scholarly journals Nuclear envelope morphology constrains diffusion and promotes asymmetric protein segregation in closed mitosis

2012 ◽  
Vol 197 (7) ◽  
pp. 921-937 ◽  
Author(s):  
Barbara Boettcher ◽  
Tatiana T. Marquez-Lago ◽  
Mathias Bayer ◽  
Eric L. Weiss ◽  
Yves Barral
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Vegetative Growth ◽  
In Silico ◽  
Mother Cell ◽  
Daughter Cell ◽  
Diffusion Barriers ◽  
Yeast Cells ◽  
Nuclear Membranes ◽  
In Silico Modeling

During vegetative growth, Saccharomyces cerevisiae cells divide asymmetrically: the mother cell buds to produce a smaller daughter cell. This daughter asymmetrically inherits the transcription factor Ace2, which activates daughter-specific transcriptional programs. In this paper, we investigate when and how this asymmetry is established and maintained. We show that Ace2 asymmetry is initiated in the elongated, but undivided, anaphase nucleus. At this stage, the nucleoplasm was highly compartmentalized; little exchange was observed for nucleoplasmic proteins between mother and bud. Using photobleaching and in silico modeling, we show that diffusion barriers compartmentalize the nuclear membranes. In contrast, the behavior of proteins in the nucleoplasm is well explained by the dumbbell shape of the anaphase nucleus. This compartmentalization of the nucleoplasm promoted Ace2 asymmetry in anaphase nuclei. Thus, our data indicate that yeast cells use the process of closed mitosis and the morphological constraints associated with it to asymmetrically segregate nucleoplasmic components.

scholarly journals Combined in silico and 19F NMR analysis of 5-fluorouracil metabolism in yeast at low ATP conditions

2019 ◽  
Vol 39 (12) ◽  
Author(s):  
Piotr H. Pawłowski ◽  
Paweł Szczęsny ◽  
Bożenna Rempoła ◽  
Anna Poznańska ◽  
Jarosław Poznański
Keyword(s):  
Oxidative Stress ◽  
Saccharomyces Cerevisiae ◽  
In Silico ◽  
31P Nmr ◽  
Yeast Cells ◽  
Atp Concentration ◽  
In Silico Modeling ◽  
Intracellular Metabolism ◽  
Hydrolysis Of ◽  
Careful Design

Abstract The cytotoxic effect of 5-fluorouracil (5-FU) on yeast cells is thought to be mainly via a misincorporation of fluoropyrimidines into both RNA and DNA, not only DNA damage via inhibition of thymidylate synthase (TYMS) by fluorodeoxyuridine monophosphate (FdUMP). However, some studies on Saccharomyces cerevisiae show a drastic decrease in ATP concentration under oxidative stress, together with a decrease in concentration of other tri- and diphosphates. This raises a question if hydrolysis of 5-fluoro-2-deoxyuridine diphosphate (FdUDP) under oxidative stress could not lead to the presence of FdUMP and the activation of so-called ‘thymine-less death’ route. We attempted to answer this question with in silico modeling of 5-FU metabolic pathways, based on new experimental results, where the stages of intracellular metabolism of 5-FU in Saccharomyces cerevisiae were tracked by a combination of 19F and 31P NMR spectroscopic study. We have identified 5-FU, its nucleosides and nucleotides, and subsequent di- and/or triphosphates. Additionally, another wide 19F signal, assigned to fluorinated unstructured short RNA, has been also identified in the spectra. The concentration of individual metabolites was found to vary substantially within hours, however, the initial steady-state was preserved only for an hour, until the ATP concentration dropped by a half, which was monitored independently via 31P NMR spectra. After that, the catabolic process leading from triphosphates through monophosphates and nucleosides back to 5-FU was observed. These results imply careful design and interpretation of studies in 5-FU metabolism in yeast.

Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae

1988 ◽  
Vol 8 (9) ◽  
pp. 3717-3725
Author(s):  
M Kornuc ◽  
R Altman ◽  
D Harrich ◽  
J Garcia ◽  
J Chao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Binding Domains ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Transactivator Protein

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

scholarly journals Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae

2010 ◽  
Vol 9 (6) ◽  
pp. 943-951 ◽  
Author(s):  
Masafumi Nishizawa ◽  
Mirai Tanigawa ◽  
Michio Hayashi ◽  
Tatsuya Maeda ◽  
Yoshiaki Yazaki ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Cellular Response ◽  
Proteolytic Processing ◽  
Yeast Cells ◽  
Nuclear Accumulation ◽  
Alkaline Stress ◽  
Content Type ◽  
Its Gene ◽  
Alkaline Conditions

ABSTRACT The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to changing environmental conditions. The Pho85 kinase, one of the yeast cyclin-dependent kinases (CDK), is known to play an important role in the cellular response to alterations in parameters such as nutrient levels and salinity. Several genes whose expression is regulated, either directly or indirectly, by the Rim101 transcription factor become constitutively activated when Pho85 function is absent,. Because Rim101 is responsible for adaptation to alkaline conditions, this observation suggests an interaction between Pho85 and Rim101 in the response to alkaline stress. We have found that Pho85 affects neither RIM101 transcription, the proteolytic processing that is required for Rim101 activation, nor Rim101 stability. Rather, Pho85 regulates the nuclear accumulation of active Rim101, possibly via phosphorylation. Additionally, we report that Pho85 and the transcription factor Pho4 are necessary for adaptation to alkaline conditions and that PTK2 activation by Pho4 is involved in this process. These findings illustrate novel roles for the regulators of the PHO system when yeast cells cope with various environmental stresses potentially threatening their survival.

Mutations in a small region of the exportin Crm1p disrupt the daughter cell-specific nuclear localization of the transcription factor Ace2p in Saccharomyces cerevisiae

2008 ◽  
Vol 100 (6) ◽  
pp. 343-354 ◽  
Author(s):  
Myriam Bourens ◽  
Waldemar Racki ◽  
Anne-Marie Bécam ◽  
Cristina Panozzo ◽  
Séverine Boulon ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Nuclear Localization ◽  
Daughter Cell ◽  
Small Region

scholarly journals SPINDLES, SPINDLE PLAQUES, AND MEIOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE (HANSEN)

1971 ◽  
Vol 50 (2) ◽  
pp. 344-361 ◽  
Author(s):  
Peter B. Moens ◽  
Ellen Rapport
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Envelope ◽  
Spore Wall ◽  
Yeast Cells ◽  
Sporulation Medium ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nuclear Membranes ◽  
Cytoplasmic Side ◽  
Opaque Body ◽  
Meiosis I

The intranuclear spindle of yeast has an electron-opaque body at each pole. These spindle plaques lie on the nuclear envelope. During mitosis the spindle elongates while the nuclear membranes remain intact. After equatorial constriction there are two daughted nuclei, each with one spindle plaque. The spindle plaque then duplicates so that two side-by-side plaques are produced. These move rapidly apart and rotate so that they bracket a stable 0.8 µm spindle. Later, during mitosis, this spindle elongates, etc. Yeast cells placed on sporulation medium soon enter meiosis. After 4 hr the spindle plaques of the more mature cells duplicate, producing a stable side-by-side arrangement. Subsequently the plaques move apart to bracket a 0.8 µm spindle which immediately starts to elongate. When this meiosis I spindle reaches its maximum length of 3–5 µm, each of the plaques at the poles of the spindle duplicates and the resulting side-by-side plaques increase in size. The nucleus does not divide. The large side-by-side plaques separate and bracket a short spindle of about 1 µm which elongates gradually to 2 or 3 µm. Thus there are two spindles within one nucleus at meiosis II. To the side of each of the four plaques a bulge forms on the nucleus. The four bulges enlarge while the original nucleus shrinks. These four developing ascospore nuclei are partially surrounded by cytoplasm and by a prospore wall which originates from the cytoplasmic side of the spindle plaque. Eventually the spore nuclei pinch off and the spore wall closes. In some of the larger yeast cells this development is completed after 8 hr on sporulation medium.

scholarly journals Adenovirus transcriptional regulatory regions are conserved in mammalian cells and Saccharomyces cerevisiae.

1988 ◽  
Vol 8 (9) ◽  
pp. 3717-3725 ◽  
Author(s):  
M Kornuc ◽  
R Altman ◽  
D Harrich ◽  
J Garcia ◽  
J Chao ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Transcriptional Regulation ◽  
Binding Sites ◽  
Mammalian Cells ◽  
Yeast Cells ◽  
Binding Domains ◽  
Cell Extracts ◽  
Dnase I Footprinting ◽  
Transactivator Protein

The adenovirus early region 3 (E3) promoter is an early viral promoter which is strongly induced by the adenovirus transactivator protein E1A. DNase I footprinting with HeLa cell extracts has identified four factor-binding domains which appear to be involved in basal and E1A-induced transcriptional regulation. These binding domains may bind TATA region-binding factors (site I), the CREB/ATF protein (site II), the AP-1 protein (site III), and nuclear factor I/CTF (site IV). Recently, it has been shown that the DNA-binding domain of transcription factor AP-1 has homology with the yeast transcription factor GCN4 and that the yeast transactivator protein GAL4 is able to stimulate transcription in HeLa cells from promoters containing GAL4-binding sites. These results suggest an evolutionary conservation of both transcription factors and the mechanisms responsible for transcriptional activation in Saccharomyces cerevisiae and higher eucaryotic organisms. To determine whether similar patterns of transcriptional regulation were seen with the E3 promoter in HeLa and yeast cells, the E3 promoter fused to the chloramphenicol acetyltransferase (cat) gene was cloned into a high-copy-number plasmid and stably introduced into yeast cells. S1 analysis revealed that similar E3 promoter mRNA start sites were found in yeast and HeLa cells. DNase I footprinting with partially purified yeast extracts revealed that four regions of the E3 promoter were protected. Several of these regions were similar to binding sites determined by using HeLa cell extracts. Oligonucleotide mutagenesis of these binding domains indicated their importance in the transcriptional regulation of the E3 promoter in yeast cells. These results suggest that similar cellular transcription factor-binding sites may be involved in the regulation of promoters in both yeast and mammalian cells.

scholarly journals Involvement of the Pleiotropic Drug Resistance Response, Protein Kinase C Signaling, and Altered Zinc Homeostasis in Resistance of Saccharomyces cerevisiae to Diclofenac

2011 ◽  
Vol 77 (17) ◽  
pp. 5973-5980 ◽  
Author(s):  
Jolanda S. van Leeuwen ◽  
Nico P. E. Vermeulen ◽  
J. Chris Vos
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Cell Wall ◽  
Transcription Factor ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Zinc Homeostasis ◽  
Yeast Cells ◽  
Pleiotropic Drug Resistance ◽  
Kinase C

ABSTRACTDiclofenac is a widely used analgesic drug that can cause serious adverse drug reactions. We usedSaccharomyces cerevisiaeas a model eukaryote with which to elucidate the molecular mechanisms of diclofenac toxicity and resistance. Although most yeast cells died during the initial diclofenac treatment, some survived and started growing again. Microarray analysis of the adapted cells identified three major processes involved in diclofenac detoxification and tolerance. In particular, pleiotropic drug resistance (PDR) genes and genes under the control of Rlm1p, a transcription factor in the protein kinase C (PKC) pathway, were upregulated in diclofenac-adapted cells. We tested if these processes or pathways were directly involved in diclofenac toxicity or resistance. Of the pleiotropic drug resistance gene products, the multidrug transporter Pdr5p was crucially important for diclofenac tolerance. Furthermore, deletion of components of the cell wall stress-responsive PKC pathway increased diclofenac toxicity, whereas incubation of cells with the cell wall stressor calcofluor white before the addition of diclofenac decreased its toxicity. Also, diclofenac induced flocculation, which might trigger the cell wall alterations. Genes involved in ribosome biogenesis and rRNA processing were downregulated, as were zinc-responsive genes. Paradoxically, deletion of the zinc-responsive transcription factor Zap1p or addition of the zinc chelator 1,10-phenanthroline significantly increased diclofenac toxicity, establishing a regulatory role for zinc in diclofenac resistance. In conclusion, we have identified three new pathways involved in diclofenac tolerance in yeast, namely, Pdr5p as the main contributor to the PDR response, cell wall signaling via the PKC pathway, and zinc homeostasis, regulated by Zap1p.

scholarly journals Ddi1 is a ubiquitin-dependent protease

2020 ◽  
Vol 117 (14) ◽  
pp. 7776-7781 ◽  
Author(s):  
Matthew C. J. Yip ◽  
Nicholas O. Bodnar ◽  
Tom A. Rapoport
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcription Factor ◽  
Enzymatic Activity ◽  
Yeast Cells ◽  
Polyubiquitin Chain ◽  
High Affinity ◽  
Helical Domain ◽  
Higher Eukaryotes ◽  
Affinity Interaction ◽  
Substrate Proteins

TheSaccharomyces cerevisiaeprotein Ddi1 and its homologs in higher eukaryotes have been proposed to serve as shuttling factors that deliver ubiquitinated substrates to the proteasome. Although Ddi1 contains both ubiquitin-interacting UBA and proteasome-interacting UBL domains, the UBL domain is atypical, as it binds ubiquitin. Furthermore, unlike other shuttling factors, Ddi1 and its homologs contain a conserved helical domain (helical domain of Ddi1, HDD) and a retroviral-like protease (RVP) domain. The RVP domain is probably responsible for cleavage of the precursor of the transcription factor Nrf1 in higher eukaryotes, which results in the up-regulation of proteasomal subunit genes. However, enzymatic activity of the RVP domain has not yet been demonstrated, and the function of Ddi1 remains poorly understood. Here, we show that Ddi1 is a ubiquitin-dependent protease, which cleaves substrate proteins only when they are tagged with long ubiquitin chains (longer than about eight ubiquitins). The RVP domain is inactive in isolation, in contrast to its retroviral counterpart. Proteolytic activity of Ddi1 requires the HDD domain and is stimulated by the UBL domain, which mediates high-affinity interaction with the polyubiquitin chain. Compromising the activity of Ddi1 in yeast cells results in the accumulation of polyubiquitinated proteins. Aside from the proteasome, Ddi1 is the only known endoprotease that acts on polyubiquitinated substrates. Ddi1 and its homologs likely cleave polyubiquitinated substrates under conditions where proteasome function is compromised.

scholarly journals High-Level Production of Lysine in the Yeast Saccharomyces cerevisiae by Rational Design of Homocitrate Synthase

2021 ◽  
Author(s):  
Shota Isogai ◽  
Tomonori Matsushita ◽  
Hiroyuki Imanishi ◽  
Jirasin Koonthongkaew ◽  
Yoichi Toyokawa ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
In Silico ◽  
Feedback Inhibition ◽  
Yeast Cells ◽  
Enzymatic Analysis ◽  
In Silico Docking ◽  
Key Enzyme ◽  
Homocitrate Synthase ◽  
High Level ◽  
Level Production

Homocitrate synthase (HCS) catalyzes the aldol condensation of 2-oxoglutarate (2-OG) and acetyl coenzyme A (AcCoA) to form homocitrate, which is the first enzyme of the lysine biosynthetic pathway in the yeast Saccharomyces cerevisiae. The HCS activity is tightly regulated via feedback inhibition by the end product lysine. Here, we designed a feedback inhibition-insensitive HCS of S. cerevisiae (ScLys20) for high-level production of lysine in yeast cells. In silico docking of the substrate 2-OG and the inhibitor lysine to ScLys20 predicted that the substitution of serine to glutamate at position 385 would be more suitable for desensitization of the lysine feedback inhibition than the substitution from serine to phenylalanine in the already-known variant Ser385Phe. Enzymatic analysis revealed that the Ser385Glu variant is far more insensitive to feedback inhibition than the Ser385Phe variant. We also found that the lysine content in yeast cells expressing the Ser385Glu variant was 4.62-fold and 1.47-fold higher than that of cells expressing the wild-type HCS and Ser385Phe variant, respectively, due to the extreme desensitization to feedback inhibition. In this study, we obtained highly feedback inhibition-insensitive HCS using in silico docking and enzymatic analysis. Our results indicate that the rational engineering of HCS for feedback-inhibition desensitization by lysine and could be useful for constructing new yeast strains with higher lysine productivity. IMPORTANCE A traditional method for screening toxic analogue-resistant mutants has been established for the breeding of microbes that produce high levels of amino acids, including lysine. However, another efficient strategy is required to further improve their productivity. Homocitrate synthase (HCS) catalyzes the first step of lysine biosynthesis in the yeast Saccharomyces cerevisiae, and its activity is subject to feedback inhibition by lysine. Here, in silico design of a key enzyme that regulates the biosynthesis of lysine was utilized to increase the productivity of lysine. We designed HCS for the high level production of lysine in yeast cells by in silico docking simulation. The engineered HCS exhibited much less sensitivity to lysine and conferred higher production of lysine than the already-known variant obtained by traditional breeding. The combination of in silico design and experimental analysis of a key enzyme will contribute to advances in metabolic engineering for the construction of industrial microorganisms.

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