correlates with ATP-driven membrane transport activity in yeast (Saccharomyces cerevisiae). Longitudinal 1H2O NMR relaxation time constant (T1) values were measured in yeast suspensions containing extracellular relaxation reagent. Two-site-exchange analysis quantified the reversible exchange kinetics as the mean intracellular water lifetime (τi), where τi−1 is the pseudo-first-order rate constant for water efflux. To modulate cellular ATP, yeast suspensions were bubbled with 95%O2/5%CO2 (O2) or 95%N2/5%CO2 (N2). ATP was high during O2, and τi−1 was 3.1 s−1 at 25°C. After changing to N2, ATP decreased and τi−1 was 1.8 s−1. The principal active yeast ion transport protein is the plasma membrane H+-ATPase. Studies using the H+-ATPase inhibitor ebselen or a yeast genetic strain with reduced H+-ATPase found reduced τi−1, notwithstanding high ATP. Steady-state water exchange correlates with H+-ATPase activity. At volume steady state, water is cycling across the plasma membrane in response to metabolic transport activity. PMID:22261073

  5. Hi-C in Budding Yeast.

    PubMed

    Belton, Jon-Matthew; Dekker, Job

    2015-07-01

    Hi-C enables simultaneous detection of interaction frequencies between all possible pairs of restriction fragments in the genome. The Hi-C method is based on chromosome conformation capture (3C), which uses formaldehyde cross-linking to fix chromatin regions that interact in three-dimensional space, irrespective of their genomic locations. In the Hi-C protocol described here, cross-linked chromatin is digested with HindIII and the ends are filled in with a nucleotide mix containing biotinylated dCTP. These fragments are ligated together, and the resulting chimeric molecules are purified and sheared to reduce length. Finally, biotinylated ligation junctions are pulled down with streptavidin-coated beads, linked to high-throughput sequencing adaptors, and amplified via polymerase chain reaction (PCR). The resolution of the Hi-C data set will depend on the depth of sequencing and choice of restriction enzyme. When sufficient sequence reads are obtained, information on chromatin interactions and chromosome conformation can be derived at single restriction fragment resolution for complete genomes.

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

    PubMed Central

    Rallis, Charalampos; López-Maury, Luis; Georgescu, Teodora; Pancaldi, Vera; Bähler, Jürg

    2014-01-01

    Summary Target 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. PMID:24463365

  7. Combining chemical genomics screens in yeast to reveal spectrum of effects of chemical inhibition of sphingolipid biosynthesis

    PubMed Central

    2009-01-01

    Background Single genome-wide screens for the effect of altered gene dosage on drug sensitivity in the model organism Saccharomyces cerevisiae provide only a partial picture of the mechanism of action of a drug. Results Using the example of the tumor cell invasion inhibitor dihydromotuporamine C, we show that a more complete picture of drug action can be obtained by combining different chemical genomics approaches – analysis of the sensitivity of ρ0 cells lacking mitochondrial DNA, drug-induced haploinsufficiency, suppression of drug sensitivity by gene overexpression and chemical-genetic synthetic lethality screening using strains deleted of nonessential genes. Killing of yeast by this chemical requires a functional mitochondrial electron-transport chain and cytochrome c heme lyase function. However, we find that it does not require genes associated with programmed cell death in yeast. The chemical also inhibits endocytosis and intracellular vesicle trafficking and interferes with vacuolar acidification in yeast and in human cancer cells. These effects can all be ascribed to inhibition of sphingolipid biosynthesis by dihydromotuporamine C. Conclusion Despite their similar conceptual basis, namely altering drug sensitivity by modifying gene dosage, each of the screening approaches provided a distinct set of information that, when integrated, revealed a more complete picture of the mechanism of action of a drug on cells. PMID:19144191

  8. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing

    PubMed Central

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  9. Novel features of the rotary catalytic mechanism revealed in the structure of yeast F1 ATPase

    SciTech Connect

    Kabaleeswaran, Venkataraman; Puri, Neeti; Walker, John E.; Leslie, Andrew G.W.; Mueller, David M.

    2010-03-08

    The crystal structure of yeast mitochondrial F{sub 1} ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the {alpha}{sub 3}{beta}{sub 3} sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F{sub 1} ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F{sub 1} ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F{sub 1} ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.

  10. Different Toxicity Mechanisms for Citrinin and Ochratoxin A Revealed by Transcriptomic Analysis in Yeast

    PubMed Central

    Vanacloig-Pedros, Elena; Proft, Markus; Pascual-Ahuir, Amparo

    2016-01-01

    Citrinin (CIT) and ochratoxin A (OTA) are important mycotoxins, which frequently co-contaminate foodstuff. In order to assess the toxicologic threat posed by the two mycotoxins separately or in combination, their biological effects were studied here using genomic transcription profiling and specific live cell gene expression reporters in yeast cells. Both CIT and OTA cause highly transient transcriptional activation of different stress genes, which is greatly enhanced by the disruption of the multidrug exporter Pdr5. Therefore, we performed genome-wide transcription profiling experiments with the pdr5 mutant in response to acute CIT, OTA, or combined CIT/OTA exposure. We found that CIT and OTA activate divergent and largely nonoverlapping gene sets in yeast. CIT mainly caused the rapid induction of antioxidant and drug extrusion-related gene functions, while OTA mainly deregulated developmental genes related with yeast sporulation and sexual reproduction, having only a minor effect on the antioxidant response. The simultaneous exposure to CIT and OTA gave rise to a genomic response, which combined the specific features of the separated mycotoxin treatments. The application of stress-specific mutants and reporter gene fusions further confirmed that both mycotoxins have divergent biological effects in cells. Our results indicate that CIT exposure causes a strong oxidative stress, which triggers a massive transcriptional antioxidant and drug extrusion response, while OTA mainly deregulates developmental genes and only marginally induces the antioxidant defense. PMID:27669300

  11. Computational analysis of translational readthrough proteins in Drosophila and yeast reveals parallels to alternative splicing.

    PubMed

    Pancsa, Rita; Macossay-Castillo, Mauricio; Kosol, Simone; Tompa, Peter

    2016-01-01

    In translational readthrough (TR) the ribosome continues extending the nascent protein beyond the first in-frame termination codon. Due to the lack of dedicated analyses of eukaryotic TR cases, the associated functional-evolutionary advantages are still unclear. Here, based on a variety of computational methods, we describe the structural and functional properties of previously proposed D. melanogaster and S. cerevisiae TR proteins and extensions. We found that in D. melanogaster TR affects long proteins in mainly regulatory roles. Their TR-extensions are structurally disordered and rich in binding motifs, which, together with their cell-type- and developmental stage-dependent inclusion, suggest that similarly to alternatively spliced exons they rewire cellular interaction networks in a temporally and spatially controlled manner. In contrast, yeast TR proteins are rather short and fulfil mainly housekeeping functions, like translation. Yeast extensions usually lack disorder and linear motifs, which precludes elucidating their functional relevance with sufficient confidence. Therefore we propose that by being much more restricted and by lacking clear functional hallmarks in yeast as opposed to fruit fly, TR shows remarkable parallels with alternative splicing. Additionally, the lack of conservation of TR extensions among orthologous TR proteins suggests that TR-mediated functions may be generally specific to lower taxonomic levels. PMID:27561673

  12. A novel single-cell screening platform reveals proteome plasticity during yeast stress responses

    PubMed Central

    Breker, Michal; Gymrek, Melissa

    2013-01-01

    Uncovering the mechanisms underlying robust responses of cells to stress is crucial for our understanding of cellular physiology. Indeed, vast amounts of data have been collected on transcriptional responses in Saccharomyces cerevisiae. However, only a handful of pioneering studies describe the dynamics of proteins in response to external stimuli, despite the fact that regulation of protein levels and localization is an essential part of such responses. Here we characterized unprecedented proteome plasticity by systematically tracking the localization and abundance of 5,330 yeast proteins at single-cell resolution under three different stress conditions (DTT, H2O2, and nitrogen starvation) using the GFP-tagged yeast library. We uncovered a unique “fingerprint” of changes for each stress and elucidated a new response arsenal for adapting to radical environments. These include bet-hedging strategies, organelle rearrangement, and redistribution of protein localizations. All data are available for download through our online database, LOQATE (localization and quantitation atlas of yeast proteome). PMID:23509072

  13. Different Toxicity Mechanisms for Citrinin and Ochratoxin A Revealed by Transcriptomic Analysis in Yeast.

    PubMed

    Vanacloig-Pedros, Elena; Proft, Markus; Pascual-Ahuir, Amparo

    2016-01-01

    Citrinin (CIT) and ochratoxin A (OTA) are important mycotoxins, which frequently co-contaminate foodstuff. In order to assess the toxicologic threat posed by the two mycotoxins separately or in combination, their biological effects were studied here using genomic transcription profiling and specific live cell gene expression reporters in yeast cells. Both CIT and OTA cause highly transient transcriptional activation of different stress genes, which is greatly enhanced by the disruption of the multidrug exporter Pdr5. Therefore, we performed genome-wide transcription profiling experiments with the pdr5 mutant in response to acute CIT, OTA, or combined CIT/OTA exposure. We found that CIT and OTA activate divergent and largely nonoverlapping gene sets in yeast. CIT mainly caused the rapid induction of antioxidant and drug extrusion-related gene functions, while OTA mainly deregulated developmental genes related with yeast sporulation and sexual reproduction, having only a minor effect on the antioxidant response. The simultaneous exposure to CIT and OTA gave rise to a genomic response, which combined the specific features of the separated mycotoxin treatments. The application of stress-specific mutants and reporter gene fusions further confirmed that both mycotoxins have divergent biological effects in cells. Our results indicate that CIT exposure causes a strong oxidative stress, which triggers a massive transcriptional antioxidant and drug extrusion response, while OTA mainly deregulates developmental genes and only marginally induces the antioxidant defense. PMID:27669300

  14. Bud Dormancy and Growth

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nearly all land plants produce ancillary meristems in the form of axillary or adventitious buds in addition to the shoot apical meristem. Outgrowth of these buds has a significant impact on plant architecture and the ability of plants to compete with neighboring plants, as well as to respond to and ...

  15. Identification of Telomerase RNAs from Filamentous Fungi Reveals Conservation with Vertebrates and Yeasts

    PubMed Central

    Kuprys, Paulius V.; Davis, Shaun M.; Hauer, Tyler M.; Meltser, Max; Tzfati, Yehuda; Kirk, Karen E.

    2013-01-01

    Telomeres are the nucleoprotein complexes at eukaryotic chromosomal ends. Telomeric DNA is synthesized by the ribonucleoprotein telomerase, which comprises a telomerase reverse transcriptase (TERT) and a telomerase RNA (TER). TER contains a template for telomeric DNA synthesis. Filamentous fungi possess extremely short and tightly regulated telomeres. Although TERT is well conserved between most organisms, TER is highly divergent and thus difficult to identify. In order to identify the TER sequence, we used the unusually long telomeric repeat sequence of Aspergillus oryzae together with reverse-transcription-PCR and identified a transcribed sequence that contains the potential template within a region predicted to be single stranded. We report the discovery of TERs from twelve other related filamentous fungi using comparative genomic analysis. These TERs exhibited strong conservation with the vertebrate template sequence, and two of these potentially use the identical template as humans. We demonstrate the existence of important processing elements required for the maturation of yeast TERs such as an Sm site, a 5′ splice site and a branch point, within the newly identified TER sequences. RNA folding programs applied to the TER sequences show the presence of secondary structures necessary for telomerase activity, such as a yeast-like template boundary, pseudoknot, and a vertebrate-like three-way junction. These telomerase RNAs identified from filamentous fungi display conserved structural elements from both yeast and vertebrate TERs. These findings not only provide insights into the structure and evolution of a complex RNA but also provide molecular tools to further study telomere dynamics in filamentous fungi. PMID:23555591

  16. Dynamics of the yeast transcriptome during wine fermentation reveals a novel fermentation stress response.

    PubMed

    Marks, Virginia D; Ho Sui, Shannan J; Erasmus, Daniel; van der Merwe, George K; Brumm, Jochen; Wasserman, Wyeth W; Bryan, Jennifer; van Vuuren, Hennie J J

    2008-02-01

    In this study, genome-wide expression analyses were used to study the response of Saccharomyces cerevisiae to stress throughout a 15-day wine fermentation. Forty per cent of the yeast genome significantly changed expression levels to mediate long-term adaptation to fermenting grape must. Among the genes that changed expression levels, a group of 223 genes was identified, which was designated as fermentation stress response (FSR) genes that were dramatically induced at various points during fermentation. FSR genes sustain high levels of induction up to the final time point and exhibited changes in expression levels ranging from four- to 80-fold. The FSR is novel; 62% of the genes involved have not been implicated in global stress responses and 28% of the FSR genes have no functional annotation. Genes involved in respiratory metabolism and gluconeogenesis were expressed during fermentation despite the presence of high concentrations of glucose. Ethanol, rather than nutrient depletion, seems to be responsible for entry of yeast cells into the stationary phase.

  17. High-Resolution Mutation Mapping Reveals Parallel Experimental Evolution in Yeast

    PubMed Central

    Segrè, Ayellet V; Murray, Andrew W

    2006-01-01

    Understanding the genetic basis of evolutionary adaptation is limited by our ability to efficiently identify the genomic locations of adaptive mutations. Here we describe a method that can quickly and precisely map the genetic basis of naturally and experimentally evolved complex traits using linkage analysis. A yeast strain that expresses the evolved trait is crossed to a distinct strain background and DNA from a large pool of progeny that express the trait of interest is hybridized to oligonucleotide microarrays that detect thousands of polymorphisms between the two strains. Adaptive mutations are detected by linkage to the polymorphisms from the evolved parent. We successfully tested our method by mapping five known genes to a precision of 0.2–24 kb (0.1–10 cM), and developed computer simulations to test the effect of different factors on mapping precision. We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose–galactose environment. All four strains had acquired one or more missense mutations in GAL80, the repressor of the galactose utilization pathway. When transferred into the ancestral strain, the gal80 mutations conferred the fitness advantage that the evolved strains show in the transition from glucose to galactose. Our results show an example of parallel adaptation caused by mutations in the same gene. PMID:16856782

  18. Defining the Essential Function of Yeast Hsf1 Reveals a Compact Transcriptional Program for Maintaining Eukaryotic Proteostasis.

    PubMed

    Solís, Eric J; Pandey, Jai P; Zheng, Xu; Jin, Dexter X; Gupta, Piyush B; Airoldi, Edoardo M; Pincus, David; Denic, Vladimir

    2016-07-01

    Despite its eponymous association with the heat shock response, yeast heat shock factor 1 (Hsf1) is essential even at low temperatures. Here we show that engineered nuclear export of Hsf1 results in cytotoxicity associated with massive protein aggregation. Genome-wide analysis revealed that Hsf1 nuclear export immediately decreased basal transcription and mRNA expression of 18 genes, which predominately encode chaperones. Strikingly, rescuing basal expression of Hsp70 and Hsp90 chaperones enabled robust cell growth in the complete absence of Hsf1. With the exception of chaperone gene induction, the vast majority of the heat shock response was Hsf1 independent. By comparative analysis of mammalian cell lines, we found that only heat shock-induced but not basal expression of chaperones is dependent on the mammalian Hsf1 homolog (HSF1). Our work reveals that yeast chaperone gene expression is an essential housekeeping mechanism and provides a roadmap for defining the function of HSF1 as a driver of oncogenesis. PMID:27320198

  19. The SPR3 gene encodes a sporulation-specific homologue of the yeast CDC3/10/11/12 family of bud neck microfilaments and is regulated by ABFI.

    PubMed

    Ozsarac, N; Bhattacharyya, M; Dawes, I W; Clancy, M J

    1995-10-16

    The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdc11p and Cdc12p), Candida albicans proteins that accumulate in the hyphal phase (CaCdc3p and CaCdc10p), mouse brain-specific (H5p) and lymphocyte (Diff6p) proteins, Drosophila melanogaster (Dm) protein Pnutp (which is localized to the cleavage furrow of dividing cells), a Diff6p homologue (DmDiff6p), and the Sc septin protein (Sep1hp), a homologue of the 10-nm filament proteins of Sc. One strongly conserved region contains a potential ATP-GTP-binding domain. Primer extension analysis revealed six major transcription start points (tsp) beginning at -142 relative to the ATG start codon. The sequence immediately upstream from the tsp contains consensus binding sites for the HAP2/3/4 and ABFI transcription factors, a T-rich sequence and two putative novel elements for mid to late sporulation, termed SPR3 and PAL. Electrophoretic mobility shift assay (EMSA) and footprint analyses demonstrated that the ABFI protein binds to a region containing the putative ABFI site in vitro, and site-directed mutagenesis showed that the ABFI motif is essential for expression of SPR3 at the appropriate stage in sporulating cells.

  20. Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation.

    PubMed

    Shen, Qi; Chen, Yue; Jin, Danfeng; Lin, Hui; Wang, Qun; Zhao, Yu-Hua

    2016-11-01

    In this work, Trichosporon fermentans CICC 1368, which has been shown to accumulate cellular lipids efficiently using industry-agricultural wastes, was subjected to preliminary genome analysis, yielding a genome size of 31.3 million bases and 12,702 predicted protein-coding genes. Our analysis also showed a high degree of gene duplications and unique genes compared with those observed in other oleaginous yeasts, with 3-4-fold more genes related to fatty acid elongation and degradation compared with those in Rhodosporidium toruloides NP11 and Yarrowia lipolytica CLIB122. Phylogenetic analysis with other oleaginous microbes suggested that the lipogenic capacity of T. fermentans was obtained during evolution after the divergence of genera. Thus, our study provided the first draft genome and comparative analysis of T. fermentans, laying the foundation for its genetic improvement to facilitate cost-effective lipid production. PMID:27664738

  1. Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation.

    PubMed

    Shen, Qi; Chen, Yue; Jin, Danfeng; Lin, Hui; Wang, Qun; Zhao, Yu-Hua

    2016-11-01

    In this work, Trichosporon fermentans CICC 1368, which has been shown to accumulate cellular lipids efficiently using industry-agricultural wastes, was subjected to preliminary genome analysis, yielding a genome size of 31.3 million bases and 12,702 predicted protein-coding genes. Our analysis also showed a high degree of gene duplications and unique genes compared with those observed in other oleaginous yeasts, with 3-4-fold more genes related to fatty acid elongation and degradation compared with those in Rhodosporidium toruloides NP11 and Yarrowia lipolytica CLIB122. Phylogenetic analysis with other oleaginous microbes suggested that the lipogenic capacity of T. fermentans was obtained during evolution after the divergence of genera. Thus, our study provided the first draft genome and comparative analysis of T. fermentans, laying the foundation for its genetic improvement to facilitate cost-effective lipid production.

  2. The Structure of the Yeast Plasma Membrane SNARE Complex Reveals Destabilizing Water Filled Cavities

    SciTech Connect

    Strop, P.; Kaiser, S.E.; Vrljic, M.; Brunger, A.T.

    2009-05-26

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form a complex that leads to membrane fusion between vesicles, organelles, and plasma membrane in all eukaryotic cells. We report the 1.7{angstrom} resolution structure of the SNARE complex that mediates exocytosis at the plasma membrane in the yeast Saccharomyces cerevisiae. Similar to its neuronal and endosomal homologues, the S. cerevisiae SNARE complex forms a parallel four-helix bundle in the center of which is an ionic layer. The S. cerevisiae SNARE complex exhibits increased helix bending near the ionic layer, contains water-filled cavities in the complex core, and exhibits reduced thermal stability relative to mammalian SNARE complexes. Mutagenesis experiments suggest that the water-filled cavities contribute to the lower stability of the S. cerevisiae complex.

  3. Yeast Genetic Analysis Reveals the Involvement of Chromatin Reassembly Factors in Repressing HIV-1 Basal Transcription

    PubMed Central

    Respaldiza, Iñaki; Rodríguez-Gil, Alfonso; Gómez-Herreros, Fernando; Jimeno-González, Silvia; Jordan, Albert; Chávez, Sebastián

    2009-01-01

    Rebound of HIV viremia after interruption of anti-retroviral therapy is due to the small population of CD4+ T cells that remain latently infected. HIV-1 transcription is the main process controlling post-integration latency. Regulation of HIV-1 transcription takes place at both initiation and elongation levels. Pausing of RNA polymerase II at the 5′ end of HIV-1 transcribed region (5′HIV-TR), which is immediately downstream of the transcription start site, plays an important role in the regulation of viral expression. The activation of HIV-1 transcription correlates with the rearrangement of a positioned nucleosome located at this region. These two facts suggest that the 5′HIV-TR contributes to inhibit basal transcription of those HIV-1 proviruses that remain latently inactive. However, little is known about the cell elements mediating the repressive role of the 5′HIV-TR. We performed a genetic analysis of this phenomenon in Saccharomyces cerevisiae after reconstructing a minimal HIV-1 transcriptional system in this yeast. Unexpectedly, we found that the critical role played by the 5′HIV-TR in maintaining low levels of basal transcription in yeast is mediated by FACT, Spt6, and Chd1, proteins so far associated with chromatin assembly and disassembly during ongoing transcription. We confirmed that this group of factors plays a role in HIV-1 postintegration latency in human cells by depleting the corresponding human orthologs with shRNAs, both in HIV latently infected cell populations and in particular single-integration clones, including a latent clone with a provirus integrated in a highly transcribed gene. Our results indicate that chromatin reassembly factors participate in the establishment of the equilibrium between activation and repression of HIV-1 when it integrates into the human genome, and they open the possibility of considering these factors as therapeutic targets of HIV-1 latency. PMID:19148280

  4. Tropical Storm Bud

    Atmospheric Science Data Center

    2013-04-19

    article title:  A Strengthening Eastern Pacific Storm     View Larger Image ... Imaging SpectroRadiometer (MISR) show then Tropical Storm Bud as it was intensifying toward hurricane status, which it acquired ...

  5. New Regulators of a High Affinity Ca2+ Influx System Revealed through a Genome-wide Screen in Yeast*

    PubMed Central

    Martin, D. Christian; Kim, Hyemin; Mackin, Nancy A.; Maldonado-Báez, Lymarie; Evangelista, Carlos C.; Beaudry, Veronica G.; Dudgeon, Drew D.; Naiman, Daniel Q.; Erdman, Scott E.; Cunningham, Kyle W.

    2011-01-01

    The bakers' yeast Saccharomyces cerevisiae utilizes a high affinity Ca2+ influx system (HACS) to survive assaults by mating pheromones, tunicamycin, and azole-class antifungal agents. HACS consists of two known subunits, Cch1 and Mid1, that are homologous and analogous to the catalytic α-subunits and regulatory α2δ-subunits of mammalian voltage-gated calcium channels, respectively. To search for additional subunits and regulators of HACS, a collection of gene knock-out mutants was screened for abnormal uptake of Ca2+ after exposure to mating pheromone or to tunicamycin. The screen revealed that Ecm7 is required for HACS function in most conditions. Cycloheximide chase experiments showed that Ecm7 was stabilized by Mid1, and Mid1 was stabilized by Cch1 in non-signaling conditions, suggesting they all interact. Ecm7 is a member of the PMP-22/EMP/MP20/Claudin superfamily of transmembrane proteins that includes γ-subunits of voltage-gated calcium channels. Eleven additional members of this superfamily were identified in yeast, but none was required for HACS activity in response to the stimuli. Remarkably, many dozens of genes involved in vesicle-mediated trafficking and protein secretion were required to prevent spontaneous activation of HACS. Taken together, the findings suggest that HACS and calcineurin monitor performance of the membrane trafficking system in yeasts and coordinate compensatory processes. Conservation of this quality control system in Candida glabrata suggests that many pathogenic species of fungi may utilize HACS and calcineurin to resist azoles and other compounds that target membrane biosynthesis. PMID:21252230

  6. Single-molecule atomic force microscopy reveals clustering of the yeast plasma-membrane sensor Wsc1.

    PubMed

    Heinisch, Jürgen J; Dupres, Vincent; Wilk, Sabrina; Jendretzki, Arne; Dufrêne, Yves F

    2010-06-14

    Signalling is a key feature of living cells which frequently involves the local clustering of specific proteins in the plasma membrane. How such protein clustering is achieved within membrane microdomains ("rafts") is an important, yet largely unsolved problem in cell biology. The plasma membrane of yeast cells represents a good model to address this issue, since it features protein domains that are sufficiently large and stable to be observed by fluorescence microscopy. Here, we demonstrate the ability of single-molecule atomic force microscopy to resolve lateral clustering of the cell integrity sensor Wsc1 in living Saccharomyces cerevisiae cells. We first localize individual wild-type sensors on the cell surface, revealing that they form clusters of approximately 200 nm size. Analyses of three different mutants indicate that the cysteine-rich domain of Wsc1 has a crucial, not yet anticipated function in sensor clustering and signalling. Clustering of Wsc1 is strongly enhanced in deionized water or at elevated temperature, suggesting its relevance in proper stress response. Using in vivo GFP-localization, we also find that non-clustering mutant sensors accumulate in the vacuole, indicating that clustering may prevent endocytosis and sensor turnover. This study represents the first in vivo single-molecule demonstration for clustering of a transmembrane protein in S. cerevisiae. Our findings indicate that in yeast, like in higher eukaryotes, signalling is coupled to the localized enrichment of sensors and receptors within membrane patches.

  7. Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation.

    PubMed

    Babazadeh, Roja; Furukawa, Takako; Hohmann, Stefan; Furukawa, Kentaro

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data. PMID:24732094

  8. Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation

    PubMed Central

    Babazadeh, Roja; Furukawa, Takako; Hohmann, Stefan; Furukawa, Kentaro

    2014-01-01

    Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data. PMID:24732094

  9. A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression.

    PubMed

    Kühl, Inge; Dujeancourt, Laurent; Gaisne, Mauricette; Herbert, Christopher J; Bonnefoy, Nathalie

    2011-10-01

    Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation.

  10. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    PubMed Central

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-01-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages. PMID:9539419

  11. Species-wide survey reveals the various flavors of intraspecific reproductive isolation in yeast.

    PubMed

    Hou, Jing; Fournier, Téo; Schacherer, Joseph

    2016-08-01

    Exploring the origin and extent of reproductive isolation within the same species is valuable to capture early events to the onset of speciation. In multiple genetic models, reproductive isolation was recently observed at the intraspecific scale, indicating that the raw potential for speciation segregates readily within populations, which could be a rule rather than an exception in a broad context. We briefly recapitulate the molecular evidence of intrinsic post-zygotic isolation in major model organisms including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster and their close relatives. We then focus on recent advances in yeast and review the genetic basis of post-zygotic isolation within and between multiple members of the Saccharomyces genus, especially in Saccharomyces cerevisiae We discuss the role of various mechanisms involved in the onset of reproductive isolation including DNA sequence divergence, chromosomal rearrangement, cytonuclear as well as nuclear-nuclear genetic incompatibilities and provide a comparative view along a continuum of genetic differentiation, which encompasses intraspecific populations, recent delineating nascent species as well as closely related sister species in the same subphylum. PMID:27288348

  12. Potassium starvation in yeast: mechanisms of homeostasis revealed by mathematical modeling.

    PubMed

    Kahm, Matthias; Navarrete, Clara; Llopis-Torregrosa, Vicent; Herrera, Rito; Barreto, Lina; Yenush, Lynne; Ariño, Joaquin; Ramos, Jose; Kschischo, Maik

    2012-01-01

    The intrinsic ability of cells to adapt to a wide range of environmental conditions is a fundamental process required for survival. Potassium is the most abundant cation in living cells and is required for essential cellular processes, including the regulation of cell volume, pH and protein synthesis. Yeast cells can grow from low micromolar to molar potassium concentrations and utilize sophisticated control mechanisms to keep the internal potassium concentration in a viable range. We developed a mathematical model for Saccharomyces cerevisiae to explore the complex interplay between biophysical forces and molecular regulation facilitating potassium homeostasis. By using a novel inference method ("the reverse tracking algorithm") we predicted and then verified experimentally that the main regulators under conditions of potassium starvation are proton fluxes responding to changes of potassium concentrations. In contrast to the prevailing view, we show that regulation of the main potassium transport systems (Trk1,2 and Nha1) in the plasma membrane is not sufficient to achieve homeostasis.

  13. Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication

    PubMed Central

    Burns, Shannon; Avena, Jennifer S; Unruh, Jay R; Yu, Zulin; Smith, Sarah E; Slaughter, Brian D; Winey, Mark; Jaspersen, Sue L

    2015-01-01

    Duplication of the yeast centrosome (called the spindle pole body, SPB) is thought to occur through a series of discrete steps that culminate in insertion of the new SPB into the nuclear envelope (NE). To better understand this process, we developed a novel two-color structured illumination microscopy with single-particle averaging (SPA-SIM) approach to study the localization of all 18 SPB components during duplication using endogenously expressed fluorescent protein derivatives. The increased resolution and quantitative intensity information obtained using this method allowed us to demonstrate that SPB duplication begins by formation of an asymmetric Sfi1 filament at mitotic exit followed by Mps1-dependent assembly of a Spc29- and Spc42-dependent complex at its tip. Our observation that proteins involved in membrane insertion, such as Mps2, Bbp1, and Ndc1, also accumulate at the new SPB early in duplication suggests that SPB assembly and NE insertion are coupled events during SPB formation in wild-type cells. DOI: http://dx.doi.org/10.7554/eLife.08586.001 PMID:26371506

  14. Genome-Wide Functional Profiling Reveals Genes Required for Tolerance to Benzene Metabolites in Yeast

    PubMed Central

    North, Matthew; Tandon, Vickram J.; Thomas, Reuben; Loguinov, Alex; Gerlovina, Inna; Hubbard, Alan E.; Zhang, Luoping; Smith, Martyn T.; Vulpe, Chris D.

    2011-01-01

    Benzene is a ubiquitous environmental contaminant and is widely used in industry. Exposure to benzene causes a number of serious health problems, including blood disorders and leukemia. Benzene undergoes complex metabolism in humans, making mechanistic determination of benzene toxicity difficult. We used a functional genomics approach to identify the genes that modulate the cellular toxicity of three of the phenolic metabolites of benzene, hydroquinone (HQ), catechol (CAT) and 1,2,4-benzenetriol (BT), in the model eukaryote Saccharomyces cerevisiae. Benzene metabolites generate oxidative and cytoskeletal stress, and tolerance requires correct regulation of iron homeostasis and the vacuolar ATPase. We have identified a conserved bZIP transcription factor, Yap3p, as important for a HQ-specific response pathway, as well as two genes that encode putative NAD(P)H:quinone oxidoreductases, PST2 and YCP4. Many of the yeast genes identified have human orthologs that may modulate human benzene toxicity in a similar manner and could play a role in benzene exposure-related disease. PMID:21912624

  15. Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication.

    PubMed

    Burns, Shannon; Avena, Jennifer S; Unruh, Jay R; Yu, Zulin; Smith, Sarah E; Slaughter, Brian D; Winey, Mark; Jaspersen, Sue L

    2015-01-01

    Duplication of the yeast centrosome (called the spindle pole body, SPB) is thought to occur through a series of discrete steps that culminate in insertion of the new SPB into the nuclear envelope (NE). To better understand this process, we developed a novel two-color structured illumination microscopy with single-particle averaging (SPA-SIM) approach to study the localization of all 18 SPB components during duplication using endogenously expressed fluorescent protein derivatives. The increased resolution and quantitative intensity information obtained using this method allowed us to demonstrate that SPB duplication begins by formation of an asymmetric Sfi1 filament at mitotic exit followed by Mps1-dependent assembly of a Spc29- and Spc42-dependent complex at its tip. Our observation that proteins involved in membrane insertion, such as Mps2, Bbp1, and Ndc1, also accumulate at the new SPB early in duplication suggests that SPB assembly and NE insertion are coupled events during SPB formation in wild-type cells. PMID:26371506

  16. Network reconstruction reveals new links between aging and calorie restriction in yeast

    PubMed Central

    Balázsi, Gábor

    2010-01-01

    Aging affects all known organisms and has been studied extensively. Yet, the underlying mechanisms are insufficiently understood, possibly due to the multiscale complexity involved in this process: the aging of multicellular organisms depends on the aging of their cells, which depends on molecular events occurring in each cell. However, the aging of unicellular populations seeded in new niches and the aging of metazoans are surprisingly similar, indicating that the multiscale aspects of aging may have been conserved since the beginnings of cellular life on Earth. This underlines the importance of aging research in unicellular organisms such as a recent study by Lorenz et al., [(2009) Proc. Natl. Acad. Sci. U.S.A. 106, 1145–1150]. In their paper, the authors combine computational network identification with extensive experimentation and literature mining to discover and validate numerous regulatory interactions among ten genes involved in the cellular response to glucose starvation. Since low levels of glucose (calorie restriction) have been known to extend the longevity of various eukaryotes, the authors test the effect of Snf1 kinase overexpression on chronological aging and discover that this key regulator of glucose repression and two of its newly discovered synergistic repressors significantly affect the chronological lifespan of baker’s yeast. PMID:21119761

  17. Genetic analysis of yeast RPA1 reveals its multiple functions in DNA metabolism.

    PubMed

    Umezu, K; Sugawara, N; Chen, C; Haber, J E; Kolodner, R D

    1998-03-01

    Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 10(4) to 10(5) times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

  18. Yeast gain-of-function mutations reveal structure-function relationships conserved among different subfamilies of transient receptor potential channels.

    PubMed

    Su, Zhenwei; Zhou, Xinliang; Haynes, W John; Loukin, Stephen H; Anishkin, Andriy; Saimi, Yoshiro; Kung, Ching

    2007-12-01

    Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.

  19. Genetic dissection of the phospholipid hydroperoxidase activity of yeast gpx3 reveals its functional importance.

    PubMed

    Avery, Angela M; Willetts, Sylvia A; Avery, Simon V

    2004-11-01

    Saccharomyces cerevisiae expresses multiple phospholipid hydroperoxide glutathione peroxidase (PHGPx)-like proteins in the absence of a classical glutathione peroxidase (cGPx), providing a unique system for dissecting the roles of these enzymes in vivo. The Gpx3 (Orp1/PHGpx3) protein transduces the hydroperoxide signal to the transcription factor Yap1, a function that could account for most GPX-dependent phenotypes. To test this hypothesis and ascertain what functions of Gpx3 can be shared by cGPx-like enzymes, we constructed a novel cGPx-like yeast enzyme, cGpx3. We confirmed that the "gap" sequences conserved among cGPxs but absent from aligned PHGPx sequences are the principal cause of the structural and functional differences of these enzymes. Peroxidase activity against a cGPx substrate was high in the cGpx3 construct, which was multimeric and had a peroxidase catalytic mechanism distinct from Gpx3; but cGpx3 was defective for phospholipid hydroperoxidase and signaling activities. cGpx3 did not complement the sensitivity to lipid peroxidation of a gpxDelta mutant, and the resistance to lipid peroxidation conferred by Gpx3 was independent of Yap1, establishing a functional role for Gpx3 phospholipid hydroperoxidase activity. Using the comparison between cGpx3 and Gpx3 in conjunction with other constructs to probe lipid peroxidation as a toxicity mechanism, we also ascertained that lipid peroxidation-dependent processes are a principal cause of cellular cadmium toxicity. The results demonstrate that phospholipid hydroperoxidase and Yap1-mediated signaling activities of Gpx3 have independent functional roles, although both functions depend on the absence of cGPx-like subunit interaction sites, and the results resolve more clearly the potential drivers of the differential selective evolution of GPx-like enzymes. PMID:15337745

  20. Common gene expression strategies revealed by genome-wide analysis in yeast

    PubMed Central

    García-Martínez, José; González-Candelas, Fernando; Pérez-Ortín, José E

    2007-01-01

    Background Gene expression is a two-step synthesis process that ends with the necessary amount of each protein required to perform its function. Since the protein is the final product, the main focus of gene regulation should be centered on it. However, because mRNA is an intermediate step and the amounts of both mRNA and protein are controlled by their synthesis and degradation rates, the desired amount of protein can be achieved following different strategies. Results In this paper we present the first comprehensive analysis of the relationships among the six variables that characterize gene expression in a living organism: transcription and translation rates, mRNA and protein amounts, and mRNA and protein stabilities. We have used previously published data from exponentially growing Saccharomyces cerevisiae cells. We show that there is a general tendency to harmonize the levels of mRNA and protein by coordinating their synthesis rates and that functionally related genes tend to have similar values for the six variables. Conclusion We propose that yeast cells use common expression strategies for genes acting in the same physiological pathways. This trend is more evident for genes coding for large and stable protein complexes, such as ribosomes or the proteasome. Hence, each functional group can be defined by a 'six variable profile' that illustrates the common strategy followed by the genes included in it. Genes encoding subunits of protein complexes show a tendency to have relatively unstable mRNAs and a less balanced profile for mRNA than for protein, suggesting a stronger regulation at the transcriptional level. PMID:17945030

  1. High-Throughput Sequencing Reveals Drastic Changes in Fungal Communities in the Phyllosphere of Norway Spruce (Picea abies) Following Invasion of the Spruce Bud Scale (Physokermes piceae).

    PubMed

    Menkis, Audrius; Marčiulynas, Adas; Gedminas, Artūras; Lynikienė, Jūratė; Povilaitienė, Aistė

    2015-11-01

    The aim of this study was to assess the diversity and composition of fungal communities in damaged and undamaged shoots of Norway spruce (Picea abies) following recent invasion of the spruce bud scale (Physokermes piceae) in Lithuania. Sampling was done in July 2013 and included 50 random lateral shoots from ten random trees in each of five visually undamaged and five damaged 40-50-year-old pure stands of P. abies. DNA was isolated from 500 individual shoots, subjected to amplification of the internal transcribed spacer of fungal ribosomal DNA (ITS rDNA), barcoded and sequenced. Clustering of 149,426 high-quality sequences resulted in 1193 non-singleton contigs of which 1039 (87.1 %) were fungal. In total, there were 893 fungal taxa in damaged shoots and 608 taxa in undamaged shoots (p < 0.0001). Furthermore, 431 (41.5 %) fungal taxa were exclusively in damaged shoots, 146 (14.0 %) were exclusively in undamaged shoots, and 462 (44.5 %) were common to both types of samples. Correspondence analysis showed that study sites representing damaged and undamaged shoots were separated from each other, indicating that in these fungal communities, these were largely different and, therefore, heavily affected by P. piceae. In conclusion, the results demonstrated that invasive alien tree pests may have a profound effect on fungal mycobiota associated with the phyllosphere of P. abies, and therefore, in addition to their direct negative effect owing physical damage of the tissue, they may also indirectly determine health, sustainability and, ultimately, distribution of the forest tree species.

  2. A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

    PubMed Central

    Arkowitz, Robert A.; Lowe, Nick

    1997-01-01

    SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain. PMID:9214378

  3. High-throughput screening of a large collection of non-conventional yeasts reveals their potential for aroma formation in food fermentation.

    PubMed

    Gamero, Amparo; Quintilla, Raquel; Groenewald, Marizeth; Alkema, Wynand; Boekhout, Teun; Hazelwood, Lucie

    2016-12-01

    Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species. PMID:27554157

  4. High-throughput screening of a large collection of non-conventional yeasts reveals their potential for aroma formation in food fermentation.

    PubMed

    Gamero, Amparo; Quintilla, Raquel; Groenewald, Marizeth; Alkema, Wynand; Boekhout, Teun; Hazelwood, Lucie

    2016-12-01

    Saccharomyces yeast species are currently the most important yeasts involved in industrial-scale food fermentations. However, there are hundreds of other yeast species poorly studied that are highly promising for flavour development, some of which have also been identified in traditional food fermentations. This work explores natural yeast biodiversity in terms of aroma formation, with a particular focus on aromas relevant for industrial fermentations such as wine and beer. Several non-Saccharomyces species produce important aroma compounds such as fusel alcohols derived from the Ehrlich pathway, acetate esters and ethyl esters in significantly higher quantities than the well-known Saccharomyces species. These species are Starmera caribaea, Hanseniaspora guilliermondii, Galactomyces geotrichum, Saccharomycopsis vini and Ambrosiozyma monospora. Certain species revealed a strain-dependent flavour profile while other species were very homogenous in their flavour profiles. Finally, characterization of a selected number of yeast species using valine or leucine as sole nitrogen sources indicates that the mechanisms of regulation of the expression of the Ehrlich pathway exist amongst non-conventional yeast species.

  5. Automated quantification of budding Saccharomyces cerevisiae using a novel image cytometry method.

    PubMed

    Laverty, Daniel J; Kury, Alexandria L; Kuksin, Dmitry; Pirani, Alnoor; Flanagan, Kevin; Chan, Leo Li-Ying

    2013-06-01

    The measurements of concentration, viability, and budding percentages of Saccharomyces cerevisiae are performed on a routine basis in the brewing and biofuel industries. Generation of these parameters is of great importance in a manufacturing setting, where they can aid in the estimation of product quality, quantity, and fermentation time of the manufacturing process. Specifically, budding percentages can be used to estimate the reproduction rate of yeast populations, which directly correlates with metabolism of polysaccharides and bioethanol production, and can be monitored to maximize production of bioethanol during fermentation. The traditional method involves manual counting using a hemacytometer, but this is time-consuming and prone to human error. In this study, we developed a novel automated method for the quantification of yeast budding percentages using Cellometer image cytometry. The automated method utilizes a dual-fluorescent nucleic acid dye to specifically stain live cells for imaging analysis of unique morphological characteristics of budding yeast. In addition, cell cycle analysis is performed as an alternative method for budding analysis. We were able to show comparable yeast budding percentages between manual and automated counting, as well as cell cycle analysis. The automated image cytometry method is used to analyze and characterize corn mash samples directly from fermenters during standard fermentation. Since concentration, viability, and budding percentages can be obtained simultaneously, the automated method can be integrated into the fermentation quality assurance protocol, which may improve the quality and efficiency of beer and bioethanol production processes.

  6. "Bud, Not Buddy."

    ERIC Educational Resources Information Center

    Brodie, Carolyn S.

    2002-01-01

    Discusses the award-winning book "Bud, Not Buddy" written by Christopher Paul Curtis. Lists different versions of the book; suggests learning activities; lists sources for biographical information and interviews with Curtis, teacher guides, professional articles, and other Depression era novels; and provides a citation for the author's Newberry…

  7. Berkeley UXO Discriminator (BUD)

    SciTech Connect

    Gasperikova, Erika; Smith, J. Torquil; Morrison, H. Frank; Becker, Alex

    2007-01-01

    The Berkeley UXO Discriminator (BUD) is an optimally designed active electromagnetic system that not only detects but also characterizes UXO. The system incorporates three orthogonal transmitters and eight pairs of differenced receivers. it has two modes of operation: (1) search mode, in which BUD moves along a profile and exclusively detects targets in its vicinity, providing target depth and horizontal location, and (2) discrimination mode, in which BUD, stationary above a target, from a single position, determines three discriminating polarizability responses together with the object location and orientation. The performance of the system is governed by a target size-depth curve. Maximum detection depth is 1.5 m. While UXO objects have a single major polarizability coincident with the long axis of the object and two equal transverse polarizabilities, scrap metal has three different principal polarizabilities. The results clearly show that there are very clear distinctions between symmetric intact UXO and irregular scrap metal, and that BUD can resolve the intrinsic polarizabilities of the target. The field survey at the Yuma Proving Ground in Arizona showed excellent results within the predicted size-depth range.

  8. Efficient Reverse Genetics Reveals Genetic Determinants of Budding and Fusogenic Differences between Nipah and Hendra Viruses and Enables Real-Time Monitoring of Viral Spread in Small Animal Models of Henipavirus Infection

    PubMed Central

    Yun, Tatyana; Park, Arnold; Hill, Terence E.; Pernet, Olivier; Beaty, Shannon M.; Juelich, Terry L.; Smith, Jennifer K.; Zhang, Lihong; Wang, Yao E.; Vigant, Frederic; Gao, Junling; Wu, Ping

    2014-01-01

    the growing numbers of novel henipavirus-like viruses. IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV) are recently emergent zoonotic and highly lethal pathogens with pandemic potential. Although differences have been observed between NiV and HeV replication and pathogenesis, the molecular basis for these differences has not been examined. In this study, we established a highly efficient system to reverse engineer changes into replication-competent NiV and HeV, which facilitated the generation of reporter-expressing viruses and recombinant NiV-HeV chimeras with substitutions in the genes responsible for viral exit (the M gene, critical for assembly and budding) and viral entry (the G [attachment] and F [fusion] genes). These chimeras revealed differences in the budding and fusogenic properties of the M and G proteins, respectively, which help explain previously observed differences between NiV and HeV. Finally, to facilitate future in vivo studies, we monitored the replication and spread of a bioluminescent reporter-expressing NiV in susceptible mice; this is the first time such in vivo imaging has been performed under BSL-4 conditions. PMID:25392218

  9. Probing Yeast Polarity with Acute, Reversible, Optogenetic Inhibition of Protein Function.

    PubMed

    Jost, Anna Payne-Tobin; Weiner, Orion D

    2015-10-16

    We recently developed a technique for rapidly and reversibly inhibiting protein function through light-inducible sequestration of proteins away from their normal sites of action. Here, we adapt this method for inducible inactivation of Bem1, a scaffold protein involved in budding yeast polarity. We find that acute inhibition of Bem1 produces profound defects in cell polarization and cell viability that are not observed in bem1Δ. By disrupting Bem1 activity at specific points in the cell cycle, we demonstrate that Bem1 is essential for the establishment of polarity and bud emergence but is dispensable for the growth of an emerged bud. By taking advantage of the reversibility of Bem1 inactivation, we show that pole size scales with cell size, and that this scaling is dependent on the actin cytoskeleton. Our experiments reveal how rapid reversible inactivation of protein function complements traditional genetic approaches. This strategy should be widely applicable to other biological contexts.

  10. Global analysis of transcriptionally engaged yeast RNA polymerase III reveals extended tRNA transcripts

    PubMed Central

    Turowski, Tomasz W.; Leśniewska, Ewa; Delan-Forino, Clementine; Sayou, Camille; Boguta, Magdalena; Tollervey, David

    2016-01-01

    RNA polymerase III (RNAPIII) synthesizes a range of highly abundant small stable RNAs, principally pre-tRNAs. Here we report the genome-wide analysis of nascent transcripts attached to RNAPIII under permissive and restrictive growth conditions. This revealed strikingly uneven polymerase distributions across transcription units, generally with a predominant 5′ peak. This peak was higher for more heavily transcribed genes, suggesting that initiation site clearance is rate-limiting during RNAPIII transcription. Down-regulation of RNAPIII transcription under stress conditions was found to be uneven; a subset of tRNA genes showed low response to nutrient shift or loss of the major transcription regulator Maf1, suggesting potential “housekeeping” roles. Many tRNA genes were found to generate long, 3′-extended forms due to read-through of the canonical poly(U) terminators. The degree of read-through was anti-correlated with the density of U-residues in the nascent tRNA, and multiple, functional terminators can be located far downstream. The steady-state levels of 3′-extended pre-tRNA transcripts are low, apparently due to targeting by the nuclear surveillance machinery, especially the RNA binding protein Nab2, cofactors for the nuclear exosome, and the 5′-exonuclease Rat1. PMID:27206856

  11. Speciation driven by hybridization and chromosomal plasticity in a wild yeast.

    PubMed

    Leducq, Jean-Baptiste; Nielly-Thibault, Lou; Charron, Guillaume; Eberlein, Chris; Verta, Jukka-Pekka; Samani, Pedram; Sylvester, Kayla; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R

    2016-01-11

    Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche.

  12. Speciation driven by hybridization and chromosomal plasticity in a wild yeast.

    PubMed

    Leducq, Jean-Baptiste; Nielly-Thibault, Lou; Charron, Guillaume; Eberlein, Chris; Verta, Jukka-Pekka; Samani, Pedram; Sylvester, Kayla; Hittinger, Chris Todd; Bell, Graham; Landry, Christian R

    2016-01-01

    Hybridization is recognized as a powerful mechanism of speciation and a driving force in generating biodiversity. However, only few multicellular species, limited to a handful of plants and animals, have been shown to fulfil all the criteria of homoploid hybrid speciation. This lack of evidence could lead to the interpretation that speciation by hybridization has a limited role in eukaryotes, particularly in single-celled organisms. Laboratory experiments have revealed that fungi such as budding yeasts can rapidly develop reproductive isolation and novel phenotypes through hybridization, showing that in principle homoploid speciation could occur in nature. Here, we report a case of homoploid hybrid speciation in natural populations of the budding yeast Saccharomyces paradoxus inhabiting the North American forests. We show that the rapid evolution of chromosome architecture and an ecological context that led to secondary contact between nascent species drove the formation of an incipient hybrid species with a potentially unique ecological niche. PMID:27571751

  13. Reconstruction of ancestral chromosome architecture and gene repertoire reveals principles of genome evolution in a model yeast genus.

    PubMed

    Vakirlis, Nikolaos; Sarilar, Véronique; Drillon, Guénola; Fleiss, Aubin; Agier, Nicolas; Meyniel, Jean-Philippe; Blanpain, Lou; Carbone, Alessandra; Devillers, Hugo; Dubois, Kenny; Gillet-Markowska, Alexandre; Graziani, Stéphane; Huu-Vang, Nguyen; Poirel, Marion; Reisser, Cyrielle; Schott, Jonathan; Schacherer, Joseph; Lafontaine, Ingrid; Llorente, Bertrand; Neuvéglise, Cécile; Fischer, Gilles

    2016-07-01

    Reconstructing genome history is complex but necessary to reveal quantitative principles governing genome evolution. Such reconstruction requires recapitulating into a single evolutionary framework the evolution of genome architecture and gene repertoire. Here, we reconstructed the genome history of the genus Lachancea that appeared to cover a continuous evolutionary range from closely related to more diverged yeast species. Our approach integrated the generation of a high-quality genome data set; the development of AnChro, a new algorithm for reconstructing ancestral genome architecture; and a comprehensive analysis of gene repertoire evolution. We found that the ancestral genome of the genus Lachancea contained eight chromosomes and about 5173 protein-coding genes. Moreover, we characterized 24 horizontal gene transfers and 159 putative gene creation events that punctuated species diversification. We retraced all chromosomal rearrangements, including gene losses, gene duplications, chromosomal inversions and translocations at single gene resolution. Gene duplications outnumbered losses and balanced rearrangements with 1503, 929, and 423 events, respectively. Gene content variations between extant species are mainly driven by differential gene losses, while gene duplications remained globally constant in all lineages. Remarkably, we discovered that balanced chromosomal rearrangements could be responsible for up to 14% of all gene losses by disrupting genes at their breakpoints. Finally, we found that nonsynonymous substitutions reached fixation at a coordinated pace with chromosomal inversions, translocations, and duplications, but not deletions. Overall, we provide a granular view of genome evolution within an entire eukaryotic genus, linking gene content, chromosome rearrangements, and protein divergence into a single evolutionary framework.

  14. Plasma Metabolomic Profiling to Reveal Antipyretic Mechanism of Shuang-Huang-Lian Injection on Yeast-Induced Pyrexia Rats

    PubMed Central

    Peng, Long; Liu, Haiyu; Zhang, Li; Bai, Xu; Wang, Yingxin; Li, Jian; Cai, Chengke

    2014-01-01

    Shuang-huang-lian injection (SHLI) is a famous Chinese patent medicine, which has been wildly used in clinic for the treatment of acute respiratory tract infection, pneumonia, influenza, etc. The existing randomized controlled trial (RCT) studies suggested that SHLI could afford a certain anti-febrile action. However, seldom does research concern the pharmacological mechanisms of SHLI. In the current study, we explored plasma metabolomic profiling technique and selected potential metabolic markers to reveal the antipyretic mechanism of SHLI on yeast-induced pyrexia rat model using UPLC-Q-TOF/MS coupled with multivariate statistical analysis and pattern recognition techniques. We discovered a significant perturbance of metabolic profile in the plasma of fever rats and obvious reversion in SHLI-administered rats. Eight potential biomarkers, i.e. 1) 3-hydeoxybutyric acid, 2) leucine, 3) 16∶0 LPC, 4) allocholic acid, 5) vitamin B2, 6) Cys-Lys-His, 7) 18∶2 LPC, and 8) 3-hydroxychola-7, 22-dien-24-oic acid, were screened out by OPLS-DA approach. Five potential perturbed metabolic pathways, i.e. 1) valine, leucine, and isoleucine biosynthesis, 2) glycerophospholipid metabolism, 3) ketone bodies synthesis and degradation, 4) bile acid biosynthesis, and 5) riboflavin metabolism, were revealed to relate to the antipyretic mechanisms of SHLI. Overall, we investigated antipyretic mechanisms of SHLI at metabolomic level for the first time, and the obtained results highlights the necessity of adopting metabolomics as a reliable tool for understanding the holism and synergism of Chinese patent drug. PMID:24940599

  15. Comparison of morphogenetic networks of filamentous fungi and yeast.

    PubMed

    Wendland, J

    2001-11-01

    Fungi generally display either of two growth modes, yeast-like or filamentous, whereas dimorphic fungi, upon environmental stimuli, are able to switch between the yeast-like and the filamentous growth mode. Signal transduction pathways have been elucidated in the budding yeast Saccharomyces cerevisiae, establishing a morphogenetic network that links cell-cycle events with cellular morphogenesis. Recent molecular genetic studies in several filamentous fungal model systems revealed key components required for distinct steps from fungal spore germination to the maintenance of polar hyphal growth, mycelium formation, and nuclear division. This allows a mechanistic comparison of yeast-like and hyphal growth and the establishment of a core model morphogenetic network for filamentous growth including signaling via the cAMP pathway, Rho modules, and cell cycle kinases. Appreciating similarities between morphogenetic networks of the unicellular yeasts and the multicellular filamentous fungi will open new research directions, help in isolating the central network components, and ultimately pave the way to elucidate the central differences (of many) that distinguish, e.g., the growth mode of filamentous fungi from that of their yeast-like relatives, the role of cAMP signaling, and nuclear division. PMID:11686673

  16. Molecular events of apical bud formation in white spruce, Picea glauca.

    PubMed

    El Kayal, Walid; Allen, Carmen C G; Ju, Chelsea J-T; Adams, Eri; King-Jones, Susanne; Zaharia, L Irina; Abrams, Suzanne R; Cooke, Janice E K

    2011-03-01

    Bud formation is an adaptive trait that temperate forest trees have acquired to facilitate seasonal synchronization. We have characterized transcriptome-level changes that occur during bud formation of white spruce [Picea glauca (Moench) Voss], a primarily determinate species in which preformed stem units contained within the apical bud constitute most of next season's growth. Microarray analysis identified 4460 differentially expressed sequences in shoot tips during short day-induced bud formation. Cluster analysis revealed distinct temporal patterns of expression, and functional classification of genes in these clusters implied molecular processes that coincide with anatomical changes occurring in the developing bud. Comparing expression profiles in developing buds under long day and short day conditions identified possible photoperiod-responsive genes that may not be essential for bud development. Several genes putatively associated with hormone signalling were identified, and hormone quantification revealed distinct profiles for abscisic acid (ABA), cytokinins, auxin and their metabolites that can be related to morphological changes to the bud. Comparison of gene expression profiles during bud formation in different tissues revealed 108 genes that are differentially expressed only in developing buds and show greater transcript abundance in developing buds than other tissues. These findings provide a temporal roadmap of bud formation in white spruce.

  17. Yeast population dynamics reveal a potential 'collaboration' between Metschnikowia pulcherrima and Saccharomyces uvarum for the production of reduced alcohol wines during Shiraz fermentation.

    PubMed

    Contreras, A; Curtin, C; Varela, C

    2015-02-01

    The wine sector is actively seeking strategies and technologies that facilitate the production of wines with lower alcohol content. One of the simplest approaches to achieve this aim would be the use of wine yeast strains which are less efficient at transforming grape sugars into ethanol; however, commercial wine yeasts have very similar ethanol yields. We recently demonstrated that Metschnikowia pulcherrima AWRI1149 was able to produce wine with reduced alcohol concentration when used in sequential inoculation with a wine strain of Saccharomyces cerevisiae. Here, different inoculation regimes were explored to study the effect of yeast population dynamics and potential yeast interactions on the metabolism of M. pulcherrima AWRI1149 during fermentation of non-sterile Shiraz must. Of all inoculation regimes tested, only ferments inoculated with M. pulcherrima AWRI1149 showed reduced ethanol concentration. Population dynamics revealed the presence of several indigenous yeast species and one of these, Saccharomyces uvarum (AWRI 2846), was able to produce wine with reduced ethanol concentration in sterile conditions. Both strains however, were inhibited when a combination of three non-Saccharomyces strains, Hanseniaspora uvarum AWRI863, Pichia kluyveri AWRI1896 and Torulaspora delbrueckii AWRI2845 were inoculated into must, indicating that the microbial community composition might impact on the growth of M. pulcherrima AWRI1149 and S. uvarum AWRI 2846. Our results indicate that mixed cultures of M. pulcherrima AWRI1149 and S. uvarum AWRI2846 enable an additional reduction of wine ethanol concentration compared to the same must fermented with either strain alone. This work thus provides a foundation to develop inoculation regimes for the successful application of non-cerevisiae yeast to the production of wines with reduced alcohol.

  18. Analysis of the Saccharomyces cerevisiae pan-genome reveals a pool of copy number variants distributed in diverse yeast strains from differing industrial environments.

    PubMed

    Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J; Pugh, Tom; Sherlock, Gavin

    2012-05-01

    Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species--i.e., its "pan-genome"--has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes-plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae.

  19. Analysis of the Saccharomyces cerevisiae pan-genome reveals a pool of copy number variants distributed in diverse yeast strains from differing industrial environments

    PubMed Central

    Dunn, Barbara; Richter, Chandra; Kvitek, Daniel J.; Pugh, Tom; Sherlock, Gavin

    2012-01-01

    Although the budding yeast Saccharomyces cerevisiae is arguably one of the most well-studied organisms on earth, the genome-wide variation within this species—i.e., its “pan-genome”—has been less explored. We created a multispecies microarray platform containing probes covering the genomes of several Saccharomyces species: S. cerevisiae, including regions not found in the standard laboratory S288c strain, as well as the mitochondrial and 2-μm circle genomes–plus S. paradoxus, S. mikatae, S. kudriavzevii, S. uvarum, S. kluyveri, and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) on 83 different S. cerevisiae strains collected across a wide range of habitats; of these, 69 were commercial wine strains, while the remaining 14 were from a diverse set of other industrial and natural environments. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole-genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae. PMID:22369888

  20. Eukaryote-to-eukaryote gene transfer events revealed by the genome sequence of the wine yeast Saccharomyces cerevisiae EC1118

    PubMed Central

    Novo, Maite; Bigey, Frédéric; Beyne, Emmanuelle; Galeote, Virginie; Gavory, Frédérick; Mallet, Sandrine; Cambon, Brigitte; Legras, Jean-Luc; Wincker, Patrick; Casaregola, Serge; Dequin, Sylvie

    2009-01-01

    Saccharomyces cerevisiae has been used for millennia in winemaking, but little is known about the selective forces acting on the wine yeast genome. We sequenced the complete genome of the diploid commercial wine yeast EC1118, resulting in an assembly of 31 scaffolds covering 97% of the S288c reference genome. The wine yeast differed strikingly from the other S. cerevisiae isolates in possessing 3 unique large regions, 2 of which were subtelomeric, the other being inserted within an EC1118 chromosome. These regions encompass 34 genes involved in key wine fermentation functions. Phylogeny and synteny analyses showed that 1 of these regions originated from a species closely related to the Saccharomyces genus, whereas the 2 other regions were of non-Saccharomyces origin. We identified Zygosaccharomyces bailii, a major contaminant of wine fermentations, as the donor species for 1 of these 2 regions. Although natural hybridization between Saccharomyces strains has been described, this report provides evidence that gene transfer may occur between Saccharomyces and non-Saccharomyces species. We show that the regions identified are frequent and differentially distributed among S. cerevisiae clades, being found almost exclusively in wine strains, suggesting acquisition through recent transfer events. Overall, these data show that the wine yeast genome is subject to constant remodeling through the contribution of exogenous genes. Our results suggest that these processes are favored by ecologic proximity and are involved in the molecular adaptation of wine yeasts to conditions of high sugar, low nitrogen, and high ethanol concentrations. PMID:19805302

  1. Crystal Structures of Mutant Forms of the Yeast F[subscript 1] ATPase Reveal Two Modes of Uncoupling

    SciTech Connect

    Arsenieva, Diana; Symersky, Jindrich; Wang, Yamin; Pagadala, Vijayakanth; Mueller, David M.

    2010-11-15

    The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F{sub 1} ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between {gamma}Leu83 and {beta}{sub DP} residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the {gamma}-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process.

  2. Transcriptome analysis of chestnut (Castanea sativa) tree buds suggests a putative role for epigenetic control of bud dormancy

    PubMed Central

    Santamaría, María Estrella; Rodríguez, Roberto; Cañal, María Jesús; Toorop, Peter E.

    2011-01-01

    Background and Aims Recent papers indicated that epigenetic control is involved in transitions in bud dormancy, purportedly controlling gene expression. The present study aimed to identify genes that are differentially expressed in dormant and non-dormant Castanea sativa buds. Methods Two suppression subtractive hybridization cDNA libraries were constructed to characterize the transcriptomes of dormant apical buds of C. sativa, and buds in which dormancy was released. Key Results A total of 512 expressed sequence tags (ESTs) were generated in a forward and reverse subtractive hybridization experiment. Classification of these ESTs into functional groups demonstrated that dormant buds were predominantly characterized by genes associated with stress response, while non-dormant buds were characterized by genes associated with energy, protein synthesis and cellular components for development and growth. ESTs for a few genes involved in different forms of epigenetic modification were found in both libraries, suggesting a role for epigenetic control in bud dormancy different from that in growth. Genes encoding histone mono-ubiquitinase HUB2 and histone acetyltransferase GCN5L were associated with dormancy, while a gene encoding histone H3 kinase AUR3 was associated with growth. Real-time RT-PCR with a selection of genes involved in epigenetic modification and stress tolerance confirmed the expression of the majority of investigated genes in various stages of bud development, revealing a cyclical expression pattern concurring with the growth seasons for most genes. However, senescing leaves also showed an increased expression of several of the genes associated with dormancy, implying pleiotropy. Furthermore, a comparison between these subtraction cDNA libraries and the poplar bud dormancy transcriptome and arabidopsis transcriptomes for seed dormancy and non-dormancy indicated a common basis for dormancy in all three systems. Conclusions Bud dormancy and non-dormancy in C

  3. Genes Required for Survival in Microgravity Revealed by Genome-Wide Yeast Deletion Collections Cultured during Spaceflight

    PubMed Central

    Nislow, Corey; Lee, Anna Y.; Allen, Patricia L.; Giaever, Guri; Smith, Andrew; Gebbia, Marinella; Stodieck, Louis S.; Hammond, Jeffrey S.; Birdsall, Holly H.; Hammond, Timothy G.

    2015-01-01

    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. PMID:25667933

  4. Detection of gamma-irradiation induced DNA damage and radioprotection of compounds in yeast using comet assay.

    PubMed

    Nemavarkar, P S; Chourasia, B K; Pasupathy, K

    2004-06-01

    The single cell gel electrophoresis assay (SCGE), a very rapid and sensitive method, has been applied to follow gamma-irradiation induced DNA damage in budding yeast, Saccharomyces cerevisiae. Spheroplasting the gamma-irradiated yeast cells by enzyme glusulase, before subjecting them to electrophoresis, resulted in a well-defined appearance of comets. Yeast comets look quite different from mammalian comets. A linear relationship was observed between the doses of irradiation and the tail moments of comets. These studies were extended to follow the action of known radio-protectors, i.e., caffeine and disulfiram. The results revealed the usefulness SCGE as applied to yeast in studies of the gamma-irradiation-induced DNA breaks and also radio-protection by chemicals at doses that are not feasible with other eukaryotes. PMID:15304956

  5. Phenotypic and chemotypic studies using Arabidopsis and yeast reveal that GHB converts to SSA and induce toxicity.

    PubMed

    Mekonnen, Dereje Worku; Ludewig, Frank

    2016-07-01

    γ-Hydroxybutyric acid (GHB) is a naturally occurring compound. It is detected in organisms such as yeasts, plants and mammals. GHB is produced from the reduction of succinic semialdehyde (SSA) by the activity of GHB dehydrogenase. Arabidopsis genome contains two GHB dehydrogenase encoding genes. The accumulation of GHB in ssadh mutants led to the speculation that GHB is the cause of aberrant phenotypes. Conversely, the accumulation of GHB in Arabidopsis plants subjected to abiotic stresses was described as a way of avoiding SSA induced damage. To resolve these contrasting views on GHB, we examined the effect of exogenous GHB and SSA on the growth of yeast and Arabidopsis plants. GHB concentrations up to 1.5 mM didn't affect shoots of Arabidopsis plants; however, root growth was inhibited. In contrast, 0.3 mM SSA has severely affected the growth of plants. Treatment of yeast wild-type strain with 10 mM SSA and 10 mM GHB didn't affect the growth. However, the growth of yeast uga2 mutant was greatly inhibited by the same concentration of SSA, but not GHB. Metabolic analysis and enzyme activity assay on native gel showed that Arabidopsis, but not yeast, possesses a GHB dehydrogenase activity that converts GHB back to SSA. The enzymatic assay has also indicated the existence of an additional GHB dehydrogenase encoding gene(s) in Arabidopsis genome. Taken together, we conclude that GHB is less toxic than SSA. Its accumulation in ssadh mutants and during abiotic stresses is a response to avoid the SSA induced damage.

  6. Phenotypic and chemotypic studies using Arabidopsis and yeast reveal that GHB converts to SSA and induce toxicity.

    PubMed

    Mekonnen, Dereje Worku; Ludewig, Frank

    2016-07-01

    γ-Hydroxybutyric acid (GHB) is a naturally occurring compound. It is detected in organisms such as yeasts, plants and mammals. GHB is produced from the reduction of succinic semialdehyde (SSA) by the activity of GHB dehydrogenase. Arabidopsis genome contains two GHB dehydrogenase encoding genes. The accumulation of GHB in ssadh mutants led to the speculation that GHB is the cause of aberrant phenotypes. Conversely, the accumulation of GHB in Arabidopsis plants subjected to abiotic stresses was described as a way of avoiding SSA induced damage. To resolve these contrasting views on GHB, we examined the effect of exogenous GHB and SSA on the growth of yeast and Arabidopsis plants. GHB concentrations up to 1.5 mM didn't affect shoots of Arabidopsis plants; however, root growth was inhibited. In contrast, 0.3 mM SSA has severely affected the growth of plants. Treatment of yeast wild-type strain with 10 mM SSA and 10 mM GHB didn't affect the growth. However, the growth of yeast uga2 mutant was greatly inhibited by the same concentration of SSA, but not GHB. Metabolic analysis and enzyme activity assay on native gel showed that Arabidopsis, but not yeast, possesses a GHB dehydrogenase activity that converts GHB back to SSA. The enzymatic assay has also indicated the existence of an additional GHB dehydrogenase encoding gene(s) in Arabidopsis genome. Taken together, we conclude that GHB is less toxic than SSA. Its accumulation in ssadh mutants and during abiotic stresses is a response to avoid the SSA induced damage. PMID:27037708

  7. A permeability barrier surrounds taste buds in lingual epithelia

    PubMed Central

    Dando, Robin; Pereira, Elizabeth; Kurian, Mani; Barro-Soria, Rene; Chaudhari, Nirupa

    2014-01-01

    Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003–1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste. PMID:25209263

  8. A permeability barrier surrounds taste buds in lingual epithelia.

    PubMed

    Dando, Robin; Pereira, Elizabeth; Kurian, Mani; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

    2015-01-01

    Epithelial tissues are characterized by specialized cell-cell junctions, typically localized to the apical regions of cells. These junctions are formed by interacting membrane proteins and by cytoskeletal and extracellular matrix components. Within the lingual epithelium, tight junctions join the apical tips of the gustatory sensory cells in taste buds. These junctions constitute a selective barrier that limits penetration of chemosensory stimuli into taste buds (Michlig et al. J Comp Neurol 502: 1003-1011, 2007). We tested the ability of chemical compounds to permeate into sensory end organs in the lingual epithelium. Our findings reveal a robust barrier that surrounds the entire body of taste buds, not limited to the apical tight junctions. This barrier prevents penetration of many, but not all, compounds, whether they are applied topically, injected into the parenchyma of the tongue, or circulating in the blood supply, into taste buds. Enzymatic treatments indicate that this barrier likely includes glycosaminoglycans, as it was disrupted by chondroitinase but, less effectively, by proteases. The barrier surrounding taste buds could also be disrupted by brief treatment of lingual tissue samples with DMSO. Brief exposure of lingual slices to DMSO did not affect the ability of taste buds within the slice to respond to chemical stimulation. The existence of a highly impermeable barrier surrounding taste buds and methods to break through this barrier may be relevant to basic research and to clinical treatments of taste.

  9. Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts

    PubMed Central

    Mattarocci, Stefano; Hafner, Lukas; Lezaja, Aleksandra; Shyian, Maksym; Shore, David

    2016-01-01

    Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions. PMID:27066066

  10. A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

    PubMed

    DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

    2014-07-15

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis.

  11. Biostimulation by methanol enables the methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. to reveal high formaldehyde biodegradation potential as well as to adapt to this toxic pollutant.

    PubMed

    Kaszycki, Paweł; Walski, Tomasz; Hachicho, Nancy; Heipieper, Hermann J

    2013-06-01

    The methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. revealed enhanced biodegradation capability of exogenously applied formaldehyde (Fd) upon biostimulation achieved by the presence of methanol, as compared to glucose. Upon growth on either of the above substrates, the strains proved to produce the activity of glutathione-dependent formaldehyde dehydrogenase-the enzyme known to control the biooxidative step of Fd detoxification. However, in the absence of methanol, the yeasts' tolerance to Fd was decreased, and the elevated sensitivity was especially pronounced for Trichosporon sp. Both strains responded to the methanol and/or Fd treatment by increasing their unsaturation index (UI) at xenobiotic levels below minimal inhibitory concentrations. This indicated that the UI changes effected from the de novo synthesis of (poly) unsaturated fatty acids carried out by viable cells. It is concluded that the yeast cell response to Fd intoxication involves stress reaction at the level of membranes. Fluidization of the lipid bilayer as promoted by methanol is suggested as a significant adaptive mechanism increasing the overall fitness enabling to cope with the formaldehyde xenobiotic via biodegradative pathway of C1-compound metabolism.

  12. A comprehensive protein–protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1

    PubMed Central

    DeMille, Desiree; Bikman, Benjamin T.; Mathis, Andrew D.; Prince, John T.; Mackay, Jordan T.; Sowa, Steven W.; Hall, Tacie D.; Grose, Julianne H.

    2014-01-01

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein–protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase–deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. PMID:24850888

  13. High-Resolution Profiling of Stationary-Phase Survival Reveals Yeast Longevity Factors and Their Genetic Interactions

    PubMed Central

    Garay, Erika; Campos, Sergio E.; González de la Cruz, Jorge; Gaspar, Ana P.; Jinich, Adrian; DeLuna, Alexander

    2014-01-01

    Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells. PMID:24586198

  14. High-resolution profiling of stationary-phase survival reveals yeast longevity factors and their genetic interactions.

    PubMed

    Garay, Erika; Campos, Sergio E; González de la Cruz, Jorge; Gaspar, Ana P; Jinich, Adrian; Deluna, Alexander

    2014-02-01

    Lifespan is influenced by a large number of conserved proteins and gene-regulatory pathways. Here, we introduce a strategy for systematically finding such longevity factors in Saccharomyces cerevisiae and scoring the genetic interactions (epistasis) among these factors. Specifically, we developed an automated competition-based assay for chronological lifespan, defined as stationary-phase survival of yeast populations, and used it to phenotype over 5,600 single- or double-gene knockouts at unprecedented quantitative resolution. We found that 14% of the viable yeast mutant strains were affected in their stationary-phase survival; the extent of true-positive chronological lifespan factors was estimated by accounting for the effects of culture aeration and adaptive regrowth. We show that lifespan extension by dietary restriction depends on the Swr1 histone-exchange complex and that a functional link between autophagy and the lipid-homeostasis factor Arv1 has an impact on cellular lifespan. Importantly, we describe the first genetic interaction network based on aging phenotypes, which successfully recapitulated the core-autophagy machinery and confirmed a role of the human tumor suppressor PTEN homologue in yeast lifespan and phosphatidylinositol phosphate metabolism. Our quantitative analysis of longevity factors and their genetic interactions provides insights into the gene-network interactions of aging cells.

  15. Genome-wide array-CGH analysis reveals YRF1 gene copy number variation that modulates genetic stability in distillery yeasts

    PubMed Central

    Adamczyk, Jagoda; Kwiatkowska, Aleksandra; Rawska, Ewa; Skoneczna, Adrianna

    2015-01-01

    Industrial yeasts, economically important microorganisms, are widely used in diverse biotechnological processes including brewing, winemaking and distilling. In contrast to a well-established genome of brewer's and wine yeast strains, the comprehensive evaluation of genomic features of distillery strains is lacking. In the present study, twenty two distillery yeast strains were subjected to electrophoretic karyotyping and array-based comparative genomic hybridization (array-CGH). The strains analyzed were assigned to the Saccharomyces sensu stricto complex and grouped into four species categories: S. bayanus, S. paradoxus, S. cerevisiae and S. kudriavzevii. The genomic diversity was mainly revealed within subtelomeric regions and the losses and/or gains of fragments of chromosomes I, III, VI and IX were the most frequently observed. Statistically significant differences in the gene copy number were documented in six functional gene categories: 1) telomere maintenance via recombination, DNA helicase activity or DNA binding, 2) maltose metabolism process, glucose transmembrane transporter activity; 3) asparagine catabolism, cellular response to nitrogen starvation, localized in cell wall-bounded periplasmic space, 4) siderophore transport, 5) response to copper ion, cadmium ion binding and 6) L-iditol 2- dehydrogenase activity. The losses of YRF1 genes (Y' element ATP-dependent helicase) were accompanied by decreased level of Y' sequences and an increase in DNA double and single strand breaks, and oxidative DNA damage in the S. paradoxus group compared to the S. bayanus group. We postulate that naturally occurring diversity in the YRF1 gene copy number may promote genetic stability in the S. bayanus group of distillery yeast strains. PMID:26384347

  16. Targeting of Chitin Synthase 3 to Polarized Growth Sites in Yeast Requires Chs5p and Myo2p

    PubMed Central

    Santos, Beatriz; Snyder, Michael

    1997-01-01

    Chitin is an essential structural component of the yeast cell wall whose deposition is regulated throughout the yeast life cycle. The temporal and spatial regulation of chitin synthesis was investigated during vegetative growth and mating of Saccharomyces cerevisiae by localization of the putative catalytic subunit of chitin synthase III, Chs3p, and its regulator, Chs5p. Immunolocalization of epitope-tagged Chs3p revealed a novel localization pattern that is cell cycledependent. Chs3p is polarized as a diffuse ring at the incipient bud site and at the neck between the mother and bud in small-budded cells; it is not found at the neck in large-budded cells containing a single nucleus. In large-budded cells undergoing cytokinesis, it reappears as a ring at the neck. In cells responding to mating pheromone, Chs3p is found throughout the projection. The appearance of Chs3p at cortical sites correlates with times that chitin synthesis is expected to occur. In addition to its localization at the incipient bud site and neck, Chs3p is also found in cytoplasmic patches in cells at different stages of the cell cycle. Epitope-tagged Chs5p also localizes to cytoplasmic patches; these patches contain Kex2p, a late Golgi-associated enzyme. Unlike Chs3p, Chs5p does not accumulate at the incipient bud site or neck. Nearly all Chs3p patches contain Chs5p, whereas some Chs5p patches lack detectable Chs3p. In the absence of Chs5p, Chs3p localizes in cytoplasmic patches, but it is no longer found at the neck or the incipient bud site, indicating that Chs5p is required for the polarization of Chs3p. Furthermore, Chs5p localization is not affected either by temperature shift or by the myo2-66 mutation, however, Chs3p polarization is affected by temperature shift and myo2-66. We suggest a model in which Chs3p polarization to cortical sites in yeast is dependent on both Chs5p and the actin cytoskeleton/Myo2p. PMID:9008706

  17. Tumor budding correlates with poor prognosis and epithelial-mesenchymal transition in tongue squamous cell carcinoma

    PubMed Central

    Wang, Cheng; Huang, Hongzhang; Huang, Zhiquan; Wang, Anxun; Chen, Xiaohua; Huang, Lei; Zhou, Xiaofeng; Liu, Xiqiang

    2011-01-01

    BACKGROUND Tumor budding is a readily detectable histopathological feature and has been recognized as an adverse prognostic factor in several human cancers. However, the prognostic value of tumor budding in tongue squamous cell carcinoma (TSCC) has not been reported. The purpose of this study is to assess the correlation of tumor budding with the clinicopathologic features, and the known molecular biomarkers (E-cadherin and Vimentin), as well as to evaluate its prognostic significance for TSCC. METHODS Archival clinical samples of 230 patients with TSCC were examined for tumor budding. Immunohistochemistry analyses were performed to examine the expression of E-cadherin and Vimentin. Statistical analyses were carried out to assess the correlation of tumor budding with clinicopathologic parameters and patient survival. The potential association between tumor budding and alterations of E-cadherin and Vimentin expression was also assessed. RESULTS Of the 230 TSCC cases examined, tumor budding was observed in 165 cases (71.7%), with a mean tumor bud count of 7.5 (range from 1 to 48 buds). High-intensity budding (≥ 5 tumor buds) was observed in 111 cases (48.3%). Statistical analysis revealed that tumor budding was associated with tumor size (P<0.05), differentiation (P<0.05), clinical stage (P<0.05), lymph node metastasis (P<0.01), and correlated with reduced overall survival. In addition, significant associations were observed among tumor budding and the deregulation of E-cadherin (P<0.001) and Vimentin (P<0.001). CONCLUSIONS Tumor budding, which associates with epithelial-mesenchymal transition, is a frequent event and appears to be an independent prognostic factor in TSCC. PMID:21481005

  18. Eukaryotic-Like Virus Budding in Archaea

    PubMed Central

    Quemin, Emmanuelle R. J.; Chlanda, Petr; Sachse, Martin; Forterre, Patrick

    2016-01-01

    ABSTRACT Similar to many eukaryotic viruses (and unlike bacteriophages), viruses infecting archaea are often encased in lipid-containing envelopes. However, the mechanisms of their morphogenesis and egress remain unexplored. Here, we used dual-axis electron tomography (ET) to characterize the morphogenesis of Sulfolobus spindle-shaped virus 1 (SSV1), the prototype of the family Fuselloviridae and representative of the most abundant archaea-specific group of viruses. Our results show that SSV1 assembly and egress are concomitant and occur at the cellular cytoplasmic membrane via a process highly reminiscent of the budding of enveloped viruses that infect eukaryotes. The viral nucleoprotein complexes are extruded in the form of previously unknown rod-shaped intermediate structures which have an envelope continuous with the host membrane. Further maturation into characteristic spindle-shaped virions takes place while virions remain attached to the cell surface. Our data also revealed the formation of constricted ring-like structures which resemble the budding necks observed prior to the ESCRT machinery-mediated membrane scission during egress of various enveloped viruses of eukaryotes. Collectively, we provide evidence that archaeal spindle-shaped viruses contain a lipid envelope acquired upon budding of the viral nucleoprotein complex through the host cytoplasmic membrane. The proposed model bears a clear resemblance to the egress strategy employed by enveloped eukaryotic viruses and raises important questions as to how the archaeal single-layered membrane composed of tetraether lipids can undergo scission. PMID:27624130

  19. A FRET-based study reveals site-specific regulation of spindle position checkpoint proteins at yeast centrosomes

    PubMed Central

    Gryaznova, Yuliya; Caydasi, Ayse Koca; Malengo, Gabriele; Sourjik, Victor; Pereira, Gislene

    2016-01-01

    The spindle position checkpoint (SPOC) is a spindle pole body (SPB, equivalent of mammalian centrosome) associated surveillance mechanism that halts mitotic exit upon spindle mis-orientation. Here, we monitored the interaction between SPB proteins and the SPOC component Bfa1 by FRET microscopy. We show that Bfa1 binds to the scaffold-protein Nud1 and the γ-tubulin receptor Spc72. Spindle misalignment specifically disrupts Bfa1-Spc72 interaction by a mechanism that requires the 14-3-3-family protein Bmh1 and the MARK/PAR-kinase Kin4. Dissociation of Bfa1 from Spc72 prevents the inhibitory phosphorylation of Bfa1 by the polo-like kinase Cdc5. We propose Spc72 as a regulatory hub that coordinates the activity of Kin4 and Cdc5 towards Bfa1. In addition, analysis of spc72∆ cells shows that a mitotic-exit-promoting dominant signal, which is triggered upon elongation of the spindle into the bud, overrides the SPOC. Our data reinforce the importance of daughter-cell-associated factors and centrosome-based regulations in mitotic exit and SPOC control. DOI: http://dx.doi.org/10.7554/eLife.14029.001 PMID:27159239

  20. Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family.

    PubMed

    Trempe, Jean-François; Šašková, Klára Grantz; Sivá, Monika; Ratcliffe, Colin D H; Veverka, Václav; Hoegl, Annabelle; Ménade, Marie; Feng, Xin; Shenker, Solomon; Svoboda, Michal; Kožíšek, Milan; Konvalinka, Jan; Gehring, Kalle

    2016-01-01

    The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains. PMID:27646017

  1. Structural studies of the yeast DNA damage-inducible protein Ddi1 reveal domain architecture of this eukaryotic protein family

    PubMed Central

    Trempe, Jean-François; Šašková, Klára Grantz; Sivá, Monika; Ratcliffe, Colin D. H.; Veverka, Václav; Hoegl, Annabelle; Ménade, Marie; Feng, Xin; Shenker, Solomon; Svoboda, Michal; Kožíšek, Milan; Konvalinka, Jan; Gehring, Kalle

    2016-01-01

    The eukaryotic Ddi1 family is defined by a conserved retroviral aspartyl protease-like (RVP) domain found in association with a ubiquitin-like (UBL) domain. Ddi1 from Saccharomyces cerevisiae additionally contains a ubiquitin-associated (UBA) domain. The substrate specificity and role of the protease domain in the biological functions of the Ddi family remain unclear. Yeast Ddi1 has been implicated in the regulation of cell cycle progression, DNA-damage repair, and exocytosis. Here, we investigated the multi-domain structure of yeast Ddi1 using X-ray crystallography, nuclear magnetic resonance, and small-angle X-ray scattering. The crystal structure of the RVP domain sheds light on a putative substrate recognition site involving a conserved loop. Isothermal titration calorimetry confirms that both UBL and UBA domains bind ubiquitin, and that Ddi1 binds K48-linked diubiquitin with enhanced affinity. The solution NMR structure of a helical domain that precedes the protease displays tertiary structure similarity to DNA-binding domains from transcription regulators. Our structural studies suggest that the helical domain could serve as a landing platform for substrates in conjunction with attached ubiquitin chains binding to the UBL and UBA domains. PMID:27646017

  2. Identification of Lethal Mutations in Yeast Threonyl-tRNA Synthetase Revealing Critical Residues in Its Human Homolog*

    PubMed Central

    Ruan, Zhi-Rong; Fang, Zhi-Peng; Ye, Qing; Lei, Hui-Yan; Eriani, Gilbert; Zhou, Xiao-Long; Wang, En-Duo

    2015-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are a group of ancient enzymes catalyzing aminoacylation and editing reactions for protein biosynthesis. Increasing evidence suggests that these critical enzymes are often associated with mammalian disorders. Therefore, complete determination of the enzymes functions is essential for informed diagnosis and treatment. Here, we show that a yeast knock-out strain for the threonyl-tRNA synthetase (ThrRS) gene is an excellent platform for such an investigation. Saccharomyces cerevisiae ThrRS has a unique modular structure containing four structural domains and a eukaryote-specific N-terminal extension. Using randomly mutated libraries of the ThrRS gene (thrS) and a genetic screen, a set of loss-of-function mutants were identified. The mutations affected the synthetic and editing activities and influenced the dimer interface. The results also highlighted the role of the N-terminal extension for enzymatic activity and protein stability. To gain insights into the pathological mechanisms induced by mutated aaRSs, we systematically introduced the loss-of-function mutations into the human cytoplasmic ThrRS gene. All mutations induced similar detrimental effects, showing that the yeast model could be used to study pathology-associated point mutations in mammalian aaRSs. PMID:25416776

  3. Genetic interaction mapping reveals a role for the SWI/SNF nucleosome remodeler in spliceosome activation in fission yeast.

    PubMed

    Patrick, Kristin L; Ryan, Colm J; Xu, Jiewei; Lipp, Jesse J; Nissen, Kelly E; Roguev, Assen; Shales, Michael; Krogan, Nevan J; Guthrie, Christine

    2015-03-01

    Although numerous regulatory connections between pre-mRNA splicing and chromatin have been demonstrated, the precise mechanisms by which chromatin factors influence spliceosome assembly and/or catalysis remain unclear. To probe the genetic network of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, we constructed an epistatic mini-array profile (E-MAP) and discovered many new connections between chromatin and splicing. Notably, the nucleosome remodeler SWI/SNF had strong genetic interactions with components of the U2 snRNP SF3 complex. Overexpression of SF3 components in ΔSWI/SNF cells led to inefficient splicing of many fission yeast introns, predominantly those with non-consensus splice sites. Deletion of SWI/SNF decreased recruitment of the splicing ATPase Prp2, suggesting that SWI/SNF promotes co-transcriptional spliceosome assembly prior to first step catalysis. Importantly, defects in SWI/SNF as well as SF3 overexpression each altered nucleosome occupancy along intron-containing genes, illustrating that the chromatin landscape both affects--and is affected by--co-transcriptional splicing.

  4. Yeast diversity in a traditional French cheese "Tomme d'orchies" reveals infrequent and frequent species with associated benefits.

    PubMed

    Ceugniez, Alexandre; Drider, Djamel; Jacques, Philippe; Coucheney, Françoise

    2015-12-01

    This study is aimed at unrevealing the yeast diversity of handmade cheese, Tomme d'orchies, produced and marketed in the north of France. A total of 185 yeast colonies were isolated from the surface and core of this cheese. From these, 80 morphologically different colonies were selected and subjected to rep-PCR analysis. The isolates were clustered into six distinct groups based on their DNA fingerprints. From each group, at least 30% of isolates were selected and identified to species level by biochemical characteristics (ID32C Api system) and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA regions. The isolates belonged to Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus, frequently isolated, and less frequently isolated Saturnispora mendoncae and Clavispora lusitaniae. Two isolates designated as Kluyveromyces lactis (isolate S-3-05) and Kluyveromyces marxianus (isolate S-2-05) were non-hemolytic, sensitive to antifungal compounds and able to inhibit the growth of pathogens including Candida albicans, Listeria monocytogenes and some bacilli.

  5. Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach

    SciTech Connect

    Han, Sangjo; Lee, Minho; Chang, Hyeshik; Nam, Miyoung; Park, Han-Oh; Kwak, Youn-Sig; Ha, Hye-jeong; Kim, Dongsup; Hwang, Sung-Ook; Hoe, Kwang-Lae; Kim, Dong-Uk

    2013-07-12

    Highlights: •The first compendium of chemical-genetic profiles form fission yeast was generated. •The first HTS of drug mode-of-action in fission yeast was performed. •The first comparative chemical genetic analysis between two yeasts was conducted. -- Abstract: Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at (http://pombe.kaist.ac.kr/compendium)

  6. Early epithelial signaling center governs tooth budding morphogenesis.

    PubMed

    Ahtiainen, Laura; Uski, Isa; Thesleff, Irma; Mikkola, Marja L

    2016-09-12

    During organogenesis, cell fate specification and patterning are regulated by signaling centers, specialized clusters of morphogen-expressing cells. In many organs, initiation of development is marked by bud formation, but the cellular mechanisms involved are ill defined. Here, we use the mouse incisor tooth as a model to study budding morphogenesis. We show that a group of nonproliferative epithelial cells emerges in the early tooth primordium and identify these cells as a signaling center. Confocal live imaging of tissue explants revealed that although these cells reorganize dynamically, they do not reenter the cell cycle or contribute to the growing tooth bud. Instead, budding is driven by proliferation of the neighboring cells. We demonstrate that the activity of the ectodysplasin/Edar/nuclear factor κB pathway is restricted to the signaling center, and its inactivation leads to fewer quiescent cells and a smaller bud. These data functionally link the signaling center size to organ size and imply that the early signaling center is a prerequisite for budding morphogenesis. PMID:27621364

  7. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant.

    PubMed

    Guo, Da-Long; Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

  8. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant

    PubMed Central

    Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape.

  9. Cryotolerance of apple tree bud is independent of endodormancy.

    PubMed

    Bilavcik, Alois; Zamecnik, Jiri; Faltus, Milos

    2015-01-01

    Increasing interest in cryopreservation of dormant buds reveals the need for better understanding of the role of dormancy in cryotolerance. Dormancy stage and low-temperature survival of vegetative apple buds (Malus domestica Borkh.), cultivars 'Sampion' and 'Spartan', collected from orchard were evaluated during three seasons contrasting in temperature and precipitation throughout the arrested plant growth period. During each season, the cultivars differed either in the onset of the endodormancy or in the length of the endodormant period. A simple relation between endodormancy of the buds and their water content was not detected. The cryosurvival of vegetative apple buds of both cultivars correlated with their cold hardening without direct regard to their particular phase of dormancy. The period of the highest bud cryotolerance after low-temperature exposure overlapped with the endodormant period in some evaluated seasons. Both cultivars had the highest cryosurvival in December and January. The presented data were compared with our previous results from a dormancy study of in vitro apple culture. Endodormancy coincided with the period of successful cryosurvival of apple buds after liquid nitrogen exposure, but as such, it was not decisive for their survival and did not limit their successful cryopreservation.

  10. Histological and Molecular Characterization of Grape Early Ripening Bud Mutant

    PubMed Central

    Yu, Yi-He; Xi, Fei-Fei; Shi, Yan-Yan; Zhang, Guo-Hai

    2016-01-01

    An early ripening bud mutant was analyzed based on the histological, SSR, and methylation-sensitive amplified polymorphism (MSAP) analysis and a layer-specific approach was used to investigate the differentiation between the bud mutant and its parent. The results showed that the thickness of leaf spongy tissue of mutant (MT) is larger than that of wild type (WT) and the differences are significant. The mean size of cell layer L2 was increased in the mutant and the difference is significant. The genetic background of bud mutant revealed by SSR analysis is highly uniform to its parent; just the variations from VVS2 SSR marker were detected in MT. The total methylation ratio of MT is lower than that of the corresponding WT. The outside methylation ratio in MT is much less than that in WT; the average inner methylation ratio in MT is larger than that in WT. The early ripening bud mutant has certain proportion demethylation in cell layer L2. All the results suggested that cell layer L2 of the early ripening bud mutant has changed from the WT. This study provided the basis for a better understanding of the characteristic features of the early ripening bud mutant in grape. PMID:27610363

  11. Budded baculovirus particle structure revisited.

    PubMed

    Wang, Qiushi; Bosch, Berend-Jan; Vlak, Just M; van Oers, Monique M; Rottier, Peter J; van Lent, Jan W M

    2016-02-01

    Baculoviruses are a group of enveloped, double-stranded DNA insect viruses with budded (BV) and occlusion-derived (ODV) virions produced during their infection cycle. BVs are commonly described as rod shaped particles with a high apical density of protein extensions (spikes) on the lipid envelope surface. However, due to the fragility of BVs the conventional purification and electron microscopy (EM) staining methods considerably distort the native viral structure. Here, we use cryo-EM analysis to reveal the near-native morphology of two intensively studied baculoviruses, Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) and Spodoptera exigua MNPV (SeMNPV), as models for BVs carrying GP64 and F as envelope fusion protein on the surface. The now well-preserved AcMNPV and SeMNPV BV particles have a remarkable elongated, ovoid shape leaving a large, lateral space between nucleocapsid (NC) and envelope. Consistent with previous findings the NC has a distinctive cap and base structure interacting tightly with the envelope. This tight interaction may explain the partial retaining of the envelope on both ends of the NC and the disappearance of the remainder of the BV envelope in the negative-staining EM images. Cryo-EM also reveals that the viral envelope contains two layers with a total thickness of ≈ 6-7 nm, which is significantly thicker than a usual biological membrane (<4 nm) as measured by X-ray scanning. Most spikes are densely clustered at the two apical ends of the virion although some envelope proteins are also found more sparsely on the lateral regions. The spikes on the surface of AcMNPV BVs appear distinctly different from those of SeMNPV. Based on our observations we propose a new near-native structural model of baculovirus BVs.

  12. Differential adaptation to multi-stressed conditions of wine fermentation revealed by variations in yeast regulatory networks

    PubMed Central

    2013-01-01

    Background Variation of gene expression can lead to phenotypic variation and have therefore been assumed to contribute the diversity of wine yeast (Saccharomyces cerevisiae) properties. However, the molecular bases of this variation of gene expression are unknown. We addressed these questions by carrying out an integrated genetical-genomic study in fermentation conditions. We report here quantitative trait loci (QTL) mapping based on expression profiling in a segregating population generated by a cross between a derivative of the popular wine strain EC1118 and the laboratory strain S288c. Results Most of the fermentation traits studied appeared to be under multi-allelic control. We mapped five phenotypic QTLs and 1465 expression QTLs. Several expression QTLs overlapped in hotspots. Among the linkages unraveled here, several were associated with metabolic processes essential for wine fermentation such as glucose sensing or nitrogen and vitamin metabolism. Variations affecting the regulation of drug detoxification and export (TPO1, PDR12 or QDR2) were linked to variation in four genes encoding transcription factors (PDR8, WAR1, YRR1 and HAP1). We demonstrated that the allelic variation of WAR1 and TPO1 affected sorbic and octanoic acid resistance, respectively. Moreover, analysis of the transcription factors phylogeny suggests they evolved with a specific adaptation of the strains to wine fermentation conditions. Unexpectedly, we found that the variation of fermentation rates was associated with a partial disomy of chromosome 16. This disomy resulted from the well known 8–16 translocation. Conclusions This large data set made it possible to decipher the effects of genetic variation on gene expression during fermentation and certain wine fermentation properties. Our findings shed a new light on the adaptation mechanisms required by yeast to cope with the multiple stresses generated by wine fermentation. In this context, the detoxification and export systems appear

  13. Complementation of the Yeast Model System Reveals that Caenorhabditis elegans OCT-1 Is a Functional Transporter of Anthracyclines

    PubMed Central

    Brosseau, Nicolas; Andreev, Emil; Ramotar, Dindial

    2015-01-01

    The yeast plasma membrane protein Agp2 belongs to the family of amino acid transporters. It acts as a regulator that controls the expression of several uptake transporter genes such as DUR3 and SAM3 encoding two high-affinity polyamine permeases. agp2Δ mutants display extreme resistance to several cationic compounds including polyamines, the anticancer agent bleomycin, and cationic antifungal peptides. We propose that Agp2 might be involved in regulating the uptake of other cationic anticancer drugs. To date, an uptake transporter has not been reported for anthracyclines, a family of chemotherapeutic agents that are used for treating adult patients with acute myeloid leukemia. Herein, we develop assay conditions to monitor the uptake of the anthracycline doxorubicin into yeast cells and demonstrate for the first time that Agp2 is required for the drug uptake. Deletion of both the DUR3 and SAM3 genes reduced doxorubicin uptake, but not the deletion of either gene alone, while the agp2Δ mutant was severely compromised, suggesting that Agp2 controls the drug uptake via Dur3 and Sam3 and at least one additional transporter. Overexpression of DUR3 or SAM3 from the endogenous promoter rescued doxorubicin uptake into the sam3Δdur3Δ double mutant, consistent with a role for these transporters in the uptake of anthracyclines. We further show by cross-species complementation analysis that expression of the Caenorhabditis elegans oct-1 gene encoding an organic cation transporter restored full doxorubicin uptake in the agp2Δ mutant. Four separate variants of CeOCT-1 derived by substituting the amino acid residues Gln15, Cys31, Gln109 and Lys300 with alanine were stably expressed, but did not mediate doxorubicin uptake into the agp2Δ mutant. Moreover, we show that overexpression of CeOCT-1 sensitized parent yeast cells to doxorubicin, suggesting that CeOCT-1 related members might be key transporters to facilitate entry of anthracyclines into human cells. PMID:26177450

  14. Complementation of the Yeast Model System Reveals that Caenorhabditis elegans OCT-1 Is a Functional Transporter of Anthracyclines.

    PubMed

    Brosseau, Nicolas; Andreev, Emil; Ramotar, Dindial

    2015-01-01

    The yeast plasma membrane protein Agp2 belongs to the family of amino acid transporters. It acts as a regulator that controls the expression of several uptake transporter genes such as DUR3 and SAM3 encoding two high-affinity polyamine permeases. agp2Δ mutants display extreme resistance to several cationic compounds including polyamines, the anticancer agent bleomycin, and cationic antifungal peptides. We propose that Agp2 might be involved in regulating the uptake of other cationic anticancer drugs. To date, an uptake transporter has not been reported for anthracyclines, a family of chemotherapeutic agents that are used for treating adult patients with acute myeloid leukemia. Herein, we develop assay conditions to monitor the uptake of the anthracycline doxorubicin into yeast cells and demonstrate for the first time that Agp2 is required for the drug uptake. Deletion of both the DUR3 and SAM3 genes reduced doxorubicin uptake, but not the deletion of either gene alone, while the agp2Δ mutant was severely compromised, suggesting that Agp2 controls the drug uptake via Dur3 and Sam3 and at least one additional transporter. Overexpression of DUR3 or SAM3 from the endogenous promoter rescued doxorubicin uptake into the sam3Δdur3Δ double mutant, consistent with a role for these transporters in the uptake of anthracyclines. We further show by cross-species complementation analysis that expression of the Caenorhabditis elegans oct-1 gene encoding an organic cation transporter restored full doxorubicin uptake in the agp2Δ mutant. Four separate variants of CeOCT-1 derived by substituting the amino acid residues Gln15, Cys31, Gln109 and Lys300 with alanine were stably expressed, but did not mediate doxorubicin uptake into the agp2Δ mutant. Moreover, we show that overexpression of CeOCT-1 sensitized parent yeast cells to doxorubicin, suggesting that CeOCT-1 related members might be key transporters to facilitate entry of anthracyclines into human cells.

  15. Analysis of aging in lager brewing yeast during serial repitching.

    PubMed

    Bühligen, Franziska; Lindner, Patrick; Fetzer, Ingo; Stahl, Frank; Scheper, Thomas; Harms, Hauke; Müller, Susann

    2014-10-10

    Serial repitching of brewing yeast inoculates is an important economic factor in the brewing industry, as their propagation is time and resource intensive. Here, we investigated whether replicative aging and/or the population distribution status changed during serial repitching in three different breweries with the same brewing yeast strain but different abiotic backgrounds and repitching regimes with varying numbers of reuses. Next to bud scar numbers the DNA content of the Saccharomyces pastorianus HEBRU cells was analyzed. Gene expression patterns were investigated using low-density microarrays with genes for aging, stress, storage compound metabolism and cell cycle. Two breweries showed a stable rejuvenation rate during serial repitching. In a third brewery the fraction of virgin cells varied, which could be explained with differing wort aeration rates. Furthermore, the number of bud scars per cell and cell size correlated in all 3 breweries throughout all runs. Transcriptome analyses revealed that from the 6th run on, mainly for the cells positive gene expression could be seen, for example up-regulation of trehalose and glycogen metabolism genes. Additionally, the cells' settling in the cone was dependent on cell size, with the lowest and the uppermost cone layers showing the highest amount of dead cells. In general, cells do not progressively age during extended serial repitching. PMID:25026460

  16. Influence of Zero-Shear on Yeast Development

    NASA Technical Reports Server (NTRS)

    McGinnis, Michael R.

    1997-01-01

    The objective of the research was to begin evaluating the effect of zero-shear on the development of the cell wall of Saccharomyces cerevisiae employing the High Aspect Rotating-Wall Vessel (HARV) NASA bioreactor. This particular yeast has enormous potential for research as a model eukaryotic system on the International Space Station, as well as the production of food stuffs' at the future lunar colony. Because the cell wall is the barrier between the cell and the environment, its form and function as influenced by microgravity is of great importance. Morphologic studies revealed that the circularity and total area of the individual yeast cells were essentially the same in both the control and test HARV's. The growth rates were also essentially the same. In zero-shear, the yeast grew in clumps consisting of rudimentary pseudohyphae in contrast to solitary budding cells in the control. Based upon mechanical and sonic shear applied to the yeast cells, those grown in zero-shear had stronger cell walls and septa. This suggests that there are structural differences, most likely related to the chitin skeleton of the cell wall. From this research further NASA support was obtained to continue the work. Investigations will deal with gene expression and ultrastructure. These will lead to a clearer assessment of the value of S. cerevisiae eukaryotic as a model for space station research.

  17. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and {gamma}-rays

    SciTech Connect

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep . E-mail: rakwal-68@aist.go.jp; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma ({gamma})-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and {gamma}-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and {gamma}-rays). Similarly, for X- and {gamma}-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and {gamma}-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-a-vis their energy levels.

  18. A conserved family of proteins facilitates nascent lipid droplet budding from the ER

    PubMed Central

    Choudhary, Vineet; Ojha, Namrata; Golden, Andy

    2015-01-01

    Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER. PMID:26504167

  19. Yeasts isolated from Algerian infants's feces revealed a burden of Candida albicans species, non-albicans Candida species and Saccharomyces cerevisiae.

    PubMed

    Seddik, Hamza Ait; Ceugniez, Alexandre; Bendali, Farida; Cudennec, Benoit; Drider, Djamel

    2016-01-01

    This study aimed at showing the yeast diversity in feces of Algerian infants, aged between 1 and 24 months, hospitalized at Bejaia hospital (northeast side of the country). Thus, 20 colonies with yeast characteristics were isolated and identified using biochemical (ID32C Api system) and molecular (sequencing of ITS1-5.8S-ITS2 region) methods. Almost all colonies isolated (19 strains) were identified as Candida spp., with predominance of Candida albicans species, and one strain was identified as Saccharomyces cerevisiae. Screening of strains with inhibitory activities unveiled the potential of Candida parapsilosis P48L1 and Candida albicans P51L1 to inhibit the growth of Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Further studies performed with these two Candida strains revealed their susceptibility to clinically used antifungal compounds and were then characterized for their cytotoxicity and hemolytic properties. On the other hand, Saccharomyces cerevisiae P9L1 isolated as well in this study was shown to be devoid of antagonism but resulted safe and overall usable as probiotic.

  20. Population Structure and Comparative Genome Hybridization of European Flor Yeast Reveal a Unique Group of Saccharomyces cerevisiae Strains with Few Gene Duplications in Their Genome

    PubMed Central

    Legras, Jean-Luc; Erny, Claude; Charpentier, Claudine

    2014-01-01

    Wine biological aging is a wine making process used to produce specific beverages in several countries in Europe, including Spain, Italy, France, and Hungary. This process involves the formation of a velum at the surface of the wine. Here, we present the first large scale comparison of all European flor strains involved in this process. We inferred the population structure of these European flor strains from their microsatellite genotype diversity and analyzed their ploidy. We show that almost all of these flor strains belong to the same cluster and are diploid, except for a few Spanish strains. Comparison of the array hybridization profile of six flor strains originating from these four countries, with that of three wine strains did not reveal any large segmental amplification. Nonetheless, some genes, including YKL221W/MCH2 and YKL222C, were amplified in the genome of four out of six flor strains. Finally, we correlated ICR1 ncRNA and FLO11 polymorphisms with flor yeast population structure, and associate the presence of wild type ICR1 and a long Flo11p with thin velum formation in a cluster of Jura strains. These results provide new insight into the diversity of flor yeast and show that combinations of different adaptive changes can lead to an increase of hydrophobicity and affect velum formation. PMID:25272156

  1. Interconnections Between RNA-Processing Pathways Revealed by a Sequencing-Based Genetic Screen for Pre-mRNA Splicing Mutants in Fission Yeast

    PubMed Central

    Larson, Amy; Fair, Benjamin Jung; Pleiss, Jeffrey A.

    2016-01-01

    Pre-mRNA splicing is an essential component of eukaryotic gene expression and is highly conserved from unicellular yeasts to humans. Here, we present the development and implementation of a sequencing-based reverse genetic screen designed to identify nonessential genes that impact pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe, an organism that shares many of the complex features of splicing in higher eukaryotes. Using a custom-designed barcoding scheme, we simultaneously queried ∼3000 mutant strains for their impact on the splicing efficiency of two endogenous pre-mRNAs. A total of 61 nonessential genes were identified whose deletions resulted in defects in pre-mRNA splicing; enriched among these were factors encoding known or predicted components of the spliceosome. Included among the candidates identified here are genes with well-characterized roles in other RNA-processing pathways, including heterochromatic silencing and 3ʹ end processing. Splicing-sensitive microarrays confirm broad splicing defects for many of these factors, revealing novel functional connections between these pathways. PMID:27172183

  2. Revisiting Mortimer’s Genome Renewal Hypothesis: Heterozygosity, Homothallism, and the Potential for Adaptation in Yeast

    PubMed Central

    Magwene, Paul M.

    2014-01-01

    In diploid organisms, the frequency and nature of sexual cycles have a major impact on genome-wide patterns of heterozygosity. Recent population genomic surveys in the budding yeast, Saccharomyces cerevisiae, have revealed surprising levels of genomic heterozygosity in what has been traditionally considered a highly inbred organism. I review evidence and hypotheses regarding the generation, maintenance, and evolutionary consequences of genomic heterozygosity in S. cerevisiae. I propose that high levels of heterozygosity in S. cerevisiae, arising from population admixture due to human domestication, coupled with selfing during rare sexual cycles, can facilitate rapid adaptation to novel environments. PMID:24277294

  3. Soft X-Ray Diffraction Microscopy of a Frozen Hydrated Yeast Cell

    DOE PAGES

    Huang, Xiaojing; Nelson, Johanna; Kirz, Janos; Lima, Enju; Marchesini, Stefano; Miao, Huijie; Neiman, Aaron M.; Shapiro, David; Steinbrener, Jan; Stewart, Andrew; et al

    2009-11-01

    We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below -170 °C, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.

  4. Inhibition of copper uptake in yeast reveals the copper transporter Ctr1p as a potential molecular target of saxitoxin

    PubMed Central

    Cusick, Kathleen D.; Minkin, Steven C.; Dodani, Sheel C.; Chang, Christopher J.; Wilhelm, Steven W.

    2012-01-01

    Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. Based on the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations couched in the context of the eco-evolutionary role this toxin may serve for species that produce it. PMID:22304436

  5. What Population Reveals about Individual Cell Identity: Single-Cell Parameter Estimation of Models of Gene Expression in Yeast

    PubMed Central

    Versari, Cristian; Cinquemani, Eugenio; Ferrari-Trecate, Giancarlo; Hersen, Pascal; Batt, Gregory

    2016-01-01

    Significant cell-to-cell heterogeneity is ubiquitously observed in isogenic cell populations. Consequently, parameters of models of intracellular processes, usually fitted to population-averaged data, should rather be fitted to individual cells to obtain a population of models of similar but non-identical individuals. Here, we propose a quantitative modeling framework that attributes specific parameter values to single cells for a standard model of gene expression. We combine high quality single-cell measurements of the response of yeast cells to repeated hyperosmotic shocks and state-of-the-art statistical inference approaches for mixed-effects models to infer multidimensional parameter distributions describing the population, and then derive specific parameters for individual cells. The analysis of single-cell parameters shows that single-cell identity (e.g. gene expression dynamics, cell size, growth rate, mother-daughter relationships) is, at least partially, captured by the parameter values of gene expression models (e.g. rates of transcription, translation and degradation). Our approach shows how to use the rich information contained into longitudinal single-cell data to infer parameters that can faithfully represent single-cell identity. PMID:26859137

  6. Next-generation sequencing analysis of lager brewing yeast strains reveals the evolutionary history of interspecies hybridization

    PubMed Central

    Okuno, Miki; Kajitani, Rei; Ryusui, Rie; Morimoto, Hiroya; Kodama, Yukiko; Itoh, Takehiko

    2016-01-01

    The lager beer yeast Saccharomyces pastorianus is considered an allopolyploid hybrid species between S. cerevisiae and S. eubayanus. Many S. pastorianus strains have been isolated and classified into two groups according to geographical origin, but this classification remains controversial. Hybridization analyses and partial PCR-based sequence data have indicated a separate origin of these two groups, whereas a recent intertranslocation analysis suggested a single origin. To clarify the evolutionary history of this species, we analysed 10 S. pastorianus strains and the S. eubayanus type strain as a likely parent by Illumina next-generation sequencing. In addition to assembling the genomes of five of the strains, we obtained information on interchromosomal translocation, ploidy, and single-nucleotide variants (SNVs). Collectively, these results indicated that the two groups of strains share S. cerevisiae haploid chromosomes. We therefore conclude that both groups of S. pastorianus strains share at least one interspecific hybridization event and originated from a common parental species and that differences in ploidy and SNVs between the groups can be explained by chromosomal deletion or loss of heterozygosity. PMID:26732986

  7. Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins.

    PubMed

    Veniamin, Simona; Sawatzky, Luanne G; Banting, Graham S; Glerum, D Moira

    2011-10-15

    A number of distinct cuproproteins of the mitochondrial inner membrane are required for the assembly of cytochrome oxidase (COX), thought to function in a "bucket brigade" fashion to provide copper to the Cu(A) and Cu(B) sites. In yeast, the loss of two these proteins, Sco1p and Cox11p, leads to respiratory deficiency and a specific inability to survive exposure to hydrogen peroxide (H(2)O(2)). Using a quantitative assay, we have identified subtle differences in the peroxide-sensitive phenotypes between sco1 and cox11 mutant strains. Interestingly, the peroxide sensitivity of the sco1 null strain can be suppressed by overexpressing either SCO2 or COX11, although overexpression of neither SCO1 nor SCO2 can rescue the cox11 null strain. We also find that overexpression of either CTT1, encoding the cytosolic catalase T, or CTA1, encoding the mitochondrial matrix catalase, suppresses the peroxide sensitivity in both the sco1 and the cox11 null mutants. Direct measurement of peroxide metabolism shows that sco1 and cox11 null strains fail to degrade a significant amount of exogenously provided H(2)O(2). Taken together, our data demonstrate that although Cox11p and Sco1p play distinct roles in COX assembly, they seem to play overlapping or related roles in peroxide metabolism that require further investigation. PMID:21821119

  8. Improved reconstitution of yeast vacuole fusion with physiological SNARE concentrations reveals an asymmetric Rab(GTP) requirement

    PubMed Central

    Zick, Michael; Wickner, William

    2016-01-01

    In vitro reconstitution of homotypic yeast vacuole fusion from purified components enables detailed study of membrane fusion mechanisms. Current reconstitutions have yet to faithfully replicate the fusion process in at least three respects: 1) The density of SNARE proteins required for fusion in vitro is substantially higher than on the organelle. 2) Substantial lysis accompanies reconstituted fusion. 3) The Rab GTPase Ypt7 is essential in vivo but often dispensable in vitro. Here we report that changes in fatty acyl chain composition dramatically lower the density of SNAREs that are required for fusion. By providing more physiological lipids with a lower phase transition temperature, we achieved efficient fusion with SNARE concentrations as low as on the native organelle. Although fused proteoliposomes became unstable at elevated SNARE concentrations, releasing their content after fusion had occurred, reconstituted proteoliposomes with substantially reduced SNARE concentrations fused without concomitant lysis. The Rab GTPase Ypt7 is essential on both membranes for proteoliposome fusion to occur at these SNARE concentrations. Strikingly, it was only critical for Ypt7 to be GTP loaded on membranes bearing the R-SNARE Nyv1, whereas the bound nucleotide of Ypt7 was irrelevant on membranes bearing the Q-SNAREs Vam3 and Vti1. PMID:27385334

  9. Genetic analysis of mlh3 mutations reveals interactions between crossover promoting factors during meiosis in baker's yeast.

    PubMed

    Sonntag Brown, Megan; Lim, Elisha; Chen, Cheng; Nishant, K T; Alani, Eric

    2013-01-01

    Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker's yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure-function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.

  10. Foamy Virus Budding and Release

    PubMed Central

    Hütter, Sylvia; Zurnic, Irena; Lindemann, Dirk

    2013-01-01

    Like all other viruses, a successful egress of functional particles from infected cells is a prerequisite for foamy virus (FV) spread within the host. The budding process of FVs involves steps, which are shared by other retroviruses, such as interaction of the capsid protein with components of cellular vacuolar protein sorting (Vps) machinery via late domains identified in some FV capsid proteins. Additionally, there are features of the FV budding strategy quite unique to the spumaretroviruses. This includes secretion of non-infectious subviral particles and a strict dependence on capsid-glycoprotein interaction for release of infectious virions from the cells. Virus-like particle release is not possible since FV capsid proteins lack a membrane-targeting signal. It is noteworthy that in experimental systems, the important capsid-glycoprotein interaction could be bypassed by fusing heterologous membrane-targeting signals to the capsid protein, thus enabling glycoprotein-independent egress. Aside from that, other systems have been developed to enable envelopment of FV capsids by heterologous Env proteins. In this review article, we will summarize the current knowledge on FV budding, the viral components and their domains involved as well as alternative and artificial ways to promote budding of FV particle structures, a feature important for alteration of target tissue tropism of FV-based gene transfer systems. PMID:23575110

  11. Glyphosate effects on the gene expression of the apical bud in soybean (Glycine max).

    PubMed

    Jiang, Ling-Xue; Jin, Long-Guo; Guo, Yong; Tao, Bo; Qiu, Li-Juan

    2013-08-01

    Glyphosate is a broad spectrum, non-selective herbicide which has been widely used for weed control. Much work has focused on elucidating the high accumulation of glyphosate in shoot apical bud (shoot apex). However, to date little is known about the molecular mechanisms of the sensitivity of shoot apical bud to glyphosate. Global gene expression profiling of the soybean apical bud response to glyphosate treatment was performed in this study. The results revealed that the glyphosate inhibited tryptophan biosynthesis of the shikimic acid pathway in the soybean apical bud, which was the target site of glyphosate. Glyphosate inhibited the expression of most of the target herbicide site genes. The promoter sequence analysis of key target genes revealed that light responsive elements were important regulators in glyphosate induction. These results will facilitate further studies of cloning genes and molecular mechanisms of glyphosate on soybean shoot apical bud.

  12. Analysis of Replication Timing Using Synchronized Budding Yeast Cultures

    PubMed Central

    Peng, Jie; Raghuraman, M.K.; Feng, Wenyi

    2015-01-01

    Eukaryotic DNA replication exhibits at once extraordinary fidelity and substantial plasticity. The importance of the apparent presence of a replication temporal program on a population level has been the subject of intense debate of late. Such debate has been, to a great extent, facilitated by methods that permit the description and analysis of replication dynamics in various model organisms, both globally and at a single-molecule level. Each of these methods provides a unique view of the replication process, and also presents challenges and questions in the interpretation of experimental observations. Thus, wider applications of these methods in different genetic backgrounds and in different organisms would doubtless enable us to better understand the execution and regulation of chromosomal DNA synthesis as well as its impact on genome maintenance. PMID:24906331

  13. Role of transcription at centromeres in budding yeast.

    PubMed

    Ohkuni, Kentaro; Kitagawa, Katsumi

    2012-01-01

    Centromeres are specialized chromosomal loci that are essential for proper chromosome segregation. Recent data show that a certain level of active transcription, regulated by transcription factors Cbf1 and Ste12, makes a direct contribution to centromere function in Saccharomyces cerevisiae. Here, we discuss the requirement and function of transcription at centromeres.

  14. Chromosome Conformation Capture Carbon Copy (5C) in Budding Yeast.

    PubMed

    Belton, Jon-Matthew; Dekker, Job

    2015-06-01

    Chromosome conformation capture carbon copy (5C) is a high-throughput method for detecting ligation products of interest in a chromosome conformation capture (3C) library. 5C uses ligation-mediated amplification (LMA) to generate carbon copies of 3C ligation product junctions using single-stranded oligonucleotide probes. This procedure produces a 5C library of short DNA molecules which represent the interactions between the corresponding restriction fragments. The 5C library can be amplified using universal primers containing the Illumina paired-end adaptor sequences for subsequent high-throughput sequencing.

  15. Monitoring Chitin Deposition During Septum Assembly in Budding Yeast.

    PubMed

    Arcones, Irene; Roncero, Cesar

    2016-01-01

    The synthesis of the septum is a critical step during cytokinesis in the fungal cell. Moreover, in Saccharomyces cerevisiae septum assembly depends mostly on the proper synthesis and deposition of chitin and, accordingly, on the timely regulation of chitin synthases. In this chapter, we will see how to follow chitin synthesis by two complementary approaches: monitoring chitin deposition in vivo at the septum by calcofluor staining and fluorescence microscopy, and measuring the chitin synthase activities responsible for this synthesis. PMID:26519305

  16. A Discrete Class of Intergenic DNA Dictates Meiotic DNA Break Hotspots in Fission Yeast

    PubMed Central

    Cam, Hugh P; Farah, Joseph A; Grewal, Shiv I. S; Smith, Gerald R

    2007-01-01

    Meiotic recombination is initiated by DNA double-strand breaks (DSBs) made by Spo11 (Rec12 in fission yeast), which becomes covalently linked to the DSB ends. Like recombination events, DSBs occur at hotspots in the genome, but the genetic factors responsible for most hotspots have remained elusive. Here we describe in fission yeast the genome-wide distribution of meiosis-specific Rec12-DNA linkages, which closely parallel DSBs measured by conventional Southern blot hybridization. Prominent DSB hotspots are located ∼65 kb apart, separated by intervals with little or no detectable breakage. Most hotspots lie within exceptionally large intergenic regions. Thus, the chromosomal architecture responsible for hotspots in fission yeast is markedly different from that of budding yeast, in which DSB hotspots are much more closely spaced and, in many regions of the genome, occur at each promoter. Our analysis in fission yeast reveals a clearly identifiable chromosomal feature that can predict the majority of recombination hotspots across a whole genome and provides a basis for searching for the chromosomal features that dictate hotspots of meiotic recombination in other organisms, including humans. PMID:17722984

  17. The multifunctional transcription factor Rap1: a regulator of yeast physiology.

    PubMed

    Azad, Gajendra Kumar; Tomar, Raghuvir Singh

    2016-01-01

    Transcription is a fundamental process that is tightly regulated by transcription factors to maintain cellular homeostasis. Transcription factors have DNA-binding domains, some of which are sequence specific, and are found throughout the eukaryotic kingdom. Recent studies have revealed the molecular mechanisms by which transcription factors perform their functions. In the budding yeast Saccharomyces cerevisiae, Rap1 (ScRap1) can either activate or repress transcription. This bimodal transcriptional activity has led to the widespread study of the mode of action of ScRap1. This review summarizes current knowledge about yeast ScRap1, including its structure, mechanisms of transcription regulation, and biological functions, and the future directions in the field.

  18. Breaking-bud pollination: a new pollination process in partially opened flowers by small bees.

    PubMed

    Yamaji, Futa; Ohsawa, Takeshi A

    2015-09-01

    Plant-pollinator interactions have usually been researched in flowers that have fully opened. However, some pollinators can visit flowers before full opening and contribute to fruit and seed sets. In this paper, we researched the pollination biology of flowers just starting to open in four field experiments. We observed the insect visitors to Lycoris sanguinea var. sanguinea for 3 years at five sites. These observations revealed that only small bees, Lasioglossum japonicum, often entered through tiny spaces between the tepals of 'breaking buds' (i.e. partially opened flowers) and collected pollen. We hypothesized that they can pollinate this species at the breaking-bud stage, when the stigma is located near the anthers. To measure the pollination effect of small bees at the breaking-bud stage, we bagged several breaking buds after small bees had visited them and examined whether these buds were pollinated. In bagging experiments, 30% of the breaking buds set fruit and seeds. Fruit-set ratios of the breaking buds did not differ significantly from those of the fully opened flowers, which had been visited by several insect species. We also counted the pollen grain numbers on the body of L. japonicum and on the anthers of randomly-selected and manipulated flowers. These experiments revealed that all of the captured bees had some pollen of target plants and that L. japonicum collected most of the pollen grains at the breaking-bud stage. Our results showed that the new pollination process, breaking-bud pollination, happened in breaking buds by L. japonicum, although there is no evidence to reveal that this is the most effective pollination method for L. sanguinea var. sanguinea. In principle, this new pollination process can occur in other flowering plants and our results are a major contribution to studies of plant-pollinator interactions.

  19. In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

    PubMed Central

    Kong, Yu; Wu, Qun; Zhang, Yan

    2014-01-01

    The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q2] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community. PMID:24727269

  20. Tropical Storms Bud and Dera

    NASA Technical Reports Server (NTRS)

    2002-01-01

    Like dancers pirouetting in opposite directions, the rotational patterns of two different tropical storms are contrasted in this pair of Multi-angle Imaging Spectroradiometer (MISR) nadir-camera images. The left-hand image is of Tropical Storm Bud, acquired on June 17, 2000 (Terra orbit 2656) as the storm was dissipating. Bud was situated in the eastern Pacific Ocean between Socorro Island and the southern tip of Baja California. South of the storm's center is a vortex pattern caused by obstruction of the prevailing flow by tiny Socorro Island. Sonora, Mexico and Baja California are visible at the top of the image. The right-hand image is of Tropical Cyclone Dera, acquired on March 12, 2001. Dera was located in the Indian Ocean, south of Madagascar. The southern end of this large island is visible in the top portion of this image. Northern hemisphere tropical storms, like Bud, rotate in a counterclockwise direction, whereas those in the southern hemisphere, such as Dera, rotate clockwise. The opposite spins are a consequence of Earth's rotation. Each image covers a swath approximately 380 kilometers wide. Image courtesy NASA/JPL/GSFC/LaRC, MISR Team

  1. Tropical Storms Bud and Dera

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Like dancers pirouetting in opposite directions, the rotational patterns of two different tropical storms are contrasted in this pair of MISR nadir-camera images.

    The left-hand image is of Tropical Storm Bud, acquired on June 17, 2000 (Terra orbit 2656) as the storm was dissipating. Bud was situated in the eastern Pacific Ocean between Socorro Island and the southern tip of Baja California. South of the storm's center is a vortex pattern caused by obstruction of the prevailing flow by tiny Socorro Island. Sonora, Mexico and Baja California are visible at the top of the image.

    The right-hand image is of Tropical Cyclone Dera, acquired on March 12, 2001 (Terra orbit 6552). Dera was located in the Indian Ocean, south of Madagascar. The southern end of this large island is visible in the top portion of this image.

    Northern hemisphere tropical storms, like Bud, rotate in a counterclockwise direction, whereas those in the southern hemisphere, such as Dera, rotate clockwise. The opposite spins are a consequence of Earth's rotation.

    Each image covers a swath approximately 380 kilometers wide.

    MISR was built and is managed by NASA's Jet Propulsion Laboratory, Pasadena, CA, for NASA's Office of Earth Science, Washington, DC. The Terra satellite is managed by NASA's Goddard Space Flight Center, Greenbelt, MD. JPL is a division of the California Institute of Technology.

  2. Structure of the Bro1 Domain Protein BROX and Functional Analyses of the ALIX Bro1 Domain in HIV-1 Budding

    SciTech Connect

    Zhai Q.; Robinson H.; Landesman M. B.; Sundquist W. I.; Hill C. P.

    2011-12-01

    Bro1 domains are elongated, banana-shaped domains that were first identified in the yeast ESCRT pathway protein, Bro1p. Humans express three Bro1 domain-containing proteins: ALIX, BROX, and HD-PTP, which function in association with the ESCRT pathway to help mediate intraluminal vesicle formation at multivesicular bodies, the abscission stage of cytokinesis, and/or enveloped virus budding. Human Bro1 domains share the ability to bind the CHMP4 subset of ESCRT-III proteins, associate with the HIV-1 NC{sup Gag} protein, and stimulate the budding of viral Gag proteins. The curved Bro1 domain structure has also been proposed to mediate membrane bending. To date, crystal structures have only been available for the related Bro1 domains from the Bro1p and ALIX proteins, and structures of additional family members should therefore aid in the identification of key structural and functional elements. We report the crystal structure of the human BROX protein, which comprises a single Bro1 domain. The Bro1 domains from BROX, Bro1p and ALIX adopt similar overall structures and share two common exposed hydrophobic surfaces. Surface 1 is located on the concave face and forms the CHMP4 binding site, whereas Surface 2 is located at the narrow end of the domain. The structures differ in that only ALIX has an extended loop that projects away from the convex face to expose the hydrophobic Phe105 side chain at its tip. Functional studies demonstrated that mutations in Surface 1, Surface 2, or Phe105 all impair the ability of ALIX to stimulate HIV-1 budding. Our studies reveal similarities in the overall folds and hydrophobic protein interaction sites of different Bro1 domains, and show that a unique extended loop contributes to the ability of ALIX to function in HIV-1 budding.

  3. Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium.

    PubMed

    Rashwan, Ahmed; Konishi, Hiroyuki; El-Sharaby, Ashraf; Kiyama, Hiroshi

    2016-01-01

    We investigated the relationship between mouse taste bud development and innervation of the soft palate. We employed scanning electron microscopy and immunohistochemistry using antibodies against protein gene product 9.5 and peripherin to detect sensory nerves, and cytokeratin 8 and α-gustducin to stain palatal taste buds. At E14, nerve fibers were observed along the medial border of the palatal shelves that tracked toward the epithelium. At E15.5, primordial stages of taste buds in the basal lamina of the soft palate first appeared. At E16, the taste buds became large spherical masses of columnar cells scattered in the soft palate basal lamina. At E17, the morphology and also the location of taste buds changed. At E18-19, some taste buds acquired a more elongated shape with a short neck, extending a variable distance from the soft palate basal lamina toward the surface epithelium. At E18, mature taste buds with taste pores and perigemmal nerve fibers were observed on the surface epithelium of the soft palate. The expression of α-gustducin was demonstrated at postnatal day 1 and the number of pored taste buds increased with age and they became pear-shaped at 8 weeks. The percent of pored fungiform-like papillae at birth was 58.3% of the whole palate; this increased to 83.8% at postnatal day 8 and reached a maximum of 95.7% at 12 weeks. The innervation of the soft palate was classified into three types of plexuses in relation to taste buds: basal nerve plexus, intragemmal and perigemmal nerve fibers. This study reveals that the nerve fibers preceded the development of taste buds in the palate of mice, and therefore the nerve fibers have roles in the initial induction of taste buds in the soft palate.

  4. The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates.

    PubMed

    Iwata, Momi; Lee, Yang; Yamashita, Tetsuo; Yagi, Takao; Iwata, So; Cameron, Alexander D; Maher, Megan J

    2012-09-18

    Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.

  5. Involvement of EARLY BUD-BREAK, an AP2/ERF Transcription Factor Gene, in Bud Break in Japanese Pear (Pyrus pyrifolia Nakai) Lateral Flower Buds: Expression, Histone Modifications and Possible Target Genes.

    PubMed

    Anh Tuan, Pham; Bai, Songling; Saito, Takanori; Imai, Tsuyoshi; Ito, Akiko; Moriguchi, Takaya

    2016-05-01

    In the Japanese pear (Pyrus pyrifolia Nakai) 'Kosui', three developmental stages of lateral flower buds have been proposed to occur during ecodormancy to the flowering phase, i.e. rapid enlargement, sprouting and flowering. Here, we report an APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor gene, named pear EARLY BUD-BREAK (PpEBB), which was highly expressed during the rapid enlargement stage occurring prior to the onset of bud break in flower buds. Gene expression analysis revealed that PpEBB expression was dramatically increased during the rapid enlargement stage in three successive growing seasons. PpEBB transcript levels peaked 1 week prior to onset of bud break in 'Kosui' potted plants treated with hydrogen cyanamide or water under forcing conditions. Chromatin immunoprecipitation-quantitative PCR showed that higher levels of active histone modifications (trimethylation of the histone H3 tail at Lys4) in the 5'-upstream and start codon regions of the PpEBB gene were associated with the induced expression level of PpEBB during the rapid enlargement stage. In addition, we provide evidence that PpEBB may interact with and regulate pear four D-type cyclin (PpCYCD3) genes during bud break in 'Kosui' lateral flower buds. PpEBB significantly increased the promoter activities of four PpCYCD3 genes in a dual-luciferase assay using tobacco leaves. Taken together, our findings uncovered aspects of the bud break regulatory mechanism in the Japanese pear and provided further evidence that the EBB family plays an important role in bud break in perennial plants. PMID:26940832

  6. Involvement of EARLY BUD-BREAK, an AP2/ERF Transcription Factor Gene, in Bud Break in Japanese Pear (Pyrus pyrifolia Nakai) Lateral Flower Buds: Expression, Histone Modifications and Possible Target Genes.

    PubMed

    Anh Tuan, Pham; Bai, Songling; Saito, Takanori; Imai, Tsuyoshi; Ito, Akiko; Moriguchi, Takaya

    2016-05-01

    In the Japanese pear (Pyrus pyrifolia Nakai) 'Kosui', three developmental stages of lateral flower buds have been proposed to occur during ecodormancy to the flowering phase, i.e. rapid enlargement, sprouting and flowering. Here, we report an APETALA2/ethylene-responsive factor (AP2/ERF) transcription factor gene, named pear EARLY BUD-BREAK (PpEBB), which was highly expressed during the rapid enlargement stage occurring prior to the onset of bud break in flower buds. Gene expression analysis revealed that PpEBB expression was dramatically increased during the rapid enlargement stage in three successive growing seasons. PpEBB transcript levels peaked 1 week prior to onset of bud break in 'Kosui' potted plants treated with hydrogen cyanamide or water under forcing conditions. Chromatin immunoprecipitation-quantitative PCR showed that higher levels of active histone modifications (trimethylation of the histone H3 tail at Lys4) in the 5'-upstream and start codon regions of the PpEBB gene were associated with the induced expression level of PpEBB during the rapid enlargement stage. In addition, we provide evidence that PpEBB may interact with and regulate pear four D-type cyclin (PpCYCD3) genes during bud break in 'Kosui' lateral flower buds. PpEBB significantly increased the promoter activities of four PpCYCD3 genes in a dual-luciferase assay using tobacco leaves. Taken together, our findings uncovered aspects of the bud break regulatory mechanism in the Japanese pear and provided further evidence that the EBB family plays an important role in bud break in perennial plants.

  7. Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

    2008-09-01

    Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

  8. Coevolutionary patterning of teeth and taste buds

    PubMed Central

    Bloomquist, Ryan F.; Parnell, Nicholas F.; Phillips, Kristine A.; Fowler, Teresa E.; Yu, Tian Y.; Sharpe, Paul T.; Streelman, J. Todd

    2015-01-01

    Teeth and taste buds are iteratively patterned structures that line the oro-pharynx of vertebrates. Biologists do not fully understand how teeth and taste buds develop from undifferentiated epithelium or how variation in organ density is regulated. These organs are typically studied independently because of their separate anatomical location in mammals: teeth on the jaw margin and taste buds on the tongue. However, in many aquatic animals like bony fishes, teeth and taste buds are colocalized one next to the other. Using genetic mapping in cichlid fishes, we identified shared loci controlling a positive correlation between tooth and taste bud densities. Genome intervals contained candidate genes expressed in tooth and taste bud fields. sfrp5 and bmper, notable for roles in Wingless (Wnt) and bone morphogenetic protein (BMP) signaling, were differentially expressed across cichlid species with divergent tooth and taste bud density, and were expressed in the development of both organs in mice. Synexpression analysis and chemical manipulation of Wnt, BMP, and Hedgehog (Hh) pathways suggest that a common cichlid oral lamina is competent to form teeth or taste buds. Wnt signaling couples tooth and taste bud density and BMP and Hh mediate distinct organ identity. Synthesizing data from fish and mouse, we suggest that the Wnt-BMP-Hh regulatory hierarchy that configures teeth and taste buds on mammalian jaws and tongues may be an evolutionary remnant inherited from ancestors wherein these organs were copatterned from common epithelium. PMID:26483492

  9. Coevolutionary patterning of teeth and taste buds.

    PubMed

    Bloomquist, Ryan F; Parnell, Nicholas F; Phillips, Kristine A; Fowler, Teresa E; Yu, Tian Y; Sharpe, Paul T; Streelman, J Todd

    2015-11-01

    Teeth and taste buds are iteratively patterned structures that line the oro-pharynx of vertebrates. Biologists do not fully understand how teeth and taste buds develop from undifferentiated epithelium or how variation in organ density is regulated. These organs are typically studied independently because of their separate anatomical location in mammals: teeth on the jaw margin and taste buds on the tongue. However, in many aquatic animals like bony fishes, teeth and taste buds are colocalized one next to the other. Using genetic mapping in cichlid fishes, we identified shared loci controlling a positive correlation between tooth and taste bud densities. Genome intervals contained candidate genes expressed in tooth and taste bud fields. sfrp5 and bmper, notable for roles in Wingless (Wnt) and bone morphogenetic protein (BMP) signaling, were differentially expressed across cichlid species with divergent tooth and taste bud density, and were expressed in the development of both organs in mice. Synexpression analysis and chemical manipulation of Wnt, BMP, and Hedgehog (Hh) pathways suggest that a common cichlid oral lamina is competent to form teeth or taste buds. Wnt signaling couples tooth and taste bud density and BMP and Hh mediate distinct organ identity. Synthesizing data from fish and mouse, we suggest that the Wnt-BMP-Hh regulatory hierarchy that configures teeth and taste buds on mammalian jaws and tongues may be an evolutionary remnant inherited from ancestors wherein these organs were copatterned from common epithelium.

  10. Structure of yeast sulfhydryl oxidase erv1 reveals electron transfer of the disulfide relay system in the mitochondrial intermembrane space.

    PubMed

    Guo, Peng-Chao; Ma, Jin-Di; Jiang, Yong-Liang; Wang, Shu-Jie; Bao, Zhang-Zhi; Yu, Xiao-Jie; Chen, Yuxing; Zhou, Cong-Zhao

    2012-10-12

    The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 Å resolution of the CTD and at 3.0 Å resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.

  11. Assigning Quantitative Function to Post-Translational Modifications Reveals Multiple Sites of Phosphorylation That Tune Yeast Pheromone Signaling Output

    SciTech Connect

    Pincus, David; Ryan, Christopher J.; Smith, Richard D.; Brent, Roger; Resnekov, Orna; Hakimi, Mohamed Ali

    2013-03-12

    Cell signaling systems transmit information by post-­translationally modifying signaling proteins, often via phosphorylation. While thousands of sites of phosphorylation have been identified in proteomic studies, the vast majority of sites have no known function. Assigning functional roles to the catalog of uncharacterized phosphorylation sites is a key research challenge. Here we present a general approach to address this challenge and apply it to a prototypical signaling pathway, the pheromone response pathway in Saccharomyces cerevisiae. The pheromone pathway includes a mitogen activated protein kinase (MAPK) cascade activated by a G-­protein coupled receptor (GPCR). We used mass spectrometry-based proteomics to identify sites whose phosphorylation changed when the system was active, and evolutionary conservation to assign priority to a list of candidate MAPK regulatory sites. We made targeted alterations in those sites, and measured the effects of the mutations on pheromone pathway output in single cells. Our work identified six new sites that quantitatively tuned system output. We developed simple computational models to find system architectures that recapitulated the quantitative phenotypes of the mutants. Our results identify a number of regulated phosphorylation events that contribute to adjust the input-­output relationship of this model eukaryotic signaling system. We believe this combined approach constitutes a general means not only to reveal modification sites required to turn a pathway on and off, but also those required for more subtle quantitative effects that tune pathway output. Our results further suggest that relatively small quantitative influences from individual regulatory phosphorylation events endow signaling systems with plasticity that evolution may exploit to quantitatively tailor signaling outcomes.

  12. Genetic interactions of yeast eukaryotic translation initiation factor 5A (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling.

    PubMed Central

    Valentini, Sandro R; Casolari, Jason M; Oliveira, Carla C; Silver, Pamela A; McBride, Anne E

    2002-01-01

    The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIF5A domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIF5A may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes. PMID:11861547

  13. High confidence proteomic analysis of yeast LDs identifies additional droplet proteins and reveals connections to dolichol synthesis and sterol acetylation[S

    PubMed Central

    Currie, Erin; Guo, Xiuling; Christiano, Romain; Chitraju, Chandramohan; Kory, Nora; Harrison, Kenneth; Haas, Joel; Walther, Tobias C.; Farese, Robert V.

    2014-01-01

    Accurate protein inventories are essential for understanding an organelle’s functions. The lipid droplet (LD) is a ubiquitous intracellular organelle with major functions in lipid storage and metabolism. LDs differ from other organelles because they are bounded by a surface monolayer, presenting unique features for protein targeting to LDs. Many proteins of varied functions have been found in purified LD fractions by proteomics. While these studies have become increasingly sensitive, it is often unclear which of the identified proteins are specific to LDs. Here we used protein correlation profiling to identify 35 proteins that specifically enrich with LD fractions of Saccharomyces cerevisiae. Of these candidates, 30 fluorophore-tagged proteins localize to LDs by microscopy, including six proteins, several with human orthologs linked to diseases, which we newly identify as LD proteins (Cab5, Rer2, Say1, Tsc10, YKL047W, and YPR147C). Two of these proteins, Say1, a sterol deacetylase, and Rer2, a cis-isoprenyl transferase, are enzymes involved in sterol and polyprenol metabolism, respectively, and we show their activities are present in LD fractions. Our results provide a highly specific list of yeast LD proteins and reveal that the vast majority of these proteins are involved in lipid metabolism. PMID:24868093

  14. Functional characterisation of novel enantioselective lipase TALipA from Trichosporon asahii MSR54: sequence comparison revealed new signature sequence AXSXG among yeast lipases.

    PubMed

    Kumari, Arti; Gupta, Rani

    2015-01-01

    A gene encoding lipase TALipA from Trichosporon asahii MSR54 was successfully isolated, cloned and expressed in Pichia pastoris X-33. It was purified to homogeneity by affinity chromatography with 1.7 purification fold. SDS-PAGE revealed it as a monomeric 27-kDa protein. Sequence comparison showed that it has close affinity with bacterial and actinobacterial lipases. It has unique oxyanion hole "GL" and conserved pentapeptide AHSMG where alanine is present instead of glycine, which is unique to yeast lipase database. The temperature and pH optima for activity were 60 °C and pH 8.0, respectively. It is thermostable with t1/2 of 68 min at 70 °C. It hydrolyzed p-np esters with better specificity on p-np palmitate, which was again confirmed during hydrolysis of triacylglyceride mixture. The enzyme was found to be regioselective during hydrolysis of triolein. It exhibited enantio preference during esterification of phenylethanol depending upon solvent used. It was S-enantioselective in 1,4-dioxane and R-selective in isopropanol and hexane. It is a magnesium-activated metalloenzyme inhibited by 10-mM EDTA. It was stable towards most of the polar and non-polar solvents.

  15. Functional characterisation of novel enantioselective lipase TALipA from Trichosporon asahii MSR54: sequence comparison revealed new signature sequence AXSXG among yeast lipases.

    PubMed

    Kumari, Arti; Gupta, Rani

    2015-01-01

    A gene encoding lipase TALipA from Trichosporon asahii MSR54 was successfully isolated, cloned and expressed in Pichia pastoris X-33. It was purified to homogeneity by affinity chromatography with 1.7 purification fold. SDS-PAGE revealed it as a monomeric 27-kDa protein. Sequence comparison showed that it has close affinity with bacterial and actinobacterial lipases. It has unique oxyanion hole "GL" and conserved pentapeptide AHSMG where alanine is present instead of glycine, which is unique to yeast lipase database. The temperature and pH optima for activity were 60 °C and pH 8.0, respectively. It is thermostable with t1/2 of 68 min at 70 °C. It hydrolyzed p-np esters with better specificity on p-np palmitate, which was again confirmed during hydrolysis of triacylglyceride mixture. The enzyme was found to be regioselective during hydrolysis of triolein. It exhibited enantio preference during esterification of phenylethanol depending upon solvent used. It was S-enantioselective in 1,4-dioxane and R-selective in isopropanol and hexane. It is a magnesium-activated metalloenzyme inhibited by 10-mM EDTA. It was stable towards most of the polar and non-polar solvents. PMID:25280633

  16. Functional adaptation between yeast actin and its cognate myosin motors.

    PubMed

    Stark, Benjamin C; Wen, Kuo-Kuang; Allingham, John S; Rubenstein, Peter A; Lord, Matthew

    2011-09-01

    We employed budding yeast and skeletal muscle actin to examine the contribution of the actin isoform to myosin motor function. While yeast and muscle actin are highly homologous, they exhibit different charge density at their N termini (a proposed myosin-binding interface). Muscle myosin-II actin-activated ATPase activity is significantly higher with muscle versus yeast actin. Whether this reflects inefficiency in the ability of yeast actin to activate myosin is not known. Here we optimized the isolation of two yeast myosins to assess actin function in a homogenous system. Yeast myosin-II (Myo1p) and myosin-V (Myo2p) accommodate the reduced N-terminal charge density of yeast actin, showing greater activity with yeast over muscle actin. Increasing the number of negative charges at the N terminus of yeast actin from two to four (as in muscle) had little effect on yeast myosin activity, while other substitutions of charged residues at the myosin interface of yeast actin reduced activity. Thus, yeast actin functions most effectively with its native myosins, which in part relies on associations mediated by its outer domain. Compared with yeast myosin-II and myosin-V, muscle myosin-II activity was very sensitive to salt. Collectively, our findings suggest differing degrees of reliance on electrostatic interactions during weak actomyosin binding in yeast versus muscle. Our study also highlights the importance of native actin isoforms when considering the function of myosins. PMID:21757693

  17. Cotton buds, momentum, and impulse

    NASA Astrophysics Data System (ADS)

    van den Berg, Ed; Nuñez, Jover; Guirit, Alfredo; van Huis, Cor

    2000-01-01

    Here is a simple experiment demonstrating impulse and momentum that was picked up from a Japanese presenter at a physics teacher conference held in Cebu City. We have not been able to trace the experiment farther and have never seen it in print. After student-author Nuñez demonstrated it during an exam on conducting demonstrations, we converted the qualitative idea into a quanitative experiment and even discovered some possibilities for student research. The lab is also suitable as homework, since it uses universally available "equipment" — cotton buds (swabs), drinking straws, and a ruler.

  18. The Prefoldin Bud27 Mediates the Assembly of the Eukaryotic RNA Polymerases in an Rpb5-Dependent Manner

    PubMed Central

    Mirón-García, María Carmen; Garrido-Godino, Ana Isabel; García-Molinero, Varinia; Hernández-Torres, Francisco; Rodríguez-Navarro, Susana; Navarro, Francisco

    2013-01-01

    The unconventional prefoldin URI/RMP, in humans, and its orthologue in yeast, Bud27, have been proposed to participate in the biogenesis of the RNA polymerases. However, this role of Bud27 has not been confirmed and is poorly elucidated. Our data help clarify the mechanisms governing biogenesis of the three eukaryotic RNA pols. We show evidence that Bud27 is the first example of a protein that participates in the biogenesis of the three eukaryotic RNA polymerases and the first example of a protein modulating their assembly instead of their nuclear transport. In addition we demonstrate that the role of Bud27 in RNA pols biogenesis depends on Rpb5. In fact, lack of BUD27 affects growth and leads to a substantial accumulation of the three RNA polymerases in the cytoplasm, defects offset by the overexpression of RPB5. Supporting this, our data demonstrate that the lack of Bud27 affects the correct assembly of Rpb5 and Rpb6 to the three RNA polymerases, suggesting that this process occurs in the cytoplasm and is a required step prior to nuclear import. Also, our data support the view that Rpb5 and Rpb6 assemble somewhat later than the rest of the complexes. Furthermore, Bud27 Rpb5-binding but not PFD-binding domain is necessary for RNA polymerases biogenesis. In agreement, we also demonstrate genetic interactions between BUD27, RPB5, and RPB6. Bud27 shuttles between the nucleus and the cytoplasm in an Xpo1-independent manner, and also independently of microtubule polarization and possibly independently of its association with the RNA pols. Our data also suggest that the role of Bud27 in RNA pols biogenesis is independent of the chaperone prefoldin (PFD) complex and of Iwr1. Finally, the role of URI seems to be conserved in humans, suggesting conserved mechanisms in RNA pols biogenesis. PMID:23459708

  19. Modes of interaction between the Arabidopsis Rab protein, Ara4, and its putative regulator molecules revealed by a yeast expression system.

    PubMed

    Ueda, T; Matsuda, N; Uchimiya, H; Nakano, A

    2000-02-01

    Ara4, a member of the Rab/Ypt GTPase family derived from Arabidopsis thaliana, causes severe growth inhibition when expressed in several yeast ypt mutants. Mutational analysis of ARA4 indicated that the Ara4 protein titrates at least three factors in yeast, including the GDP dissociation inhibitor (GDI). The coexpression of AtGDI1 (Arabidopsis GDI) suppressed the growth defect caused by Ara4 in yeast ypt1, suggesting that Ara4 and AtGDI1 interact in yeast to compensate for the titration of yeast GDI. We screened an Arabidopsis cDNA library for other suppressors that may also interact with Ara4 physiologically. A novel suppressor, SAY1, encoded a hydrophilic protein with two putative coiled-coil regions, which showed partial similarity to the yeast Vps27 protein. To understand the structural requirements of Ara4 for interacting with these molecules, we examined whether AtGDI1 and SAY1 could suppress the growth defect of ypt1 caused by various mutant versions of ARA4. The results indicated that the interaction between Ara4 and AtGDI1 depends on the conserved C-terminal Cys-motif and Thr44 in the effector domain of Ara4. In contrast, neither of these motifs is necessary for the interaction between Say1 and Ara4. This approach provides a powerful method to dissect complex interactions between a GTPase and its regulators.

  20. [Impact of TDZ and NAA on adventitious bud induction and cluster bud multiplication in Tulipa edulis].

    PubMed

    Zhu, Li-Fang; Xu, Chao; Zhu, Zai-Biao; Yang, He-Tong; Guo, Qiao-Sheng; Xu, Hong-jian; Ma, Hong-Jian; Zhao, Gui-Hua

    2014-08-01

    To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established. PMID:25509282

  1. [Impact of TDZ and NAA on adventitious bud induction and cluster bud multiplication in Tulipa edulis].

    PubMed

    Zhu, Li-Fang; Xu, Chao; Zhu, Zai-Biao; Yang, He-Tong; Guo, Qiao-Sheng; Xu, Hong-jian; Ma, Hong-Jian; Zhao, Gui-Hua

    2014-08-01

    To explore the method of explants directly induced bud and establish the tissue culture system of mutiple shoot by means of direct organogenesis, core bud and daughter bulbs (the top of bud stem expanded to form daughter bulb) of T. edulis were used as explants and treated with thidiazuron (TDZ) and 1-naphthlcetic acid (NAA). The results showed that the optimal medium for bud inducted form core bud and daughter bulb were MS + TDZ 2.0 mg x L(-1) + NAA 4.0 mg x L(-1) and MS +TDZ 2.0 mg x L(-1) + NAA 2.0 mg x L(-1) respectively, both of them had a bud induction rate of 72.92%, 79.22%. The optimal medium for cluster buds multiplication was MS + TDZ 0.2 mg x L(-1) + NAA 0.2 mg x L(-1), and proliferation coefficient was 2.23. After proliferation, cluster buds rooting occurred on MS medium with IBA 1.0 mg x L(-1) and the rooting rate was 52.6%, three to five seedlings in each plant. Using core bud and daughter bulb of T. edulis, the optimum medium for adventitious bud directly inducted from daughter bulb, core bud and cluster bud multiplication were screened out and the tissue culture system of multiple shoot by means of direct organogenesis was established.

  2. Bud-Localization of CLB2 mRNA Can Constitute a Growth Rate Dependent Daughter Sizer.

    PubMed

    Spiesser, Thomas W; Kühn, Clemens; Krantz, Marcus; Klipp, Edda

    2015-04-01

    Maintenance of cellular size is a fundamental systems level process that requires balancing of cell growth with proliferation. This is achieved via the cell division cycle, which is driven by the sequential accumulation and destruction of cyclins. The regulatory network around these cyclins, particularly in G1, has been interpreted as a size control network in budding yeast, and cell size as being decisive for the START transition. However, it is not clear why disruptions in the G1 network may lead to altered size rather than loss of size control, or why the S-G2-M duration also depends on nutrients. With a mathematical population model comprised of individually growing cells, we show that cyclin translation would suffice to explain the observed growth rate dependence of cell volume at START. Moreover, we assess the impact of the observed bud-localisation of the G2 cyclin CLB2 mRNA, and find that localised cyclin translation could provide an efficient mechanism for measuring the biosynthetic capacity in specific compartments: The mother in G1, and the growing bud in G2. Hence, iteration of the same principle can ensure that the mother cell is strong enough to grow a bud, and that the bud is strong enough for independent life. Cell sizes emerge in the model, which predicts that a single CDK-cyclin pair per growth phase suffices for size control in budding yeast, despite the necessity of the cell cycle network around the cyclins to integrate other cues. Size control seems to be exerted twice, where the G2/M control affects bud size through bud-localized translation of CLB2 mRNA, explaining the dependence of the S-G2-M duration on nutrients. Taken together, our findings suggest that cell size is an emergent rather than a regulatory property of the network linking growth and proliferation. PMID:25910075

  3. Functional analysis of the TFIID-specific yeast TAF4 (yTAF(II)48) reveals an unexpected organization of its histone-fold domain.

    PubMed

    Thuault, Sylvie; Gangloff, Yann-Gaël; Kirchner, Jay; Sanders, Steven; Werten, Sebastiaan; Romier, Christophe; Weil, P Anthony; Davidson, Irwin

    2002-11-22

    Yeast TFIID comprises the TATA binding protein and 14 TBP-associated factors (TAF(II)s), nine of which contain histone-fold domains (HFDs). The C-terminal region of the TFIID-specific yTAF4 (yTAF(II)48) containing the HFD shares strong sequence similarity with Drosophila (d)TAF4 (dTAF(II)110) and human TAF4 (hTAF(II)135). A structure/function analysis of yTAF4 demonstrates that the HFD, a short conserved C-terminal domain (CCTD), and the region separating them are all required for yTAF4 function. Temperature-sensitive mutations in the yTAF4 HFD alpha2 helix or the CCTD can be suppressed upon overexpression of yTAF12 (yTAF(II)68). Moreover, coexpression in Escherichia coli indicates direct yTAF4-yTAF12 heterodimerization optimally requires both the yTAF4 HFD and CCTD. The x-ray crystal structure of the orthologous hTAF4-hTAF12 histone-like heterodimer indicates that the alpha3 region within the predicted TAF4 HFD is unstructured and does not correspond to the bona fide alpha3 helix. Our functional and biochemical analysis of yTAF4, rather provides strong evidence that the HFD alpha3 helix of the TAF4 family lies within the CCTD. These results reveal an unexpected and novel HFD organization in which the alpha3 helix is separated from the alpha2 helix by an extended loop containing a conserved functional domain. PMID:12237303

  4. Mutational analysis of the intramembranous H10 loop of yeast Nhx1 reveals a critical role in ion homoeostasis and vesicle trafficking.

    PubMed

    Mukherjee, Sanchita; Kallay, Laura; Brett, Christopher L; Rao, Rajini

    2006-08-15

    Yeast Nhx1 [Na+(K+)/H+ exchanger 1] is an intracellular Na+(K+)/H+ exchanger, localizing to the late endosome where it is important for ion homoeostasis and vesicle trafficking. Phylogenetic analysis of NHE (Na+/H+ exchanger) sequences has identified orthologous proteins, including HsNHE6 (human NHE6), HsNHE7 and HsNHE9 of unknown physiological role. These appear distinct from well-studied mammalian plasma membrane isoforms (NHE1-NHE5). To explore the differences between plasma membrane and intracellular NHEs and understand the link between ion homoeostasis and vesicle trafficking, we examined the consequence of replacing residues in the intramembranous H10 loop of Nhx1 between transmembrane segments 9 and 10. The critical role for the carboxy group of Glu355 in ion transport is consistent with the invariance of this residue in all NHEs. Surprisingly, residues specifically conserved in the intracellular isoforms (such as Phe357 and Tyr361) could not be replaced with closely similar residues (leucine and phenylalanine) found in the plasma membrane isoforms without loss of function, revealing unexpected side chain specificity. The trafficking phenotypes of all Nhx1 mutants, including hygromycin-sensitivity and missorting of carboxypeptidase Y, were found to directly correlate with pH homoeostasis defects and could be proportionately corrected by titration with weak base. The present study demonstrates the importance of the H10 loop of the NHE family, highlights the differences between plasma membrane and intracellular isoforms and shows that trafficking defects are tightly coupled with pH homoeostasis.

  5. Insights into the Life Cycle of Yeasts from the CTG Clade Revealed by the Analysis of the Millerozyma (Pichia) farinosa Species Complex

    PubMed Central

    Jacques, Noémie; Leh-Louis, Véronique; Sacerdot, Christine; Casaregola, Serge

    2012-01-01

    Among ascomycetous yeasts, the CTG clade is so-called because its constituent species translate CTG as serine instead of leucine. Though the biology of certain pathogenic species such as Candida albicans has been much studied, little is known about the life cycles of non-pathogen species of the CTG clade. Taking advantage of the recently obtained sequence of the biotechnological Millerozyma (Pichiasorbitophila) farinosa strain CBS 7064, we used MLST to better define phylogenic relationships between most of the Millerozyma farinosa strains available in public collections. This led to the constitution of four phylogenetic clades diverging from 8% to 15% at the DNA level and possibly constituting a species complex (M. farinosa) and to the proposal of two new species:Millerozyma miso sp. nov. CBS 2004T ( = CLIB 1230T) and Candida pseudofarinosa sp. nov.NCYC 386T( = CLIB 1231T).Further analysis showed that M. farinosa isolates exist as haploid and inter-clade hybrids. Despite the sequence divergence between the clades, secondary contacts after reproductive isolation were evidenced, as revealed by both introgression and mitochondria transfer between clades. We also showed that the inter-clade hybrids do sporulate to generate mainly viable vegetative diploid spores that are not the result of meiosis, and very rarely aneuploid spores possibly through the loss of heterozygosity during sporulation. Taken together, these results show that in this part of the CTG clade, non-Mendelian genetic exchanges occur at high rates through hybridization between divergent strainsfrom distinct clades and subsequent massive loss of heterozygosity. This combination of mechanisms could constitute an alternative sexuality leading to an unsuspected biodiversity. PMID:22574125

  6. Bud23 methylates G1575 of 18S rRNA and is required for efficient nuclear export of pre-40S subunits.

    PubMed

    White, Joshua; Li, Zhihua; Sardana, Richa; Bujnicki, Janusz M; Marcotte, Edward M; Johnson, Arlen W

    2008-05-01

    BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5' internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Delta mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.

  7. Mapping nucleosome resolution chromosome folding in yeast by Micro-C

    PubMed Central

    Hsieh, Tsung-Han S.; Weiner, Assaf; Lajoie, Bryan; Dekker, Job; Friedman, Nir; Rando, Oliver J.

    2015-01-01

    SUMMARY We describe a Hi-C based method, Micro-C, in which micrococcal nuclease is used instead of restriction enzymes to fragment chromatin, enabling nucleosome resolution chromosome folding maps. Analysis of Micro-C maps for budding yeast reveals abundant self-associating domains similar to those reported in other species, but not previously observed in yeast. These structures, far shorter than topologically-associating domains in mammals, typically encompass one to five genes in yeast. Strong boundaries between self-associating domains occur at promoters of highly transcribed genes and regions of rapid histone turnover that are typically bound by the RSC chromatin-remodeling complex. Investigation of chromosome folding in mutants confirms roles for RSC, “gene looping” factor Ssu72, Mediator, H3K56 acetyltransferase Rtt109, and the N-terminal tail of H4 in folding of the yeast genome. This approach provides detailed structural maps of a eukaryotic genome, and our findings provide insights into the machinery underlying chromosome compaction. PMID:26119342

  8. Identification of Chemical-Genetic Interactions via Parallel Analysis of Barcoded Yeast Strains.

    PubMed

    Suresh, Sundari; Schlecht, Ulrich; Xu, Weihong; Miranda, Molly; Davis, Ronald W; Nislow, Corey; Giaever, Guri; St Onge, Robert P

    2016-01-01

    The Yeast Knockout Collection is a complete set of gene deletion strains for the budding yeast, Saccharomyces cerevisiae In each strain, one of approximately 6000 open-reading frames is replaced with a dominant selectable marker flanked by two DNA barcodes. These barcodes, which are unique to each gene, allow the growth of thousands of strains to be individually measured from a single pooled culture. The collection, and other resources that followed, has ushered in a new era in chemical biology, enabling unbiased and systematic identification of chemical-genetic interactions (CGIs) with remarkable ease. CGIs link bioactive compounds to biological processes, and hence can reveal the mechanism of action of growth-inhibitory compounds in vivo, including those of antifungal, antibiotic, and anticancer drugs. The chemogenomic profiling method described here measures the sensitivity induced in yeast heterozygous and homozygous deletion strains in the presence of a chemical inhibitor of growth (termed haploinsufficiency profiling and homozygous profiling, respectively, or HIPHOP). The protocol is both scalable and amenable to automation. After competitive growth of yeast knockout collection cultures, with and without chemical inhibitors, CGIs can be identified and quantified using either array- or sequencing-based approaches as described here. PMID:27587778

  9. Identification of Chemical-Genetic Interactions via Parallel Analysis of Barcoded Yeast Strains.

    PubMed

    Suresh, Sundari; Schlecht, Ulrich; Xu, Weihong; Miranda, Molly; Davis, Ronald W; Nislow, Corey; Giaever, Guri; St Onge, Robert P

    2016-09-01

    The Yeast Knockout Collection is a complete set of gene deletion strains for the budding yeast, Saccharomyces cerevisiae In each strain, one of approximately 6000 open-reading frames is replaced with a dominant selectable marker flanked by two DNA barcodes. These barcodes, which are unique to each gene, allow the growth of thousands of strains to be individually measured from a single pooled culture. The collection, and other resources that followed, has ushered in a new era in chemical biology, enabling unbiased and systematic identification of chemical-genetic interactions (CGIs) with remarkable ease. CGIs link bioactive compounds to biological processes, and hence can reveal the mechanism of action of growth-inhibitory compounds in vivo, including those of antifungal, antibiotic, and anticancer drugs. The chemogenomic profiling method described here measures the sensitivity induced in yeast heterozygous and homozygous deletion strains in the presence of a chemical inhibitor of growth (termed haploinsufficiency profiling and homozygous profiling, respectively, or HIPHOP). The protocol is both scalable and amenable to automation. After competitive growth of yeast knockout collection cultures, with and without chemical inhibitors, CGIs can be identified and quantified using either array- or sequencing-based approaches as described here.

  10. Embryonic origin of amphibian taste buds.

    PubMed

    Barlow, L A; Northcutt, R G

    1995-05-01

    Despite numerous descriptive studies, the embryonic origin of vertebrate taste buds has never been experimentally determined. A number of different alternatives have been suggested for taste bud origins, including epibranchial placodes, the neural crest, and the local epithelium of the oropharyngeal cavity. The role of a series of epibranchial placodes and the cephalic neural crest, which together give rise to the cranial nerves innervating taste buds, was examined with regard to the development of oropharyngeal taste buds in an ambystomatid salamander, the axolotl. When pigmented placodal ectoderm or neural folds were grafted isotopically and isochronically into nonpigmented host embryos, known derivatives of each tissue contained pigmented cells, but labeled taste buds were never encountered. Thus, neither epibranchial placodes nor neural crest contribute cells to taste buds during embryogenesis. The majority of the oropharyngeal cavity of ambystomatid salamanders is lined by an endodermal epithelium. In order to demonstrate conclusively that taste buds arise from this local epithelium, the presumptive cephalic endoderm of early axolotl gastrulae was microinjected with the lipophilic dye, DiI. In the oropharyngeal epithelium of all larvae examined, both taste buds and general epithelial cells were labeled with DiI, indicating their common endodermal origin. Our findings are novel in that this is the first experimental demonstration of the endodermal origin of a vertebrate sensory receptor cell class. PMID:7750643

  11. Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated, sorted and matured in the fission yeast Schizosaccharomyces pombe.

    PubMed

    Simeon, A; Egner, R; Gascon, S; Suarez-Rendueles, P

    1995-03-01

    Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPYsc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 mu derived plasmid. Immunoblot analysis revealed that CPYsc produced in the fission yeast has a higher molecular mass than mature CPYsc produced by the budding yeast. CPYsc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPYsc produced by S. cerevisiae. CPYsc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPYsc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.

  12. Ultrastructural localization of anionic sites on the surface of yeast, hyphal and germ-tube forming cells of Candida albicans.

    PubMed

    Horisberger, M; Clerc, M F

    1988-08-01

    The cell wall of Candida albicans contains chitin, beta-glucans and phosphorylated mannoproteins, and possesses a fuzzy coat which is thought to play a role in pathogenicity, phagocytosis, and adherence of this dimorphic yeast. Using scanning electron microscopy and the gold method, mannoproteins were detected on the whole surface of blastoconidia including the bud scars, but chitin was absent even after alpha-mannosidase treatment of the cells. The presence of surface beta-(1----6)glucan (but not beta(1----3)glucan) was observed only after extensive alpha-mannosidase and alkaline phosphatase treatments of blastoconidia. Using transmission and scanning electron microscopy, the locations of anionic sites were revealed by polycationic colloidal gold-chitosan complexes on the surface of blastoconidia, germ tubes and hyphae. Anionic sites were dispersed evenly over the surface of blastoconidia bearing bud scars. Depending upon the growth conditions, anionic sites could be detected on emerging buds and young cells. However, bud scars were always free of marking. When germ-tube formation was induced, anionic sites were present at different densities on all cell surfaces, the highest density being observed on cells with bud scars. Anionic sites were detected at a remarkably high density on all hyphal surfaces. An apical concentration of anionic sites was observed on germ tubes and hyphae. The distribution of anionic sites was not modified by endoglucosaminidase treatment of blastoconidia, germ tubes and hyphae. The anionic sites were associated with the fuzzy coat. As the hyphal form is regarded as possessing the greatest invasiveness, it is suggested that anionic sites play an important role in establishing tissue colonization by this human pathogen. PMID:3053174

  13. Acetylated H4 histone and genomic DNA methylation patterns during bud set and bud burst in Castanea sativa.

    PubMed

    Santamaría, Ma Estrella; Hasbún, Rodrigo; Valera, Ma José; Meijón, Mónica; Valledor, Luis; Rodríguez, Jose L; Toorop, Peter E; Cañal, Ma Jesús; Rodríguez, Roberto

    2009-09-01

    The relationships between genomic DNA cytosine methylation, histone H4 acetylation and bud dormancy in Castanea sativa are described. Acetylated H4 histone and genomic DNA methylation patterns showed opposite abundance patterns during bud set and bud burst. Increased and decreased methylation levels in the apical buds coincided with bud set and bud burst, respectively. Intermediate axillary buds were characterized by constant levels of DNA methylation during burst of apical buds and reduced fluctuation in DNA methylation throughout the year, which coincided with the absence of macro-morphological changes. Furthermore, acetylated histone H4 (AcH4) levels from apical buds were higher during bud burst than during bud set, as was demonstrated by immunodetection. Results were validated with three additional C. sativa provenances. Thus, global DNA methylation and AcH4 levels showed opposite patterns and coincided with changes in bud dormancy in C. sativa.

  14. Membrane targeting of the yeast exocyst complex.

    PubMed

    Pleskot, Roman; Cwiklik, Lukasz; Jungwirth, Pavel; Žárský, Viktor; Potocký, Martin

    2015-07-01

    The exocytosis is a process of fusion of secretory vesicles with plasma membrane, which plays a prominent role in many crucial cellular processes, e.g. secretion of neurotransmitters, cytokinesis or yeast budding. Prior to the SNARE-mediated fusion, the initial contact of secretory vesicle with the target membrane is mediated by an evolutionary conserved vesicle tethering protein complex, the exocyst. In all eukaryotic cells, the exocyst is composed of eight subunits - Sec5, Sec6, Sec8, Sec10, Sec15, Exo84 and two membrane-targeting landmark subunits Sec3 and Exo70, which have been described to directly interact with phosphatidylinositol (4,5)-bisphosphate (PIP2) of the plasma membrane. In this work, we utilized coarse-grained molecular dynamics simulations to elucidate structural details of the interaction of yeast Sec3p and Exo70p with lipid bilayers containing PIP2. We found that PIP2 is coordinated by the positively charged pocket of N-terminal part of Sec3p, which folds into unique Pleckstrin homology domain. Conversely, Exo70p interacts with the lipid bilayer by several binding sites distributed along the structure of this exocyst subunit. Moreover, we observed that the interaction of Exo70p with the membrane causes clustering of PIP2 in the adjacent leaflet. We further revealed that PIP2 is required for the correct positioning of small GTPase Rho1p, a direct Sec3p interactor, prior to the formation of the functional Rho1p-exocyst-membrane assembly. Our results show the critical importance of the plasma membrane pool of PIP2 for the exocyst function and suggest that specific interaction with acidic phospholipids represents an ancestral mechanism for the exocyst regulation.

  15. Non-structural carbohydrate status in Norway spruce buds in the context of annual bud structural development as affected by acidic pollution.

    PubMed

    Svobodová; Lipavská; Albrechtová

    2000-06-01

    The present study focused on changes in the annual dynamics of the contents of non-structural saccharides (NSS) of Norway spruce vegetative buds related to their structural development under the effect of acidic pollution during the year 1995. Two types of material were analysed: (1) 4-year-old trees treated for 2 years by simulated acid rain (SAR; pH 2.9 and 3.9), and (2) 40-60-year-old trees growing in natural mountain stands exhibiting different degrees of macroscopic damage. Our study revealed that the dynamics of the NSS content reflected the major morphogenetic and developmental changes occurring during the annual bud developmental cycle. No systematic changes in the annual dynamics of NSS content were observed in buds from both mountain sites, or as a consequence of the SAR. The total sugar content of bud tissues was composed of a combination of five main sugar components: sucrose, glucose, fructose, raffinose family oligosaccharides (RFO; combination of raffinose and stachyose), and a pinitol fraction (PF) probably of cyclitols with pinitol as a main member. The dynamics of individual sugar components also reflected possible carbohydrate mediated bud frost protection. Interesting results were obtained from buds in dormant state. In dormant buds of the SAR experiment the higher value of the ratio PF:RFO of the pinitol fraction and raffinose family oligosaccharides followed the higher dose of SAR treatment. When evaluating the ratio from both types of material we assumed that changes in PF:RFO ratio corresponded to early stages of damage or acute metabolic reaction. Thus, we suggest the ratio PF:RFO as a possible non-specific metabolic marker of early bud stress reaction which is, among other stress factors, sensitive to increasing load of acidic pollutants.

  16. Cell contact-dependent mechanisms specify taste bud pattern during a critical period early in embryonic development.

    PubMed

    Parker, Mark A; Bell, Melanie L; Barlow, Linda A

    2004-08-01

    After gastrulation, the pharyngeal endoderm is specified to give rise to taste receptor organs without further signaling from other embryonic tissues. We hypothesized that intercellular signaling might be responsible for the specification of taste buds. To test if and when this signaling was occurring, intercellular contacts were transiently disrupted in cultures of pharyngeal endoderm from axolotl embryos, and the number, size, and distribution of taste buds analyzed. Disruption of cell contacts at progressive time points, from neurula to late tail bud stages, revealed a critical period, during mid-tail bud stages, when disruption of cell contacts resulted in a significant increase in taste bud number and size. The spatial distribution of taste buds was also altered; taste buds were more clustered in explants disrupted during the critical period. These effects were not due to general alterations in mitosis and apoptosis. Rather, at least three aspects of taste bud patterning, i.e., number, size, and distribution, are governed by mechanisms dependent on normal cell contacts during a concise time window. Furthermore, our findings are consistent with specification of taste buds by means of lateral inhibitory signaling, which we hypothesize results from cell contact-dependent or short-range diffusible signals. PMID:15254897

  17. Cytokinins Are Initial Targets of Light in the Control of Bud Outgrowth1[OPEN

    PubMed Central

    Girault, Tiffanie; Barbier, François; Péron, Thomas; Pěnčík, Aleš; Sakr, Soulaiman; Lothier, Jérémy

    2016-01-01

    Bud outgrowth is controlled by environmental and endogenous factors. Through the use of the photosynthesis inhibitor norflurazon and of masking experiments, evidence is given here that light acts mainly as a morphogenic signal in the triggering of bud outgrowth and that initial steps in the light signaling pathway involve cytokinins (CKs). Indeed, in rose (Rosa hybrida), inhibition of bud outgrowth by darkness is suppressed solely by the application of CKs. In contrast, application of sugars has a limited effect. Exposure of plants to white light (WL) induces a rapid (after 3–6 h of WL exposure) up-regulation of CK synthesis (RhIPT3 and RhIPT5), of CK activation (RhLOG8), and of CK putative transporter RhPUP5 genes and to the repression of the CK degradation RhCKX1 gene in the node. This leads to the accumulation of CKs in the node within 6 h and in the bud at 24 h and to the triggering of bud outgrowth. Molecular analysis of genes involved in major mechanisms of bud outgrowth (strigolactone signaling [RwMAX2], metabolism and transport of auxin [RhPIN1, RhYUC1, and RhTAR1], regulation of sugar sink strength [RhVI, RhSUSY, RhSUC2, and RhSWEET10], and cell division and expansion [RhEXP and RhPCNA]) reveal that, when supplied in darkness, CKs up-regulate their expression as rapidly and as intensely as WL. Additionally, up-regulation of CKs by WL promotes xylem flux toward the bud, as evidenced by Methylene Blue accumulation in the bud after CK treatment in the dark. Altogether, these results suggest that CKs are initial components of the light signaling pathway that controls the initiation of bud outgrowth. PMID:27462085

  18. Cytokinins Are Initial Targets of Light in the Control of Bud Outgrowth.

    PubMed

    Roman, Hanaé; Girault, Tiffanie; Barbier, François; Péron, Thomas; Brouard, Nathalie; Pěnčík, Aleš; Novák, Ondřej; Vian, Alain; Sakr, Soulaiman; Lothier, Jérémy; Le Gourrierec, José; Leduc, Nathalie

    2016-09-01

    Bud outgrowth is controlled by environmental and endogenous factors. Through the use of the photosynthesis inhibitor norflurazon and of masking experiments, evidence is given here that light acts mainly as a morphogenic signal in the triggering of bud outgrowth and that initial steps in the light signaling pathway involve cytokinins (CKs). Indeed, in rose (Rosa hybrida), inhibition of bud outgrowth by darkness is suppressed solely by the application of CKs. In contrast, application of sugars has a limited effect. Exposure of plants to white light (WL) induces a rapid (after 3-6 h of WL exposure) up-regulation of CK synthesis (RhIPT3 and RhIPT5), of CK activation (RhLOG8), and of CK putative transporter RhPUP5 genes and to the repression of the CK degradation RhCKX1 gene in the node. This leads to the accumulation of CKs in the node within 6 h and in the bud at 24 h and to the triggering of bud outgrowth. Molecular analysis of genes involved in major mechanisms of bud outgrowth (strigolactone signaling [RwMAX2], metabolism and transport of auxin [RhPIN1, RhYUC1, and RhTAR1], regulation of sugar sink strength [RhVI, RhSUSY, RhSUC2, and RhSWEET10], and cell division and expansion [RhEXP and RhPCNA]) reveal that, when supplied in darkness, CKs up-regulate their expression as rapidly and as intensely as WL Additionally, up-regulation of CKs by WL promotes xylem flux toward the bud, as evidenced by Methylene Blue accumulation in the bud after CK treatment in the dark. Altogether, these results suggest that CKs are initial components of the light signaling pathway that controls the initiation of bud outgrowth. PMID:27462085

  19. Genome and transcriptome analyses reveal that MAPK- and phosphatidylinositol-signaling pathways mediate tolerance to 5-hydroxymethyl-2-furaldehyde for industrial yeast Saccharomyces cerevisiae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The industrial ethanologenic yeast Saccharomyces cerevisiae is a promising biocatalyst for next-generation advanced biofuels applications including lignocellulose-to-ethanol conversion. Here we present the first insight into the genomic background of NRRL Y-12632, a type strain from a worldwide coll...

  20. The metabolism beyond programmed cell death in yeast

    PubMed Central

    Ring, Julia; Sommer, Cornelia; Carmona-Gutierrez, Didac; Ruckenstuhl, Christoph; Eisenberg, Tobias; Madeo, Frank

    2012-01-01

    A cell's reaction to any change in the endogenous or exogenous conditions often involves a complex response that eventually either leads to cell adaptation and survival or to the initiation and execution of (programmed) cell death. The molecular decision whether to live or die, while depending on a cell's genome, is fundamentally influenced by its actual metabolic status. Thus, the collection of all metabolites present in a biological system at a certain time point (the so-called metabolome) defines its physiological, developmental and pathological state and determines its fate during changing and stressful conditions. The budding yeast Saccharomyces cerevisiae is a unicellular organism that allows to easily modify and monitor conditions affecting the cell's metabolome, for instance through a simple change of the nutrition source. Such changes can be used to mimic and study (patho)physiological scenarios, including caloric restriction and longevity, the Warburg effect in cancer cells or changes in mitochondrial mass affecting cell death. In addition, disruption of single genes or generation of respiratory deficiency (via abrogation of mitochondrial DNA) assists in revealing connections between metabolism and apoptosis. In this minireview, we discuss recent studies using the potential of the yeast model to provide new insights into the processes of stress defense, cell death and longevity. PMID:22480867

  1. Gravity-induced buds formation from protonemata apical cells in the mosses

    NASA Astrophysics Data System (ADS)

    Kyyak, Natalia; Khorkavtsiv, Yaroslava

    The acceleration of moss protonemata development after the exit it to light from darkness is important gravidependent morphogenetic manifestation of the moss protonemata. The accelerated development of mosses shows in transformation of apical protonemata cells into the gametophores buds (Ripetskyj et al., 1999). In order to establish, that such reaction on gravitation is general property of gravisensity species, or its typical only for single moss species, experiments with the following moss species - Bryum intermedium (Ludw.) Brig., Bryum caespiticium Hedw., Bryum argenteum Hedw., Dicranodontium denudatum (Brid.) Britt. were carried out. All these species in response to influence of gravitation were capable to form rich bunches of gravitropical protonemata in darkness, that testified to their gravisensity. After the transference of Petri dishes with gravitropical protonemata from darkness on light was revealed, that in 3 of the investigated species the gametophores buds were absent. Only B. argenteum has reacted to action of gravitation by buds formation from apical cells of the gravitropical protonemata. With the purpose of strengthening of buds formation process, the experiments with action of exogenous kinetin (in concentration of 10 (-6) M) were carried out. Kinetin essentially stimulated apical buds formation of B. argenteum. The quantity of apical buds has increased almost in three times in comparison with the control. Besides, on separate stolons a few (3-4) buds from one apical cell were formed. Experimentally was established, that the gametophores buds formation in mosses is controlled by phytohormones (Bopp, 1985; Demkiv et al., 1991). In conditions of gravity influence its essentially accelerated. Probably, gravity essentially strengthened acropetal transport of phytohormones and formation of attractive center in the protonemata apical cell. Our investigations have allowed to make the conclusion, that gravi-dependent formation of the apical buds is

  2. Counting Yeast.

    ERIC Educational Resources Information Center

    Bealer, Jonathan; Welton, Briana

    1998-01-01

    Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

  3. Yeast Infections

    MedlinePlus

    Candida is the scientific name for yeast. It is a fungus that lives almost everywhere, including in ... infection that causes white patches in your mouth Candida esophagitis is thrush that spreads to your esophagus, ...

  4. The dormant buds of Rhabdopleura compacta (Hemichordata).

    PubMed

    Dilly, P N

    1975-06-13

    Rhabdopleura has an overwintering stage that consists of two layers of cells surrounding a central yolk mass. This cellular part is surrounded by a thick electron dense capsule which is secreted by the bud itself. The capsule is probably impervious and protective to its contents. Blood vessels join the buds to the zooids of the colony. They form the probable route of transfer of yolk from the zooids to the dormant bud. The capsule of the dormant bud has some structural features in common with the black stolon of the adult zooids. The black stolon is probably formed in a manner similar to that which made the fusellar fabric of the periderm of fossil graptolities. PMID:1149105

  5. Supercooling in Overwintering Azalea Flower Buds 1

    PubMed Central

    George, Milon F.; Burke, Michael J.; Weiser, Conrad J.

    1974-01-01

    Differential thermal analysis and nuclear magnetic resonance spectroscopy experiments on whole flower buds and excised floral primordia of azalea (Rhododendron kosterianum, Schneid.) proved that supercooling is the mode of freezing resistance (avoidance) of azalea flower primordia. Increase in the linewidth of nuclear magnetic resonance spectra for water upon thawing supports the view that injury to the primordia occurs at the moment of freezing. Nonliving primordia freeze at the same temperatures as living primordia, indicating that morphological features of primordial tissues are a key factor in freezing avoidance of dormant azalea flower primordia. Differential thermal analyses was used to study the relationship of cooling rate to the freezing points of floral primordia in whole flower buds. At a cooling rate of 8.5 C per hour, primordia in whole buds froze at about the same subfreezing temperatures as did excised primordia cooled at 37 C per hour. At more rapid cooling rates primordia in intact buds froze at higher temperatures. PMID:16658832

  6. The dormant buds of Rhabdopleura compacta (Hemichordata).

    PubMed

    Dilly, P N

    1975-06-13

    Rhabdopleura has an overwintering stage that consists of two layers of cells surrounding a central yolk mass. This cellular part is surrounded by a thick electron dense capsule which is secreted by the bud itself. The capsule is probably impervious and protective to its contents. Blood vessels join the buds to the zooids of the colony. They form the probable route of transfer of yolk from the zooids to the dormant bud. The capsule of the dormant bud has some structural features in common with the black stolon of the adult zooids. The black stolon is probably formed in a manner similar to that which made the fusellar fabric of the periderm of fossil graptolities.

  7. Yeast killer systems.

    PubMed Central

    Magliani, W; Conti, S; Gerloni, M; Bertolotti, D; Polonelli, L

    1997-01-01

    The killer phenomenon in yeasts has been revealed to be a multicentric model for molecular biologists, virologists, phytopathologists, epidemiologists, industrial and medical microbiologists, mycologists, and pharmacologists. The surprisingly widespread occurrence of the killer phenomenon among taxonomically unrelated microorganisms, including prokaryotic and eukaryotic pathogens, has engendered a new interest in its biological significance as well as its theoretical and practical applications. The search for therapeutic opportunities by using yeast killer systems has conceptually opened new avenues for the prevention and control of life-threatening fungal diseases through the idiotypic network that is apparently exploited by the immune system in the course of natural infections. In this review, the biology, ecology, epidemiology, therapeutics, serology, and idiotypy of yeast killer systems are discussed. PMID:9227858

  8. Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA-protein interactions.

    PubMed

    Khanova, Elena; Esakova, Olga; Perederina, Anna; Berezin, Igor; Krasilnikov, Andrey S

    2012-04-01

    Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

  9. Function of a plant stress-induced gene, HVA22. Synthetic enhancement screen with its yeast homolog reveals its role in vesicular traffic.

    PubMed

    Brands, Alex; Ho, Tuan-hua David

    2002-11-01

    Expression of the barley (Hordeum vulgare) HVA22 gene is induced by environmental stresses, such as dehydration, salinity, and extreme temperatures, and by a plant stress hormone, abscisic acid. Genes sharing high level of sequence similarities with HVA22 exist in diverse eukaryotic organisms, including animals, plants, and fungi, but not in any prokaryotic organisms. The yeast (Saccharomyces cerevisiae) HVA22 homolog, Yop1p, has been shown to interact with the GTPase-interacting protein, Yip1p. Deletion of YOP1 led to only a modest reduction of the stationary phase titer at 37C. A synthetic enhancement mutant screen was performed in the yop1 deletion background to identify genes interacting with YOP1. The open reading frame YOR165W (renamed SEY1 for synthetic enhancement of YOP1) was identified as a YOP1-dependent complementation gene. The yeast SEY1 is a homolog of the Arabidopsis RHD3 gene whose mutations cause the accumulation of transport vesicles near the tips of defective root hairs. The yeast double mutant of yop1 and sey1 is defective in vesicular traffic as evidenced by the accumulation of transport vesicles and the decrease in invertase secretion. Based on these observations, we suggest that Yop1p/HVA22 regulates vesicular traffic in stressed cells either to facilitate membrane turnover, or to decrease unnecessary secretion.

  10. Properties of peach flower buds which facilitate supercooling.

    PubMed

    Ashworth, E N

    1982-11-01

    Water in dormant peach (Prunus persica [L.] Batsch. var. ;Harbrite') flower buds deep supercooled. Both supercooling and the freezing of water within the bud axis and primordium as distinct components depended on the viability of the bud axis tissue. The viability of the primordium was not critical. Supercooling was prevented by wounding buds with a dissecting needle, indicating that bud structural features were important. Bud morphological features appeared to prevent the propagation of ice through the vascular tissue and into the primordium. In dormant buds, procambial cells had not yet differentiated into xylem vessel elements. Xylem continuity between the bud primordium and adjacent tissues did not appear to be established until buds had deacclimated. It was concluded that structural, morphological, and physiological features of the bud facilitated supercooling.

  11. Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid

    PubMed Central

    Aihara, Eitaro; Mahe, Maxime M.; Schumacher, Michael A.; Matthis, Andrea L.; Feng, Rui; Ren, Wenwen; Noah, Taeko K.; Matsu-ura, Toru; Moore, Sean R.; Hong, Christian I.; Zavros, Yana; Herness, Scott; Shroyer, Noah F.; Iwatsuki, Ken; Jiang, Peihua; Helmrath, Michael A.; Montrose, Marshall H.

    2015-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5+) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5+ cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration. PMID:26597788

  12. Characterization of stem/progenitor cell cycle using murine circumvallate papilla taste bud organoid.

    PubMed

    Aihara, Eitaro; Mahe, Maxime M; Schumacher, Michael A; Matthis, Andrea L; Feng, Rui; Ren, Wenwen; Noah, Taeko K; Matsu-ura, Toru; Moore, Sean R; Hong, Christian I; Zavros, Yana; Herness, Scott; Shroyer, Noah F; Iwatsuki, Ken; Jiang, Peihua; Helmrath, Michael A; Montrose, Marshall H

    2015-01-01

    Leucine-rich repeat-containing G-protein coupled receptor 5-expressing (Lgr5(+)) cells have been identified as stem/progenitor cells in the circumvallate papillae, and single cultured Lgr5(+) cells give rise to taste cells. Here we use circumvallate papilla tissue to establish a three-dimensional culture system (taste bud organoids) that develops phenotypic characteristics similar to native tissue, including a multilayered epithelium containing stem/progenitor in the outer layers and taste cells in the inner layers. Furthermore, characterization of the cell cycle of the taste bud progenitor niche reveals striking dynamics of taste bud development and regeneration. Using this taste bud organoid culture system and FUCCI2 transgenic mice, we identify the stem/progenitor cells have at least 5 distinct cell cycle populations by tracking within 24-hour synchronized oscillations of proliferation. Additionally, we demonstrate that stem/progenitor cells have motility to form taste bud organoids. Taste bud organoids provides a system for elucidating mechanisms of taste signaling, disease modeling, and taste tissue regeneration. PMID:26597788

  13. Expression and function of myc during asexual reproduction of the budding ascidian Polyandrocarpa misakiensis.

    PubMed

    Fujiwara, Shigeki; Isozaki, Takaomi; Mori, Kyoko; Kawamura, Kazuo

    2011-12-01

    The budding ascidian Polyandrocarpa misakiensis proliferates asexually by budding. The atrial epithelium is a multipotent but differentiated tissue, which transdifferentiates into various tissues and organs after the bud separates from the parental body. We isolated cDNA clones homologous to the myc proto-oncogene from P. misakiensis. The cDNA, named Pm-myc, encoded a polypeptide of 639 amino acid residues, containing Myc-specific functional motifs, Myc box I and Myc box II, and the basic helix-loop-helix domain. Expression of Pm-myc was observed in the atrial epithelium in the organ-forming region of the developing bud, where the epithelial cells dedifferentiate and re-enter the cell cycle. The expression was also observed in fibroblast-like cells, which are known to participate in the organogenesis together with the epithelial cells. Unexpectedly, the atrial epithelium expressed Pm-myc more than one day before the dedifferentiation. The organogenesis was disturbed by Pm-myc-specific double-stranded RNA. In situ hybridization revealed that Pm-myc-positive fibroblast-like cells disappeared around the organ primordium of the dsRNA-treated bud. The results suggest that the mesenchymal-epithelial transition of fibroblast-like cells is important for the organogenesis in this budding ascidian species.

  14. Eighteen new oleaginous yeast species.

    PubMed

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes.

  15. Eighteen new oleaginous yeast species.

    PubMed

    Garay, Luis A; Sitepu, Irnayuli R; Cajka, Tomas; Chandra, Idelia; Shi, Sandy; Lin, Ting; German, J Bruce; Fiehn, Oliver; Boundy-Mills, Kyria L

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes. PMID:27072563

  16. Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

    PubMed Central

    Lee, I-Ju; Wang, Ning; Hu, Wen; Schott, Kersey; Bähler, Jürg; Giddings, Thomas H.; Pringle, John R.; Du, Li-Lin; Wu, Jian-Qiu

    2014-01-01

    Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly. PMID:25031431

  17. Predicting the fission yeast protein interaction network.

    PubMed

    Pancaldi, Vera; Saraç, Omer S; Rallis, Charalampos; McLean, Janel R; Převorovský, Martin; Gould, Kathleen; Beyer, Andreas; Bähler, Jürg

    2012-04-01

    A systems-level understanding of biological processes and information flow requires the mapping of cellular component interactions, among which protein-protein interactions are particularly important. Fission yeast (Schizosaccharomyces pombe) is a valuable model organism for which no systematic protein-interaction data are available. We exploited gene and protein properties, global genome regulation datasets, and conservation of interactions between budding and fission yeast to predict fission yeast protein interactions in silico. We have extensively tested our method in three ways: first, by predicting with 70-80% accuracy a selected high-confidence test set; second, by recapitulating interactions between members of the well-characterized SAGA co-activator complex; and third, by verifying predicted interactions of the Cbf11 transcription factor using mass spectrometry of TAP-purified protein complexes. Given the importance of the pathway in cell physiology and human disease, we explore the predicted sub-networks centered on the Tor1/2 kinases. Moreover, we predict the histidine kinases Mak1/2/3 to be vital hubs in the fission yeast stress response network, and we suggest interactors of argonaute 1, the principal component of the siRNA-mediated gene silencing pathway, lost in budding yeast but preserved in S. pombe. Of the new high-quality interactions that were discovered after we started this work, 73% were found in our predictions. Even though any predicted interactome is imperfect, the protein network presented here can provide a valuable basis to explore biological processes and to guide wet-lab experiments in fission yeast and beyond. Our predicted protein interactions are freely available through PInt, an online resource on our website (www.bahlerlab.info/PInt).

  18. Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection.

    PubMed

    Li, Yi-Ke; Yang, Juan-Mei; Huang, Yi-Bo; Ren, Dong-Dong; Chi, Fang-Lu

    2015-06-01

    The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.

  19. Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection

    PubMed Central

    Li, Yi-ke; Yang, Juan-mei; Huang, Yi-bo; Ren, Dong-dong; Chi, Fang-lu

    2015-01-01

    The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds. PMID:26199619

  20. Ubiquitin depletion and dominant-negative VPS4 inhibit rhabdovirus budding without affecting alphavirus budding.

    PubMed

    Taylor, Gwen M; Hanson, Phyllis I; Kielian, Margaret

    2007-12-01

    The budding reactions of a number of enveloped viruses use the cellular machinery involved in the formation of the luminal vesicles of endosomal multivesicular bodies (MVB). Budding of these viruses is dependent on the presence of specific late-domain motifs in membrane-associated viral proteins. Such budding reactions usually involve ubiquitin and are blocked by expression of an ATPase-deficient form of VPS4, a cellular AAA+ ATPase believed to be required late in the MVB pathway for the disassembly/release of the MVB machinery. Here we examined the role of the MVB pathway in the budding of the late-domain-containing rhabdovirus vesicular stomatitis virus (VSV) and the alphavirus Semliki Forest virus (SFV). We tested early and late steps in the MVB pathway by depleting ubiquitin with the proteasome inhibitor MG-132 and by using cell lines inducibly expressing VPS4A or VPS4B protein. As previously shown, VSV budding was strongly dependent on ubiquitin. In contrast to the findings of previous studies with VPS4A, expression of ATPase-deficient mutants of either VPS4A or VPS4B inhibited VSV budding. Inhibition by VPS4 required the presence of the PPPY late domain on the VSV matrix protein and resulted in the accumulation of nonreleased VSV particles at the plasma membrane. In contrast, SFV budding was independent of both ubiquitin and the activity of VPS4, perhaps reflecting the important role of the highly organized envelope protein lattice during alphavirus budding.

  1. Preformation in vegetative buds of Prunus persica: factors influencing number of leaf primordia in overwintering buds.

    PubMed

    Gordon, D; Damiano, C; DeJong, T M

    2006-04-01

    We investigated the influence of bud position, cultivar, tree age, tree carbohydrate status, sampling date, drought and light exposure on the number of leaf primordia formed in dormant vegetative peach buds (Prunus persica (L.) Batsch) relative to the number of primordia formed after bud break (neoformed). During winter dormancy, vegetative peach buds from California and Italy were dissected and the number of leaf primordia recorded. Between leaf drop and bud break, the number of leaf primordia doubled from about five to about 10. Parent shoot length, number of nodes on the parent shoot, cross-sectional area of the parent shoot, bud position along the parent shoot and bud cross-sectional area were correlated with the number of leaf primordia. Previous season light exposure, drought and tree carbohydrate status did not affect the number of leaf primordia present. The number of leaf primordia differed significantly among peach varieties and tree ages at leaf drop, but not at bud break. Our results indicate that neoformation accounted for all shoot growth beyond about 10 nodes. The predominance of neoformed shoot growth in peach allows this species great plasticity in its response to current-season conditions. PMID:16414932

  2. Yeast Droplets

    NASA Astrophysics Data System (ADS)

    Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

    2002-11-01

    It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

  3. Revisiting the yeast PPR proteins--application of an Iterative Hidden Markov Model algorithm reveals new members of the rapidly evolving family.

    PubMed

    Lipinski, Kamil A; Puchta, Olga; Surendranath, Vineeth; Kudla, Marek; Golik, Pawel

    2011-10-01

    Pentatricopeptide repeat (PPR) proteins are the largest known RNA-binding protein family, and are found in all eukaryotes, being particularly abundant in higher plants. PPR proteins localize mostly to mitochondria and chloroplasts, and many were shown to modulate organellar genome expression on the posttranscriptional level. Although the genomes of land plants encode hundreds of PPR proteins, only a few have been identified in Fungi and Metazoa. As the current PPR motif profiles are built mainly on the basis of the predominant plant sequences, they are unlikely to be optimal for detecting fungal and animal members of the family, and many putative PPR proteins in these genomes may remain undetected. In order to verify this hypothesis, we designed a hidden Markov model-based bioinformatic tool called Supervised Clustering-based Iterative Phylogenetic Hidden Markov Model algorithm for the Evaluation of tandem Repeat motif families (SCIPHER) using sequence data from orthologous clusters from available yeast genomes. This approach allowed us to assign 12 new proteins in Saccharomyces cerevisiae to the PPR family. Similarly, in other yeast species, we obtained a 5-fold increase in the detection of PPR motifs, compared with the previous tools. All the newly identified S. cerevisiae PPR proteins localize in the mitochondrion and are a part of the RNA processing interaction network. Furthermore, the yeast PPR proteins seem to undergo an accelerated divergent evolution. Analysis of single and double amino acid substitutions in the Dmr1 protein of S. cerevisiae suggests that cooperative interactions between motifs and pseudoreversion could be the force driving this rapid evolution.

  4. Structural basis for endosomal recruitment of ESCRT-I by ESCRT-0 in yeast

    SciTech Connect

    Ren, Xuefeng; Hurley, James H.

    2011-10-28

    The ESCRT-0 and ESCRT-I complexes coordinate the clustering of ubiquitinated cargo with intralumenal budding of the endosomal membrane, two essential steps in vacuolar/lysosomal protein sorting from yeast to humans. The 1.85-{angstrom} crystal structure of interacting regions of the yeast ESCRT-0 and ESCRT-I complexes reveals that PSDP motifs of the Vps27 ESCRT-0 subunit bind to a novel electropositive N-terminal site on the UEV domain of the ESCRT-I subunit Vps23 centred on Trp16. This novel site is completely different from the C-terminal part of the human UEV domain that binds to P(S/T)AP motifs of human ESCRT-0 and HIV-1 Gag. Disruption of the novel PSDP-binding site eliminates the interaction in vitro and blocks enrichment of Vps23 in endosome-related class E compartments in yeast cells. However, this site is non-essential for sorting of the ESCRT cargo Cps1. Taken together, these results show how a conserved motif/domain pair can evolve to use strikingly different binding modes in different organisms.

  5. Photoperiod and temperature responses of bud swelling and bud burst in four temperate forest tree species.

    PubMed

    Basler, David; Körner, Christian

    2014-04-01

    Spring phenology of temperate forest trees is optimized to maximize the length of the growing season while minimizing the risk of freezing damage. The release from winter dormancy is environmentally mediated by species-specific responses to temperature and photoperiod. We investigated the response of early spring phenology to temperature and photoperiod at different stages of dormancy release in cuttings from four temperate tree species in controlled environments. By tracking bud development, we were able to identify the onset of bud swelling and bud growth in Acer pseudoplatanus L., Fagus sylvatica L., Quercus petraea (Mattuschka) Liebl. and Picea abies (L.) H. Karst. At a given early stage of dormancy release, the onset and duration of the bud swelling prior to bud burst are driven by concurrent temperature and photoperiod, while the maximum growth rate is temperature dependent only, except for Fagus, where long photoperiods also increased bud growth rates. Similarly, the later bud burst was controlled by temperature and photoperiod (in the photoperiod sensitive species Fagus, Quercus and Picea). We conclude that photoperiod is involved in the release of dormancy during the ecodormancy phase and may influence bud burst in trees that have experienced sufficient chilling. This study explored and documented the early bud swelling period that precedes and defines later phenological stages such as canopy greening in conventional phenological works. It is the early bud growth resumption that needs to be understood in order to arrive at a causal interpretation and modelling of tree phenology at a large scale. Classic spring phenology events mark visible endpoints of a cascade of processes as evidenced here. PMID:24713858

  6. Photoperiod and temperature responses of bud swelling and bud burst in four temperate forest tree species.

    PubMed

    Basler, David; Körner, Christian

    2014-04-01

    Spring phenology of temperate forest trees is optimized to maximize the length of the growing season while minimizing the risk of freezing damage. The release from winter dormancy is environmentally mediated by species-specific responses to temperature and photoperiod. We investigated the response of early spring phenology to temperature and photoperiod at different stages of dormancy release in cuttings from four temperate tree species in controlled environments. By tracking bud development, we were able to identify the onset of bud swelling and bud growth in Acer pseudoplatanus L., Fagus sylvatica L., Quercus petraea (Mattuschka) Liebl. and Picea abies (L.) H. Karst. At a given early stage of dormancy release, the onset and duration of the bud swelling prior to bud burst are driven by concurrent temperature and photoperiod, while the maximum growth rate is temperature dependent only, except for Fagus, where long photoperiods also increased bud growth rates. Similarly, the later bud burst was controlled by temperature and photoperiod (in the photoperiod sensitive species Fagus, Quercus and Picea). We conclude that photoperiod is involved in the release of dormancy during the ecodormancy phase and may influence bud burst in trees that have experienced sufficient chilling. This study explored and documented the early bud swelling period that precedes and defines later phenological stages such as canopy greening in conventional phenological works. It is the early bud growth resumption that needs to be understood in order to arrive at a causal interpretation and modelling of tree phenology at a large scale. Classic spring phenology events mark visible endpoints of a cascade of processes as evidenced here.

  7. Posttranscriptional Control of Gene Expression in Yeast

    PubMed Central

    McCarthy, John E. G.

    1998-01-01

    Studies of the budding yeast Saccharomyces cerevisiae have greatly advanced our understanding of the posttranscriptional steps of eukaryotic gene expression. Given the wide range of experimental tools applicable to S. cerevisiae and the recent determination of its complete genomic sequence, many of the key challenges of the posttranscriptional control field can be tackled particularly effectively by using this organism. This article reviews the current knowledge of the cellular components and mechanisms related to translation and mRNA decay, with the emphasis on the molecular basis for rate control and gene regulation. Recent progress in characterizing translation factors and their protein-protein and RNA-protein interactions has been rapid. Against the background of a growing body of structural information, the review discusses the thermodynamic and kinetic principles that govern the translation process. As in prokaryotic systems, translational initiation is a key point of control. Modulation of the activities of translational initiation factors imposes global regulation in the cell, while structural features of particular 5′ untranslated regions, such as upstream open reading frames and effector binding sites, allow for gene-specific regulation. Recent data have revealed many new details of the molecular mechanisms involved while providing insight into the functional overlaps and molecular networking that are apparently a key feature of evolving cellular systems. An overall picture of the mechanisms governing mRNA decay has only very recently begun to develop. The latest work has revealed new information about the mRNA decay pathways, the components of the mRNA degradation machinery, and the way in which these might relate to the translation apparatus. Overall, major challenges still to be addressed include the task of relating principles of posttranscriptional control to cellular compartmentalization and polysome structure and the role of molecular channelling

  8. Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveals chaperone-dependent regulation of ribonucleotide reductase

    PubMed Central

    Truman, Andrew W.; Kristjansdottir, Kolbrun; Wolfgeher, Donald; Ricco, Natalia; Mayampurath, Anoop; Volchenboum, Samuel L.; Clotet, Josep; Kron, Stephen J.

    2015-01-01

    The highly conserved molecular chaperones Hsp90 and Hsp70 are indispensible for folding and maturation of a significant fraction of the proteome, including many proteins involved in signal transduction and stress response. To examine the dynamics of chaperone-client interactions after DNA damage, we applied quantitative affinity-purification mass spectrometry (AP-MS) proteomics to characterize interactomes of the yeast Hsp70 isoform Ssa1 and Hsp90 isoform Hsp82 before and after exposure to methyl methanesulfonate. Of 256 proteins identified and quantified via 16O/18O labeling and LC-MS/MS, 142 are novel Hsp70/90 interactors. Nearly all interactions remained unchanged or decreased after DNA damage, but 5 proteins increased interactions with Ssa1 and/or Hsp82, including the ribonucleotide reductase (RNR) subunit Rnr4. Inhibiting Hsp70 or 90 chaperone activity destabilized Rnr4 in yeast and its vertebrate homolog hRMM2 in breast cancer cells. In turn, pre-treatment of cancer cells with chaperone inhibitors sensitized cells to the RNR inhibitor gemcitabine, suggesting a novel chemotherapy strategy. All MS data have been deposited in the ProteomeXchange with identifier PXD001284. PMID:25452130

  9. Proteogenomic Discovery of a Small, Novel Protein in Yeast Reveals a Strategy for the Detection of Unannotated Short Open Reading Frames.

    PubMed

    Yagoub, Daniel; Tay, Aidan P; Chen, Zhiliang; Hamey, Joshua J; Cai, Curtis; Chia, Samantha Z; Hart-Smith, Gene; Wilkins, Marc R

    2015-12-01

    In recent years, proteomic data have contributed to genome annotation efforts, most notably in humans and mice, and spawned a field termed "proteogenomics". Yeast, in contrast with higher eukaryotes, has a small genome, which has lent itself to simpler ORF prediction. Despite this, continual advances in mass spectrometry suggest that proteomics should be able to improve genome annotation even in this well-characterized species. Here we applied a proteogenomics workflow to yeast to identify novel protein-coding genes. Specific databases were generated, from intergenic regions of the genome, which were then queried with MS/MS data. This suggested the existence of several putative novel ORFs of <100 codons, one of which we chose to validate. Synthetic peptides, RNA-Seq analysis, and evidence of evolutionary conservation allowed for the unequivocal definition of a new protein of 78 amino acids encoded on chromosome X, which we dub YJR107C-A. It encodes a new type of domain, which ab initio modeling suggests as predominantly α-helical. We show that this gene is nonessential for growth; however, deletion increases sensitivity to osmotic stress. Finally, from the above discovery process, we discuss a generalizable strategy for the identification of short ORFs and small proteins, many of which are likely to be undiscovered. PMID:26554900

  10. Multiway real-time PCR gene expression profiling in yeast Saccharomyces cerevisiae reveals altered transcriptional response of ADH-genes to glucose stimuli

    PubMed Central

    Ståhlberg, Anders; Elbing, Karin; Andrade-Garda, José Manuel; Sjögreen, Björn; Forootan, Amin; Kubista, Mikael

    2008-01-01

    Background The large sensitivity, high reproducibility and essentially unlimited dynamic range of real-time PCR to measure gene expression in complex samples provides the opportunity for powerful multivariate and multiway studies of biological phenomena. In multiway studies samples are characterized by their expression profiles to monitor changes over time, effect of treatment, drug dosage etc. Here we perform a multiway study of the temporal response of four yeast Saccharomyces cerevisiae strains with different glucose uptake rates upon altered metabolic conditions. Results We measured the expression of 18 genes as function of time after addition of glucose to four strains of yeast grown in ethanol. The data are analyzed by matrix-augmented PCA, which is a generalization of PCA for 3-way data, and the results are confirmed by hierarchical clustering and clustering by Kohonen self-organizing map. Our approach identifies gene groups that respond similarly to the change of nutrient, and genes that behave differently in mutant strains. Of particular interest is our finding that ADH4 and ADH6 show a behavior typical of glucose-induced genes, while ADH3 and ADH5 are repressed after glucose addition. Conclusion Multiway real-time PCR gene expression profiling is a powerful technique which can be utilized to characterize functions of new genes by, for example, comparing their temporal response after perturbation in different genetic variants of the studied subject. The technique also identifies genes that show perturbed expression in specific strains. PMID:18412983

  11. Virus Budding and the ESCRT Pathway

    PubMed Central

    Votteler, Jörg; Sundquist, Wesley I.

    2013-01-01

    Enveloped viruses escape infected cells by budding through limiting membranes. In the decade since the discovery that the Human Immunodeficiency Virus (HIV) recruits cellular ESCRT (endosomal sorting complexes required for transport) machinery to facilitate viral budding, this pathway has emerged as the major escape route for enveloped viruses. In cells, the ESCRT pathway catalyzes the analogous membrane fission events required for the abscission stage of cytokinesis and for a series of “reverse topology” vesiculation events. Studies of enveloped virus budding are therefore providing insights into the complex cellular mechanisms of cell division and membrane protein trafficking (and vice versa). Here, we review how viruses mimic cellular recruiting signals to usurp the ESCRT pathway, discuss mechanistic models for ESCRT pathway functions, and highlight important research frontiers. PMID:24034610

  12. Dental cell sheet biomimetic tooth bud model.

    PubMed

    Monteiro, Nelson; Smith, Elizabeth E; Angstadt, Shantel; Zhang, Weibo; Khademhosseini, Ali; Yelick, Pamela C

    2016-11-01

    Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) - dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 10(6)/cm(2)) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation.

  13. Dental cell sheet biomimetic tooth bud model.

    PubMed

    Monteiro, Nelson; Smith, Elizabeth E; Angstadt, Shantel; Zhang, Weibo; Khademhosseini, Ali; Yelick, Pamela C

    2016-11-01

    Tissue engineering and regenerative medicine technologies offer promising