93 results on '"Diauxic shift"'
Search Results
2. Xrn1 Exoribonuclease—An Intrinsic Marker of Yeast Population Growth
- Author
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Tomas Grousl and Tomas Vomastek
- Subjects
xrn1 ,p-bodies ,eisosomes ,diauxic shift ,yeast ,Environmental sciences ,GE1-350 ,Microbiology ,QR1-502 - Abstract
Background: Xrn1 exoribonuclease is the major mRNA degradation enzyme in Saccharomyces cerevisiae. In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown. Methods: We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δxrn1 cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy. Results: We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy. Conclusions: The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated.
- Published
- 2024
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- View/download PDF
3. Natural Variation in Diauxic Shift between Patagonian Saccharomyces eubayanus Strains
- Author
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Jennifer Molinet, Juan I. Eizaguirre, Pablo Quintrel, Nicolás Bellora, Carlos A. Villarroel, Pablo Villarreal, José Benavides-Parra, Roberto F. Nespolo, Diego Libkind, and Francisco A. Cubillos
- Subjects
Saccharomyces eubayanus ,wild strains ,beer ,RNA-seq ,ATAC-seq ,diauxic shift ,Microbiology ,QR1-502 - Abstract
ABSTRACT The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ, hap5Δ, and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.
- Published
- 2022
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4. H3 Lysine 4 Methylation Is Required for Full Activation of Genes Involved in α-Ketoglutarate Availability in the Nucleus of Yeast Cells after Diauxic Shift
- Author
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Elena Di Nisio, Svetlana Danovska, Livia Condemi, Angela Cirigliano, Teresa Rinaldi, Valerio Licursi, and Rodolfo Negri
- Subjects
H3K4 tri-methylation ,diauxic shift ,transcriptional regulation ,Microbiology ,QR1-502 - Abstract
We show that in S. cerevisiae the metabolic diauxic shift is associated with a H3 lysine 4 tri-methylation (H3K4me3) increase which involves a significant fraction of transcriptionally induced genes which are required for the metabolic changes, suggesting a role for histone methylation in their transcriptional regulation. We show that histone H3K4me3 around the start site correlates with transcriptional induction in some of these genes. Among the methylation-induced genes are IDP2 and ODC1, which regulate the nuclear availability of α-ketoglutarate, which, as a cofactor for Jhd2 demethylase, regulates H3K4 tri-methylation. We propose that this feedback circuit could be used to regulate the nuclear α-ketoglutarate pool concentration. We also show that yeast cells adapt to the absence of Jhd2 by decreasing Set1 methylation activity.
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- 2023
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5. Data-driven multiscale modeling reveals the role of metabolic coupling for the spatio-temporal growth dynamics of yeast colonies
- Author
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Jukka Intosalmi, Adrian C. Scott, Michelle Hays, Nicholas Flann, Olli Yli-Harja, Harri Lähdesmäki, Aimée M. Dudley, and Alexander Skupin
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Multicellular systems ,Multiscale modeling ,Yeast colony ,Metabolic coupling ,Diauxic shift ,Markov chain Monte Carlo ,Cytology ,QH573-671 - Abstract
Abstract Background Multicellular entities like mammalian tissues or microbial biofilms typically exhibit complex spatial arrangements that are adapted to their specific functions or environments. These structures result from intercellular signaling as well as from the interaction with the environment that allow cells of the same genotype to differentiate into well-organized communities of diversified cells. Despite its importance, our understanding how this cell–cell and metabolic coupling lead to functionally optimized structures is still limited. Results Here, we present a data-driven spatial framework to computationally investigate the development of yeast colonies as such a multicellular structure in dependence on metabolic capacity. For this purpose, we first developed and parameterized a dynamic cell state and growth model for yeast based on on experimental data from homogeneous liquid media conditions. The inferred model is subsequently used in a spatially coarse-grained model for colony development to investigate the effect of metabolic coupling by calibrating spatial parameters from experimental time-course data of colony growth using state-of-the-art statistical techniques for model uncertainty and parameter estimations. The model is finally validated by independent experimental data of an alternative yeast strain with distinct metabolic characteristics and illustrates the impact of metabolic coupling for structure formation. Conclusions We introduce a novel model for yeast colony formation, present a statistical methodology for model calibration in a data-driven manner, and demonstrate how the established model can be used to generate predictions across scales by validation against independent measurements of genetically distinct yeast strains.
- Published
- 2019
- Full Text
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6. Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift.
- Author
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Di Bartolomeo, Francesca, Malina, Carl, Campbell, Kate, Mormino, Maurizio, Fuchs, Johannes, Vorontsov, Egor, Gustafsson, Claes M., and Nielsen, Jens
- Subjects
- *
CELL respiration , *YEAST , *BIOSYNTHESIS , *SACCHAROMYCES cerevisiae , *MITOCHONDRIAL physiology - Abstract
Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
7. Data-driven multiscale modeling reveals the role of metabolic coupling for the spatio-temporal growth dynamics of yeast colonies.
- Author
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Intosalmi, Jukka, Scott, Adrian C., Hays, Michelle, Flann, Nicholas, Yli-Harja, Olli, Lähdesmäki, Harri, Dudley, Aimée M., and Skupin, Alexander
- Subjects
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MONTE Carlo method , *MULTISCALE modeling , *YEAST , *SPATIAL arrangement , *MARKOV chain Monte Carlo - Abstract
Background: Multicellular entities like mammalian tissues or microbial biofilms typically exhibit complex spatial arrangements that are adapted to their specific functions or environments. These structures result from intercellular signaling as well as from the interaction with the environment that allow cells of the same genotype to differentiate into well-organized communities of diversified cells. Despite its importance, our understanding how this cell-cell and metabolic coupling lead to functionally optimized structures is still limited. Results: Here, we present a data-driven spatial framework to computationally investigate the development of yeast colonies as such a multicellular structure in dependence on metabolic capacity. For this purpose, we first developed and parameterized a dynamic cell state and growth model for yeast based on on experimental data from homogeneous liquid media conditions. The inferred model is subsequently used in a spatially coarse-grained model for colony development to investigate the effect of metabolic coupling by calibrating spatial parameters from experimental time-course data of colony growth using state-of-the-art statistical techniques for model uncertainty and parameter estimations. The model is finally validated by independent experimental data of an alternative yeast strain with distinct metabolic characteristics and illustrates the impact of metabolic coupling for structure formation. Conclusions: We introduce a novel model for yeast colony formation, present a statistical methodology for model calibration in a data-driven manner, and demonstrate how the established model can be used to generate predictions across scales by validation against independent measurements of genetically distinct yeast strains. [ABSTRACT FROM AUTHOR]
- Published
- 2019
8. Xrn1 Exoribonuclease-An Intrinsic Marker of Yeast Population Growth.
- Author
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Grousl T and Vomastek T
- Subjects
- Population Growth, RNA, Messenger metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Exoribonucleases genetics, Exoribonucleases metabolism
- Abstract
Background: Xrn1 exoribonuclease is the major mRNA degradation enzyme in Saccharomyces cerevisiae. In exponentially growing cells, Xrn1 is localised in the yeast cells and directs the degradation of mRNA molecules. Xrn1 is gradually deposited and presumably inactivated in the processing bodies (P-bodies) as the yeast population ages. Xrn1 can also localise to the membrane compartment of the arginine permease Can1/eisosome compartment at the yeast plasma membrane. This localisation correlates with the metabolic (diauxic) shift from glucose fermentation to respiration, although the relevance of this Xrn1 localisation remains unknown., Methods: We monitored the growth rates and morphology of Xrn1-green fluorescent protein (GFP) cells compared to wild-type and Δ xrn1 cells and observed the Xrn1-GFP localisation pattern in different media types for up to 72 hours using fluorescence microscopy., Results: We present the dynamic changes in the localisation of Xrn1 as a versatile tool for monitoring the growth of yeast populations at the single-cell level using fluorescence microscopy., Conclusions: The dynamic changes in the localisation of Xrn1 can be a versatile tool for monitoring the growth of yeast populations at the single-cell level. Simultaneously, Xrn1 localisation outside of P-bodies in post-diauxic cells supports its storage and cytoprotective function, yet the role of P-bodies in cell metabolism has still not yet been entirely elucidated., Competing Interests: The authors declare no conflict of interest., (© 2024 The Author(s). Published by IMR Press.)
- Published
- 2024
- Full Text
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9. Systems Biology: Developments and Applications
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Kumar, Rahul, Lahtvee, Petri-Jaan, Nielsen, Jens, Piškur, Jure, editor, and Compagno, Concetta, editor
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- 2014
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10. Closed-loop cycles of experiment design, execution, and learning accelerate systems biology model development in yeast.
- Author
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Coutant, Anthony, Roper, Katherine, Trejo-Banos, Daniel, Bouthinon, Dominique, Carpenter, Martin, Grzebyta, Jacek, Santini, Guillaume, Soldano, Henry, Elati, Mohamed, Ramon, Jan, Rouveirol, Celine, Soldatova, Larisa N., and King, Ross D.
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SYSTEMS biology , *DEVELOPMENTAL biology , *EXPERIMENTAL design , *CELL transformation , *SEMANTIC Web - Abstract
One of the most challenging tasks in modern science is the development of systems biology models: Existing models are often very complex but generally have low predictive performance. The construction of high-fidelity models will require hundreds/thousands of cycles of model improvement, yet few current systems biology research studies complete even a single cycle. We combined multiple software tools with integrated laboratory robotics to execute three cycles of model improvement of the prototypical eukaryotic cellular transformation, the yeast (Saccharomyces cerevisiae) diauxic shift. In the first cycle, a model outperforming the best previous diauxic shift model was developed using bioinformatic and systems biology tools. In the second cycle, the model was further improved using automatically planned experiments. In the third cycle, hypothesis-led experiments improved the model to a greater extent than achieved using high-throughput experiments. All of the experiments were formalized and communicated to a cloud laboratory automation system (Eve) for automatic execution, and the results stored on the semantic web for reuse. The final model adds a substantial amount of knowledge about the yeast diauxic shift: 92 genes (+45%), and 1,048 interactions (+147%). This knowledge is also relevant to understanding cancer, the immune system, and aging. We conclude that systems biology software tools can be combined and integrated with laboratory robots in closed-loop cycles. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
11. Monitoring ADP/ATP ratio in yeast cells using the fluorescent-protein reporter PercevalHR.
- Author
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Nguyen, Phuong Thi Mai, Ishiwata-Kimata, Yuki, and Kimata, Yukio
- Subjects
- *
ADENOSINE diphosphate , *YEAST , *FLUORESCENT proteins - Abstract
PercevalHR (Perceval High Resolution) is an artificially designed fluorescent protein, which changes its excitation spectrum based on the ADP/ATP ratio of the environment. Here we demonstrated that PercevalHR can be used for monitoring energy status of Saccharomyces cerevisiae cells, which are affected by diauxic shift and mitochondria inhibition, in a non-invasive and non-destructive manner. Yeast cells producing PercevalHR emit green fluorescence, excitation spectrum of which varies dependently on cellular ATP/ATP ratio. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
12. Induction and relocalization of telomeric repeat-containing RNAs during diauxic shift in budding yeast.
- Author
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Perez-Romero, Carmina Angelica, Lalonde, Maxime, Chartrand, Pascal, and Cusanelli, Emilio
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TELOMERES , *HETEROCHROMATIC genes , *SACCHAROMYCES cerevisiae , *NON-coding RNA , *FLUORESCENCE in situ hybridization - Abstract
Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response. [ABSTRACT FROM AUTHOR]
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- 2018
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13. Gene Regulatory Networks
- Author
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Dewey, T. Gregory, Galas, David J., Koonin, Eugene V., Wolf, Yuri I., and Karev, Georgy P.
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- 2006
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14. Improving the Identification of Differentially Expressed Genes in cDNA Microarray Experiments
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Ultsch, Alfred, Bock, H.-H., editor, Gaul, W., editor, Vichi, M., editor, Arabie, Ph., editor, Baier, D., editor, Critchley, F., editor, Decker, R., editor, Diday, E., editor, Greenacre, M., editor, Lauro, C., editor, Meulman, J., editor, Monari, P., editor, Nishisato, S., editor, Ohsumi, N., editor, Opitz, O., editor, Ritter, G., editor, Schader, M., editor, Weihs, C., editor, Weihs, Claus, editor, and Gaul, Wolfgang, editor
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- 2005
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15. The Proteasome Lid Triggers COP9 Signalosome Activity during the Transition of Sachharomyces cerevisiae Cells into Quiescence
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Laylan Bramasole, Abhishek Sinha, Dana Harshuk, Angela Cirigliano, Gurevich Sylvia, Zanlin Yu, Rinat Lift Carmeli, Michael H. Glickman, Teresa Rinaldi, and Elah Pick
- Subjects
26S proteasome ,proteasome lid ,Rpn11 ,Cdc53 ,Cullin ,SCF (Skp, Cullin, F-box containing complex) ,NEDD8 (neural precursor cell expressed developmentally down-regulated 8) ,Rub1 (Related ubiquitin 1) ,CSN (COP9 signalosome) ,Saccharomyces cerevisiae ,diauxic shift ,budding yeast ,Microbiology ,QR1-502 - Abstract
The class of Cullin−RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8−Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1−CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53−Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes.
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- 2019
- Full Text
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16. Oxidative stress responses in yeast
- Author
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Toledano, Michel B., Delaunay, Agnes, Biteau, Benoit, Spector, Daniel, Azevedo, Dulce, Hohmann, Stefan, editor, and Mager, Willem H., editor
- Published
- 2003
- Full Text
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17. Gene Expression Data Mining and Analysis
- Author
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Brazma, Alvis, Robinson, Alan, Vilo, Jaak, and Jordan, B. R., editor
- Published
- 2001
- Full Text
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18. Caloric Restriction Extends Yeast Chronological Life Span by Optimizing the Snf1 (AMPK) Signaling Pathway.
- Author
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Wierman, Margaret B., Maqani, Nazif, Strickler, Erika, Mingguang Li, and Smith, Jeffrey S.
- Subjects
- *
PROTEIN kinases , *ADENOSINE monophosphate , *YEAST , *LIFE spans , *ENERGY metabolism , *LOW-calorie diet - Abstract
AMP-activated protein kinase (AMPK) and the homologous yeast SNF1 complex are key regulators of energy metabolism that counteract nutrient deficiency and ATP depletion by phosphorylating multiple enzymes and transcription factors that maintain energetic homeostasis. AMPK/SNF1 also promotes longevity in several model organisms, including yeast. Here we investigate the role of yeast SNF1 in mediating the extension of chronological life span (CLS) by caloric restriction (CR). We find that SNF1 activity is required throughout the transition of log phase to stationary phase (diauxic shift) for effective CLS extension. CR expands the period of maximal SNF1 activation beyond the diauxic shift, as indicated by Sak1-dependent T210 phosphorylation of the Snf1 catalytic α-subunit. A concomitant increase in ADP is consistent with SNF1 activation by ADP in vivo. Downstream of SNF1, the Cat8 and Adr1 transcription factors are required for full CR-induced CLS extension, implicating an alternative carbon source utilization for acetyl coenzyme A (acetyl-CoA) production and gluconeogenesis. Indeed, CR increased acetyl-CoA levels during the diauxic shift, along with expression of both acetyl-CoA synthetase genes ACS1 and ACS2. We conclude that CR maximizes Snf1 activity throughout and beyond the diauxic shift, thus optimizing the coordination of nucleocytosolic acetyl-CoA production with massive reorganization of the transcriptome and respiratory metabolism. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
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19. Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift
- Author
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Egor Vorontsov, Francesca Di Bartolomeo, Maurizio Mormino, Kate Campbell, Claes M. Gustafsson, Johannes Fuchs, Jens Nielsen, and Carl Malina
- Subjects
Saccharomyces cerevisiae Proteins ,Proteome ,Cellular respiration ,Saccharomyces cerevisiae ,Cell Respiration ,Mitochondrion ,Mass Spectrometry ,Mitochondrial Proteins ,chemistry.chemical_compound ,Biosynthesis ,Gene Expression Regulation, Fungal ,Multidisciplinary ,biology ,Ethanol ,Chemistry ,Systems Biology ,Absolute proteomics ,Diauxic shift ,diauxic shift ,Metabolism ,Biological Sciences ,biology.organism_classification ,Yeast ,Mitochondria ,Glucose ,Biochemistry ,Fermentation ,absolute proteomics ,Function (biology) - Abstract
Significance This work offers a unique portrayal of yeast mitochondria through the characterization of its absolute proteome. The study of biophysical changes in the mitochondrial network associated with proteome profiling, throughout yeast growth and the transition from fermentative to respiratory metabolism, lays out the crucial role this organelle has in balancing the overall metabolic status of the cell. Using proteomic mass spectrometry, state of the art fluorescence microscopy, and lipidomics analysis, these data provide a highly quantitative description of key mitochondrial processes across three states of metabolism. In particular, the work highlights the significant contribution of functional and structural remodeling occurring during the diauxic shift of this subcellular organelle., Saccharomyces cerevisiae constitutes a popular eukaryal model for research on mitochondrial physiology. Being Crabtree-positive, this yeast has evolved the ability to ferment glucose to ethanol and respire ethanol once glucose is consumed. Its transition phase from fermentative to respiratory metabolism, known as the diauxic shift, is reflected by dramatic rearrangements of mitochondrial function and structure. To date, the metabolic adaptations that occur during the diauxic shift have not been fully characterized at the organelle level. In this study, the absolute proteome of mitochondria was quantified alongside precise parametrization of biophysical properties associated with the mitochondrial network using state-of-the-art optical-imaging techniques. This allowed the determination of absolute protein abundances at a subcellular level. By tracking the transformation of mitochondrial mass and volume, alongside changes in the absolute mitochondrial proteome allocation, we could quantify how mitochondria balance their dual role as a biosynthetic hub as well as a center for cellular respiration. Furthermore, our findings suggest that in the transition from a fermentative to a respiratory metabolism, the diauxic shift represents the stage where major structural and functional reorganizations in mitochondrial metabolism occur. This metabolic transition, initiated at the mitochondria level, is then extended to the rest of the yeast cell.
- Published
- 2020
20. Monitoring ADP/ATP ratio in yeast cells using the fluorescent-protein reporter PercevalHR
- Author
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Yuki Ishiwata-Kimata, Phuong Thi Mai Nguyen, and Yukio Kimata
- Subjects
0301 basic medicine ,ADP ,Saccharomyces cerevisiae ,High resolution ,Mitochondrion ,yeast ,Applied Microbiology and Biotechnology ,Biochemistry ,Fluorescence ,Analytical Chemistry ,03 medical and health sciences ,0302 clinical medicine ,Adenosine Triphosphate ,Fluorescent protein ,Molecular Biology ,Fluorescent reporter ,biology ,Chemistry ,Organic Chemistry ,diauxic shift ,General Medicine ,biology.organism_classification ,Yeast ,Mitochondria ,ATP ,Adenosine Diphosphate ,Luminescent Proteins ,030104 developmental biology ,Biophysics ,ATP–ADP translocase ,030217 neurology & neurosurgery ,Biotechnology - Abstract
PercevalHR (Perceval High Resolution) is an artificially designed fluorescent protein, which changes its excitation spectrum based on the ADP/ATP ratio of the environment. Here we demonstrated that PercevalHR can be used for monitoring energy status of Saccharomyces cerevisiae cells, which are affected by diauxic shift and mitochondria inhibition, in a non-invasive and non-destructive manner.
- Published
- 2019
21. Detecting shifts in gene regulatory networks during time-course experiments at single-time-point temporal resolution.
- Author
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Takenaka, Yoichi, Seno, Shigeto, and Matsuda, Hideo
- Subjects
- *
GENE regulatory networks , *BIOINFORMATICS , *ORDINARY differential equations , *GENETIC regulation , *GENE expression , *GRAPHICAL modeling (Statistics) - Abstract
Comprehensively understanding the dynamics of biological systems is one of the greatest challenges in biology. Vastly improved biological technologies have provided vast amounts of information that must be understood by bioinformatics and systems biology researchers. Gene regulations have been frequently modeled by ordinary differential equations or graphical models based on time-course gene expression profiles. The state-of-the-art computational approaches for analyzing gene regulations assume that their models are same throughout time-course experiments. However, these approaches cannot easily analyze transient changes at a time point, such as diauxic shift. We propose a score that analyzes the gene regulations at each time point. The score is based on the information gains of information criterion values. The method detects the shifts in gene regulatory networks (GRNs) during time-course experiments with single-time-point resolution. The effectiveness of the method is evaluated on the diauxic shift from glucose to lactose in Escherichia coli. Gene regulation shifts were detected at two time points: the first corresponding to the time at which the growth of E. coli ceased and the second corresponding to the end of the experiment, when the nutrient sources (glucose and lactose) had become exhausted. According to these results, the proposed score and method can appropriately detect the time of gene regulation shifts. The method based on the proposed score provides a new tool for analyzing dynamic biological systems. Because the score value indicates the strength of gene regulation at each time point in a gene expression profile, it can potentially infer hidden GRNs from time-course experiments. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
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22. Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae.
- Author
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Williams, Thomas C., Espinosa, Monica I., Nielsen, Lars K., and Vickers, Claudia E.
- Subjects
- *
GENE expression , *RNA interference , *SACCHAROMYCES cerevisiae , *SUCROSE , *SYNTHETIC biology - Abstract
Background: Engineering dynamic, environmentally- and temporally-responsive control of gene expression is one of the principle objectives in the field of synthetic biology. Dynamic regulation is desirable because many engineered functions conflict with endogenous processes which have evolved to facilitate growth and survival, and minimising conflict between growth and production phases can improve product titres in microbial cell factories. There are a limited number of mechanisms that enable dynamic regulation in yeast, and fewer still that are appropriate for application in an industrial setting. Results: To address this problem we have identified promoters that are repressed during growth on glucose, and activated during growth on sucrose. Catabolite repression and preferential glucose utilisation allows active growth on glucose before switching to production on sucrose. Using sucrose as an activator of gene expression circumvents the need for expensive inducer compounds and enables gene expression to be triggered during growth on a fermentable, high energy-yield carbon source. The ability to fine-tune the timing and population density at which gene expression is activated from the SUC2 promoter was demonstrated by varying the ratio of glucose to sucrose in the growth medium. Finally, we demonstrated that the system could also be used to repress gene expression (a process also required for many engineering projects). We used the glucose/sucrose system to control a heterologous RNA interference module and dynamically repress the expression of a constitutively regulated GFP gene. Conclusions: The low noise levels and high dynamic range of the SUC2 promoter make it a promising option for implementing dynamic regulation in yeast. The capacity to repress gene expression using RNA interference makes the system highly versatile, with great potential for metabolic engineering applications. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
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23. Hierarchy of non-glucose sugars in Escherichia coli.
- Author
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Aidelberg, Guy, Towbin, Benjamin D., Rothschild, Daphna, Dekel, Erez, Bren, Anat, and Alon, Uri
- Subjects
- *
ESCHERICHIA coli physiology , *GLUCOSE , *BIOTECHNOLOGY , *NATURE , *FLUORESCENCE , *CATALYST supports - Abstract
Background Understanding how cells make decisions, and why they make the decisions they make, is of fundamental interest in systems biology. To address this, we study the decisions made by E. coli on which genes to express when presented with two different sugars. It is well-known that glucose, E. coli's preferred carbon source, represses the uptake of other sugars by means of global and gene-specific mechanisms. However, less is known about the utilization of glucose-free sugar mixtures which are found in the natural environment of E. coli and in biotechnology. Results Here, we combine experiment and theory to map the choices of E. coli among 6 different non-glucose carbon sources. We used robotic assays and fluorescence reporter strains to make precise measurements of promoter activity and growth rate in all pairs of these sugars. We find that the sugars can be ranked in a hierarchy: in a mixture of a higher and a lower sugar, the lower sugar system shows reduced promoter activity. The hierarchy corresponds to the growth rate supported by each sugar- the faster the growth rate, the higher the sugar on the hierarchy. The hierarchy is 'soft' in the sense that the lower sugar promoters are not completely repressed. Measurement of the activity of the master regulator CRP-cAMP shows that the hierarchy can be quantitatively explained based on differential activation of the promoters by CRP-cAMP. Comparing sugar system activation as a function of time in sugar pair mixtures at sub-saturating concentrations, we find cases of sequential activation, and also cases of simultaneous expression of both systems. Such simultaneous expression is not predicted by simple models of growth rate optimization, which predict only sequential activation. We extend these models by suggesting multi-objective optimization for both growing rapidly now and preparing the cell for future growth on the poorer sugar. Conclusion We find a defined hierarchy of sugar utilization, which can be quantitatively explained by differential activation by the master regulator cAMP-CRP. The present approach can be used to understand cell decisions when presented with mixtures of conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2014
- Full Text
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24. Natural Variation in Diauxic Shift between Patagonian Saccharomyces eubayanus Strains.
- Author
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Molinet J, Eizaguirre JI, Quintrel P, Bellora N, Villarroel CA, Villarreal P, Benavides-Parra J, Nespolo RF, Libkind D, and Cubillos FA
- Subjects
- Beer, Glucose, Chromatin, Saccharomyces cerevisiae genetics, Maltose metabolism
- Abstract
The study of natural variation can untap novel alleles with immense value for biotechnological applications. Saccharomyces eubayanus Patagonian isolates exhibit differences in the diauxic shift between glucose and maltose, representing a suitable model to study their natural genetic variation for novel strains for brewing. However, little is known about the genetic variants and chromatin regulators responsible for these differences. Here, we show how genome-wide chromatin accessibility and gene expression differences underlie distinct diauxic shift profiles in S. eubayanus. We identified two strains with a rapid diauxic shift between glucose and maltose (CL467.1 and CBS12357) and one strain with a remarkably low fermentation efficiency and longer lag phase during diauxic shift (QC18). This is associated in the QC18 strain with lower transcriptional activity and chromatin accessibility of specific genes of maltose metabolism and higher expression levels of glucose transporters. These differences are governed by the HAP complex, which differentially regulates gene expression depending on the genetic background. We found in the QC18 strain a contrasting phenotype to those phenotypes described in S. cerevisiae, where hap4Δ , hap5Δ , and cin5Δ knockouts significantly improved the QC18 growth rate in the glucose-maltose shift. The most profound effects were found between CIN5 allelic variants, suggesting that Cin5p could strongly activate a repressor of the diauxic shift in the QC18 strain but not necessarily in the other strains. The differences between strains could originate from the tree host from which the strains were obtained, which might determine the sugar source preference and the brewing potential of the strain. IMPORTANCE The diauxic shift has been studied in budding yeast under laboratory conditions; however, few studies have addressed the diauxic shift between carbon sources under fermentative conditions. Here, we study the transcriptional and chromatin structure differences that explain the natural variation in fermentative capacity and efficiency during diauxic shift of natural isolates of S. eubayanus. Our results show how natural genetic variants in transcription factors impact sugar consumption preferences between strains. These variants have different effects depending on the genetic background, with a contrasting phenotype to those phenotypes previously described in S. cerevisiae. Our study shows how relatively simple genetic/molecular modifications/editing in the lab can facilitate the study of natural variations of microorganisms for the brewing industry.
- Published
- 2022
- Full Text
- View/download PDF
25. Régulation de l’expression et de la localisation des ARN TLC1 et TERRA en réponse à différents stress génomiques chez la levure
- Author
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Lalonde, Maxime and Chartrand, Pascal
- Subjects
transition diauxique ,long non-coding RNA ,localisation de l’ARN ,long ARN non-codant ,TERRA ,diauxic shift ,télomère ,TLC1 RNA ,cohésion ,ARN TLC1 ,DNA damage ,télomérase ,dommage à l’ADN ,Saccharomyce cerevisiae - Abstract
Les télomères forment la structure qui coiffe les extrémités des chromosomes. Ils sont essentiels pour protéger l’intégrité génomique. À cause du problème de fin de réplication, les télomères raccourcissent à chaque division cellulaire, menant à l’arrêt du cycle cellulaire, à la sénescence et à la mort cellulaire. Pour contrevenir au raccourcissement des télomères, les cellules immortalisées et hautement prolifératives, ainsi que la plupart des eucaryotes unicellulaires tels que Saccharomyces cerevisiae, expriment la télomérase, un complexe ribonucléoprotéique enzymatique qui rallonge les télomères. Pour permettre le maintien de la longueur des télomères et assurer l’intégrité du génome, plusieurs régulateurs contrôlent le recrutement et l’activité de la télomérase, s’assurant du ciblage précis de l’activité de la télomérase à ses substrats. Aux télomères, un dérèglement des mécanismes de régulation de la télomérase peut mener au raccourcissement des télomères, à des fusions de chromosomes et au développement d’un potentiel cancéreux. La télomérase peut aussi agir aux cassures d’ADN où son activité se traduit par l’ajout de novo d’un télomère et conduit à la perte de matériel génétique, à l’instabilité génomique et possiblement à la mort cellulaire. Son recrutement et son activité y sont donc inhibés. Les mécanismes par lesquels la cellule régule l’activité de la télomérase aux télomères et aux cassures d’ADN restent encore peu connus. De plus, en réponse à certains stress, ces mécanismes peuvent être altérés. Les travaux présentés dans cette thèse ont pour but d’étudier les régulateurs de l’activité de la télomérase et l’impact de certains stress cellulaires sur cette régulation. Dans la première partie, nous avons étudié la localisation de l’ARN TLC1, la sous-unité ARN de la télomérase, à travers le cycle cellulaire chez S. cerevisiae. Alors que cet ARN est majoritairement dans le nucléoplasme en G1/S, il démontre une accumulation nucléolaire en phase G2/M du cycle cellulaire. Chez la levure, la réparation des cassures d’ADN se fait majoritairement par recombinaison homologue et est exclue du nucléole. Dans ce contexte, nous avons formulé l’hypothèse que l’accumulation de l’ARN TLC1 au nucléole en G2/M constitue un mécanisme par lequel l’ajout de novo de télomère est inhibé aux cassures d’ADN. Nous avons fixé comme buts de caractériser les mécanismes régulant l’accumulation nucléolaire de l’ARN TCL1 et d’étudier comment la présence de dommage à l’ADN influence cette régulation. Nous avons pu montrer que la localisation nucléolaire de l’ARN TLC1 dépend de l’hélicase Pif1, de la protéine de la recombinaison homologue Rad52 et que la présence de dommage à l’ADN et l’absence de Rad52 influence le trafic nucléaire de cet ARN. Dans ces conditions, la protéine de la recombinaison homologue Rad51 permet l’accumulation de Cdc13 aux cassures et favorise l’accumulation de l’ARN TLC1 au nucléoplasme et aux cassures d’ADN. Cette accumulation est dépendante de la SUMO ligase Siz1 et mène à une augmentation d’ajout de novo de télomère aux sites de cassures d’ADN. Pour pouvoir quantifier l’augmentation d’ajout de novo de télomère, nous avons développé une nouvelle approche basée sur le séquençage haut-débit de type Illumina pour identifier et quantifier les événements d’ajout de novo de télomère sur le génome entier de manière non-biaisée. Dans la deuxième partie de la thèse, nous avons étudié les mécanismes contrôlant l’expression d’un régulateur de la télomérase nommé TERRA (telomeric repeats containing RNA). TERRA est un long ARN non-codant qui est transcrit à partir des régions sous-télomériques jusqu’aux répétitions télomériques. Chez S. cerevisiae, l’expression de TERRA est inhibée au niveau de sa transcription par le complexe SIR et au niveau de sa dégradation par l’exonucléase Rat1. Pourtant, les télomères courts expriment TERRA à des niveaux élevés. Cette augmentation de l’expression de TERRA permet de concentrer et de cibler l’activité de la télomérase aux télomères courts. En étudiant l’expression de TERRA, nous avons remarqué que les télomères exprimant cet ARN démontrent une perte prématurée de leur cohésion en phase S du cycle cellulaire. Nous pensons que l’organisation structurelle des télomères et, plus particulièrement, la cohésion télomérique participe à la régulation de l’expression de TERRA. De plus, plusieurs groupes ont montré que l’expression de TERRA était régulée en réponse à plusieurs stress, de façon indépendante de la taille des télomères. Dans ce contexte, nous formulons l’hypothèse que le stress oxydatif et les changements métaboliques induits durant la transition diauxique influence l’expression de TERRA. Pour cette partie de la thèse, nous avions comme but d’étudier comment l’expression de TERRA étaient régulé par les changements métaboliques comme la transition diauxique et d’étudier le rôle joué par le complexe de la cohésine dans la régulation de l’expression de TERRA. Nous avons montré que les télomères courts montrent une perte de cohésion prématurée en début de phase S, ce qui favorise l’expression de TERRA en cis. Alors qu’une perte de fonction partielle de la cohésine résulte en une augmentation de l’expression de TERRA, la rétention forcée de cohésine à un télomère court réprime sa transcription. Cette perte de cohésion aux télomères courts est dépendante de Sir4 mais indépendante de Sir2, ce qui suggère que le rôle de Sir4 dans l’ancrage des télomères à la membrane nucléaire pourrait être impliqué dans ce phénomène. Nous avons également montré que la transcription de TERRA est induite durant la transition diauxique, une phase de croissance cellulaire où, suite à la déplétion du glucose, les cellules adaptent leur métabolisme en faveur de la respiration oxydative. Cette augmentation d’expression coïncide avec l’accumulation cytoplasmique de TERRA. Ensemble, les travaux présentés dans cette thèse explorent les liens entre les stress cellulaires tels que les dommages à l’ADN, le raccourcissement télomérique, le stress oxydatif et le métabolisme cellulaire, et leur impact sur le trafic de la télomérase et l’expression de son régulateur TERRA., Telomeres constitute the structure at the end of linear chromosomes which is essential to protect genome integrity. Due to the end-replication problem, telomeres get shorter with every cell division, leading to cell cycle arrest, senescence and cell death. To counteract telomere shortening, highly proliferative cells and most unicellular eukaryotes, like Saccharomyces cerevisiae, express telomerase, a ribonucleoprotein enzyme that elongates telomeres. Many regulatory pathways affect telomerase activity and recruitment to assure precise targeting of telomerase activity to its proper substrate, the telomeres. Impairing these pathways can lead to telomere shortening, end-to-end chromosome fusions and immortalization. Telomerase can also be recruited at double strand breaks (DSBs), where its activity leads to de novo telomere additions which induce genomic instability, loss of genetic information and possibly cell death. For this reason, telomerase recruitment and activity is strongly inhibited at DSB. However, the mechanisms behind this regulation are still poorly understood. Furthermore, many cellular stresses affect telomerase regulation at telomeres and DSBs. Our goal is to study the regulation of telomerase activity and the impact of cellular stresses on this regulation. In the first part of this thesis, we looked at the cell cycle localization of the Saccharomyces cerevisiae RNA subunit of the telomerase, TLC1 RNA. While TLC1 RNA is mostly in the nucleoplasm in G1/S, it accumulates in the nucleolus in G2/M. In yeast, the most common DSB repair pathway is homologous recombination (HR). As HR is mostly excluded from the nucleolus in G2/M, we propose that the accumulation of TLC1 RNA in the nucleolus in G2/M may represent a regulatory pathway that repress de novo telomere addition by physically separating telomerase from sites of DNA repair by HR. We aim to characterize the mechanisms by which TLC1 RNA localization is regulated and how the presence of DSB affects this trafficking. We were able to show that the nucleolar localization of TLC1 RNA is dependent on the Pif1 helicase and on the HR protein Rad52. Furthermore, we showed that the presence of DSBs and the absence of Rad52 alter the nuclear trafficking of TLC1 RNA. In these conditions, Rad51 favors the accumulation of Cdc13 at DSBs and promotes the nucleoplasmic accumulation of TLC1 RNA. This accumulation is dependent on the SUMO ligase Siz1 and leads to an increased addition of de novo telomere at DNA breaks. In order to identify de novo telomere addition events genome-wide, we developed an unbiased genome-wide technique based on Illumina sequencing of genomic DNA. In the second part of this thesis, we studied another regulator of telomerase activity, the long non-coding RNA (lncRNA) TERRA (telomeric repeats-containing RNA), which is transcribed from subtelomeric regions through the telomeric tracts. In S. cerevisiae, TERRA expression is controlled at the transcriptional level by the SIR complex and its degradation by the exonuclease Rat1. Nevertheless, short telomeres escape transcriptional inhibition and degradation to express TERRA at higher levels. TERRA serves as a regulator of telomerase, allowing the concentration and the targeting of telomerase activity to short telomeres. While studying TERRA expression, we observed that TERRA-expressing telomeres display a premature S-phase loss of cohesion. We propose that cohesin and telomere cohesion are regulators of TERRA expression. In addition, other groups have shown that TERRA expression was regulated in response to different cellular stress. This regulation seems to be independent from telomere length. In these contexts, we propose that oxidative stress and metabolic changes induced during the diauxic shift affect TERRA expression. We aim to study how the diauxic shift affects TERRA expression and study the role of cohesin in regulating TERRA expression. We were able to show that telomere cohesion inhibits TERRA expression and that short telomeres display a premature loss of cohesion to allow TERRA expression. This loss of cohesion is dependent on Sir4 and probably on Sir4-mediated telomere anchoring at the nuclear membrane. Additionally, we showed that TERRA transcription is increased during the diauxic shift, when yeast cells switch from fermentative glycolysis to oxidative respiration. Yeast cells in this phase also display a cytoplasmic accumulation of TERRA molecules. Altogether, the articles presented in this thesis explore the interplay between cellular stresses such as DNA damage, telomere shortening, oxidative stress and respiratory metabolism, and their roles in the regulation of the localisation and expression of TLC1 RNA and TERRA.
- Published
- 2020
26. Temporal system‐level organization of the switch from glycolytic to gluconeogenic operation in yeast
- Author
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Guillermo G Zampar, Anne Kümmel, Jennifer Ewald, Stefan Jol, Bastian Niebel, Paola Picotti, Ruedi Aebersold, Uwe Sauer, Nicola Zamboni, and Matthias Heinemann
- Subjects
diauxic shift ,fluxome ,metabolome ,proteome ,Saccharomyces cerevisiae ,Biology (General) ,QH301-705.5 ,Medicine (General) ,R5-920 - Abstract
The diauxic shift in Saccharomyces cerevisiae is an ideal model to study how eukaryotic cells readjust their metabolism from glycolytic to gluconeogenic operation. In this work, we generated time‐resolved physiological data, quantitative metabolome (69 intracellular metabolites) and proteome (72 enzymes) profiles. We found that the diauxic shift is accomplished by three key events that are temporally organized: (i) a reduction in the glycolytic flux and the production of storage compounds before glucose depletion, mediated by downregulation of phosphofructokinase and pyruvate kinase reactions; (ii) upon glucose exhaustion, the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions; and (iii) in the later stages of the adaptation, the shutting down of the pentose phosphate pathway with a change in NADPH regeneration. Moreover, we identified the transcription factors associated with the observed changes in protein abundances. Taken together, our results represent an important contribution toward a systems‐level understanding of how this adaptation is realized.
- Published
- 2013
- Full Text
- View/download PDF
27. Harvesting yeast (Saccharomyces cerevisiae) at different physiological phases significantly affects its functionality in bread dough fermentation.
- Author
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Rezaei, Mohammad N., Dornez, Emmie, Jacobs, Pieter, Parsi, Anali, Verstrepen, Kevin J., and Courtin, Christophe M.
- Subjects
- *
YEAST , *SACCHAROMYCES cerevisiae , *BAKED products , *FOOD fermentation , *LEAVENING agents , *RHEOLOGY (Biology) , *METABOLITES - Abstract
Abstract: Fermentation of sugars into CO2, ethanol and secondary metabolites by baker's yeast (Saccharomyces cerevisiae) during bread making leads to leavening of dough and changes in dough rheology. The aim of this study was to increase our understanding of the impact of yeast on dough related aspects by investigating the effect of harvesting yeast at seven different points of the growth profile on its fermentation performance, metabolite production, and the effect on critical dough fermentation parameters, such as gas retention potential. The yeast cells harvested during the diauxic shift and post-diauxic growth phase showed a higher fermentation rate and, consequently, higher maximum dough height than yeast cells harvested in the exponential or stationary growth phase. The results further demonstrate that the onset of CO2 loss from fermenting dough is correlated with the fermentation rate of yeast, but not with the amount of CO2 that accumulated up to the onset point. Analysis of the yeast metabolites produced in dough yielded a possible explanation for this observation, as they are produced in different levels depending on physiological phase and in concentrations that can influence dough matrix properties. Together, our results demonstrate a strong effect of yeast physiology at the time of harvest on subsequent dough fermentation performance, and hint at an important role of yeast metabolites on the subsequent gas holding capacity. [Copyright &y& Elsevier]
- Published
- 2014
- Full Text
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28. Data-driven multiscale modeling reveals the role of metabolic coupling for the spatio-temporal growth dynamics of yeast colonies
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Harri Lähdesmäki, Alexander Skupin, Jukka Intosalmi, Nicholas S. Flann, Aimée M. Dudley, Olli Yli-Harja, Michelle Hays, Adrian C. Scott, Tampere University, BioMediTech, Research group: Computational Systems Biology, Centre of Excellence in Molecular Systems Immunology and Physiology Research Group, SyMMys, Pacific Northwest Research Institute, University of Washington, Utah State University, Helsinki Institute for Information Technology (HIIT), University of Luxembourg, Department of Computer Science, Aalto-yliopisto, Aalto University, and BioMed Central Ltd.
- Subjects
Computer science ,Saccharomyces cerevisiae ,Biology ,01 natural sciences ,Models, Biological ,Data-driven ,symbols.namesake ,010104 statistics & probability ,03 medical and health sciences ,Spatio-Temporal Analysis ,Multiscale modeling ,Computer Simulation ,lcsh:QH573-671 ,0101 mathematics ,Molecular Biology ,030304 developmental biology ,Microbial Biofilms ,Bayesian optimization ,Metabolic coupling ,0303 health sciences ,Yeast colony ,Computer Sciences ,030306 microbiology ,lcsh:Cytology ,Dynamics (mechanics) ,Diauxic shift ,Markov chain Monte Carlo ,217 Medical engineering ,Cell Biology ,Yeast ,Multicellular organism ,Coupling (computer programming) ,Homogeneous ,Multicellular systems ,symbols ,Biological system ,Research Article - Abstract
MotivationMulticellular entities, such as mammalian tissues or microbial biofilms, typically exhibit complex spatial arrangements that are adapted to their specific functions or environments. These structures result from intercellular signaling as well as from the interaction with the environment that allow cells of the same genotype to differentiate into well-organized communities of diversified cells. Despite its importance, our understanding on how cell–cell and metabolic coupling produce functionally optimized structures is still limited.ResultsHere, we present a data-driven spatial framework to computationally investigate the development of one multicellular structure, yeast colonies. Using experimental growth data from homogeneous liquid media conditions, we develop and parameterize a dynamic cell state and growth model. We then use the resulting model in a coarse-grained spatial model, which we calibrate using experimental time-course data of colony growth. Throughout the model development process, we use state-of-the-art statistical techniques to handle the uncertainty of model structure and parameterization. Further, we validate the model predictions against independent experimental data and illustrate how metabolic coupling plays a central role in colony formation.AvailabilityExperimental data and a computational implementation to reproduce the results are available athttp://research.cs.aalto.fi/csb/software/multiscale/code.zip.Contactjukka.intosalmi@aalto.fi,alexander.skupin@uni.lu
- Published
- 2019
29. Temporal system-level organization of the switch from glycolytic to gluconeogenic operation in yeast.
- Author
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Zampar, Guillermo G., Kümmel, Anne, Ewald, Jennifer, Jol, Stefan, Niebel, Bastian, Picotti, Paola, Aebersold, Ruedi, Sauer, Uwe, Zamboni, Nicola, and Heinemann, Matthias
- Abstract
The diauxic shift in Saccharomyces cerevisiae is an ideal model to study how eukaryotic cells readjust their metabolism from glycolytic to gluconeogenic operation. In this work, we generated time-resolved physiological data, quantitative metabolome (69 intracellular metabolites) and proteome (72 enzymes) profiles.We found that the diauxic shift is accomplished by three key events that are temporally organized: (i) a reduction in the glycolytic flux and the production of storage compounds before glucose depletion, mediated by downregulation of phosphofructokinase and pyruvate kinase reactions; (ii) upon glucose exhaustion, the reversion of carbon flow through glycolysis and onset of the glyoxylate cycle operation triggered by an increased expression of the enzymes that catalyze the malate synthase and cytosolic citrate synthase reactions; and (iii) in the later stages of the adaptation, the shutting down of the pentose phosphate pathway with a change in NADPH regeneration. Moreover,we identified the transcription factors associated with the observed changes in protein abundances. Taken together, our results represent an important contribution toward a systems-level understanding of how this adaptation is realized. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
30. A Microfluidic Platform for Tracking Individual Cell Dynamics during an Unperturbed Nutrients Exhaustion.
- Author
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Aspert T, Jacquel B, and Charvin G
- Abstract
Microorganisms have evolved adaptive strategies to respond to the autonomous degradation of their environment. Indeed, a growing culture progressively exhausts nutrients from its media and modifies its composition. Yet, how single cells react to these modifications remains difficult to study since it requires population-scale growth experiments to allow cell proliferation to have a collective impact on the environment, while monitoring the same individuals exposed to this environment for days. For this purpose, we have previously described an integrated microfluidic pipeline, based on continuous separation of the cells from the media and subsequent perfusion of the filtered media in an observation chamber containing isolated single cells. Here, we provide a detailed protocol to implement this methodology, including the setting up of the microfluidic system and the processing of timelapse images., Competing Interests: Competing interests The authors declare no competing interests., (Copyright © The Authors; exclusive licensee Bio-protocol LLC.)
- Published
- 2022
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31. Metabolic alterations in yeast lacking copper–zinc superoxide dismutase
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Sehati, Sadaf, Clement, Matthew H.S., Martins, Jake, Xu, Lei, Longo, Valter D., Valentine, Joan S., and Gralla, Edith B.
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- *
YEAST , *METABOLIC regulation , *COPPER , *ZINC , *SUPEROXIDE dismutase , *METHIONINE , *REACTIVE oxygen species , *GLUTATHIONE , *OXIDATIVE stress - Abstract
Abstract: Yeast lacking copper–zinc superoxide dismutase (sod1∆) have a number of oxygen-dependent defects, including auxotrophies for lysine and methionine and sensitivity to oxygen. Here we report additional defects in metabolic regulation. Under standard growth conditions with glucose as the carbon source, yeast undergo glucose repression in which mitochondrial respiration is deemphasized, energy is mainly derived from glycolysis, and ethanol is produced. When glucose is depleted, the diauxic shift is activated, in which mitochondrial respiration is reemphasized and stress resistance increases. We find that both of these programs are adversely affected by the lack of Sod1p. Key events in the diauxic shift do not occur and sod1∆ cells do not utilize ethanol and stop growing. The ability to shift to growth on ethanol is gradually lost as time in culture increases. In early stages of culture, sod1∆ cells consume more oxygen and have more mitochondrial mass than wild-type cells, indicating that glucose repression is not fully activated. These changes are at least partially dependent on the activity of the Hap2,3,4,5 complex, as indicated by CYC1–lacZ reporter assays. These changes may indicate a role for superoxide in metabolic signaling and regulation and/or a role for glucose derepression in defense against oxidative stress. [Copyright &y& Elsevier]
- Published
- 2011
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32. On-line high performance liquid chromatography measurements of extracellular metabolites in an aerobic batch yeast ( Saccharomyces cerevisiae) culture.
- Author
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Tohmola, Niina, Ahtinen, Jouni, Pitkänen, Juha-Pekka, Parviainen, Ville, Joenväärä, Sakari, Hautamäki, Mika, Lindroos, Peter, Mäkinen, Jarno, and Renkonen, Risto
- Abstract
We constructed a bioprocess environment enabling automatic sampling from a bioreactor combined with a compact on-line high performance liquid chromatography (HPLC) unit. This setup allowed us to measure extracellular glucose, ethanol, glycerol, and acetate concentrations automatically at 5 min intervals during the cultivation. This environment also provides mechanical measurement of the optical density (OD) of cells and enables us to collect and store (−35°C) samples for further off-line analyses. Among the available devices, the performance of the sampling-analysis unit is by far the best with regard to speed and number of analytes. Both the sampling and analysis phases are easily controlled by software; thus, providing a unique environment to perform various bioprocess activity tasks, whether they would be cell line screening or optimisation of conditions for growth and productivity. Complex research set-ups can be created and continuous automated measurements empower long-term cultivations with a time series. We provide evidence for the applicability of this environment by performing three comparable batch cultivations with Saccharomyces cerevisiae yeast and show that both the on-line sampling and analysis modes produce reliable data for further use in the monitoring and controlling of bioprocesses. On-line data provided new insight into the dynamics of the diauxic shift during aerobic glucose batch cultivation. When cell growth and carbon dioxide production ceased for the first time during the diauxic shift, acetate accumulation and consumption of the remaining glucose below 0.15 g/L continued to occur for 1 h. At the same time, glycerol and ethanol began to be consumed. Samples were also collected during cultivation for later analysis of intracellular metabolites and to collect more valuable information about metabolism. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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33. Effect of different glucose concentrations on proteome of Saccharomyces cerevisiae
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Francesca, Guidi, Francesca, Magherini, Tania, Gamberi, Marina, Borro, Maurizio, Simmaco, and Alessandra, Modesti
- Subjects
- *
GLUCOSE , *SACCHAROMYCES cerevisiae , *PROTEOMICS , *CARBON , *FERMENTATION , *BACTERIAL metabolism , *OXIDATIVE stress - Abstract
Abstract: We performed a proteomic study to understand how Saccharomyces cerevisiae adapts its metabolism during the exponential growth on three different concentrations of glucose; this information will be necessary to understand yeast carbon metabolism in different environments. We induced a natural diauxic shift by growing yeast cells in glucose restriction thus having a fast and complete glucose exhaustion. We noticed differential expressions of groups of proteins. Cells in high glucose have a decreased growth rate during the initial phase of fermentation; in glucose restriction and in high glucose we found an over-expression of a protein (Peroxiredoxin) involved in protection against oxidative stress insult. The information obtained in our study validates the application of a proteomic approach for the identification of the molecular bases of environmental variations such as fermentation in high glucose and during a naturally induced diauxic shift. [Copyright &y& Elsevier]
- Published
- 2010
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34. Complementation of coenzyme Q-deficient yeast by coenzyme Q analogues requires the isoprenoid side chain.
- Author
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James, Andrew M., Cochem, Helena M., Murai, Masatoshi, Miyoshi, Hideto, and Murphy, Michael P.
- Subjects
- *
UBIQUINONES , *MITOCHONDRIA formation , *YEAST fungi biotechnology , *OXIDATIVE stress , *GLYCERIN ,ISOPENTENOID synthesis - Abstract
The ubiquinone coenzyme Q (CoQ) is synthesized in mitochondria with a large, hydrophobic isoprenoid side chain. It functions in mitochondrial respiration as well as protecting membranes from oxidative damage. Yeast that cannot synthesize CoQ ( ΔCoQ) are viable, but cannot grow on nonfermentable carbon sources, unless supplied with ubiquinone. Previously we demonstrated that the isoprenoid side chain of the exogenous ubiquinone was important for growth of a ΔCoQ strain on the nonfermentable substrate glycerol [James AM et al. (2005) J Biol Chem 280, 21295–21312]. In the present study we investigated the structural requirements of exogenously supplied CoQ2 for growth on glycerol and found that the first double bond of the initial isoprenoid unit is essential for utilization of respiratory substrates. As CoQ2 analogues that did not complement growth on glycerol supported respiration in isolated mitochondria, discrimination does not occur via the respiratory chain complexes. The endogenous form of CoQ in yeast (CoQ6) is extremely hydrophobic and transported to mitochondria via the endocytic pathway when supplied exogenously. We found that CoQ2 does not require this pathway when supplied exogenously and the pathway is unlikely to be responsible for the structural discrimination observed. Interestingly, decylQ, an analogue unable to support growth on glycerol, is not toxic, but antagonizes growth of ΔCoQ yeast in the presence of exogenous CoQ2. Using a ΔCoQ double-knockout library we identified a number of genes that decrease the ability of yeast to grow on exogenous CoQ. Here we suggest that CoQ or its redox state may be a signal for growth during the shift to respiration. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
35. A phenotypic study of TFS1 mutants differentially altered in the inhibition of Ira2p or CPY.
- Author
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Gombault, Aurélie, Warringer, Jonas, Caesar, Robert, Godin, Fabienne, Vallée, Béatrice, Doudeau, Michel, Chautard, Hélène, Blomberg, Anders, and Bénédetti, Hélène
- Subjects
- *
YEAST , *PHENOTYPES , *GENETIC mutation , *SACCHAROMYCES cerevisiae , *PROTEINS - Abstract
The Saccharomyces cerevisiae protein Tfs1p is known as a dual protein. On the one hand, it inhibits the carboxypeptidase Y protease, and on the other, it inhibits Ira2p, a GTPase-activating protein of Ras. We managed to dissect precise areas of Tfs1p specifically involved in only one of those functions. Based on these data, specific Tfs1p point mutants affected in only one of these two functions were constructed. In order to obtain insights on the physiological role of these functions, systematic phenotypic tests were performed on strains expressing these specific Tfs1p mutants. The results obtained demonstrate that the inhibition of Ira2p by Tfs1p is the predominant function under the conditions tested. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
36. Saccharomyces cerevisiae Hsp31p, a stress response protein conferring protection against reactive oxygen species
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Skoneczna, Adrianna, Miciałkiewicz, Arkadiusz, and Skoneczny, Marek
- Subjects
- *
SACCHAROMYCES cerevisiae , *REACTIVE oxygen species , *OXIDATIVE stress , *AMINO acids - Abstract
Abstract: The Saccharomyces cerevisiae HSP31 (YDR533c) gene encodes a protein that belongs to the DJ-1/PfpI family and its function is unknown. Homologs to Hsp31p polypeptide can be found in organisms from all systematic groups of eukaryotes and prokaryotes, and the functions of the vast majority of them are also hypothetical. One of the homologs is human protein DJ-1. Various amino acid substitutions within this protein correlate with early onset hereditary Parkinson''s disease. The deletion of the HSP31 gene displays no apparent phenotype under standard growth conditions, but a thorough functional analysis of S. cerevisiae revealed that its absence makes the cells sensitive to a subset of reactive oxygen species (ROS) generators. HSP31 is induced under conditions of oxidative stress in a YAP1-dependent manner. Similar to other stress response genes, it is also induced in the postdiauxic phase of growth and this induction is YAP1-independent. The patterns of sensitivities to various ROS generators of the hsp31Δ strain and the strain with the deletion of SOD1, another gene defending the cell against ROS, are different. We postulate that Hsp31p protects the cell against oxidative stress and complements other stress protection systems within the cell. [Copyright &y& Elsevier]
- Published
- 2007
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37. Glucose deprivation mediates interaction between CTDK-I and Snf1 in Saccharomyces cerevisiae
- Author
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Van Driessche, Benoit, Coddens, Séverine, Van Mullem, Vincent, and Vandenhaute, Jean
- Subjects
- *
GLUCOSE , *SUCROSE , *SACCHAROMYCES cerevisiae , *DNA polymerases - Abstract
Abstract: Ctk1 is a kinase involved in transcriptional control. We show in the two-hybrid system that Ctk1 interacts with Snf1, a kinase regulating glucose-dependent genes. Co-purification experiments confirmed the two-hybrid interaction but only when cells were grown at low glucose concentrations. Deletion of Ctk1 or its associated partners, Ctk2 and Ctk3, conferred synthetic lethality with null mutants of Snf1 or Snf1-associated proteins. Northern blot analysis suggested that Ctk1 and Snf1 act together in vivo to regulate GSY2. These findings support the view that Ctk1 interacts with Snf1 in a functional module involved in the cellular response to glucose limitation. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
- View/download PDF
38. Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase.
- Author
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Werner, Thomas, Amrhein, Nikolaus, and Freimoser, Florian
- Subjects
- *
POLYPHOSPHATES , *SACCHAROMYCES cerevisiae , *GLUCOSE , *PHOSPHATES , *CELLS , *YEAST - Abstract
Inorganic polyphosphate (iPoP)—linear chains of up to hundreds of phosphate residues—is ubiquitous in nature and appears to be involved in many different cellular processes. In Saccharomyces cerevisiae, iPoP has been detected in high concentrations, especially after transfer of phosphate-deprived cells to a high-phosphate medium. Here, the dynamics of iPoP synthesis in yeast as a function of the growth phase as well as glucose and phosphate availability have been investigated. To address this question, a simple, fast and novel method for the quantification of iPoP from yeast was developed. Both the iPoP content during growth and the iPoP “overplus” were highest towards the end of the exponential phase, when glucose became limiting. Accumulation of iPoP during growth required excess of free phosphate, while the iPoP “overplus” was only observed after the shift from low- to high-phosphate medium. The newly developed iPoP quantification method and the knowledge about the dynamics of iPoP content during growth made it possible to define specific growth conditions for the analysis of iPoP levels. These experimental procedures will be essential for the large-scale analysis of various mutant strains or the comparison of different growth conditions. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
39. Glutathione, but not transcription factor Yap1, is required for carbon source-dependent resistance to oxidative stress in Saccharomyces cerevisiae.
- Author
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Maris, Angel F., Kern, Ana Lúcia, Picada, Jaqueline N., Boccardi, Fabiane, Brendel, Martin, and Henriques, João A. P.
- Subjects
GLUTATHIONE ,TRANSCRIPTION factors ,GLUCOSE ,SUCROSE ,ALCOHOL ,HYDROGEN peroxide - Abstract
Resistance of haploid yeast to hydrogen peroxide and to tert-butylhydroperoxide strongly increases when 4% glucose is replaced by glycerol or ethanol as the carbon source of the complex medium. Using a GSH1-promoter-lacZ-fusion reporter construct we could demonstrate that GSH1 is one of the genes that are up-regulated during the shift from fermentative to oxidative metabolism. A gsh1 mutant did not exhibit respiratory growth resistance to H
2 O2 , whereas it was only slightly impaired in acquiring resistance against t-BOOH in the same experimental conditions. An isogenic Δyap1 mutant, although more sensitive to oxidative stress than the wild-type (WT), could increase resistance to both peroxides by a similar factor as observed for the WT when shifted from 4% glucose to a non-fermentable carbon source. This indicates that in this case induction of resistance to oxidative stress is independent from Yap1 and from the Yap1-mediated stress response via the STRE motif. [ABSTRACT FROM AUTHOR]- Published
- 2000
- Full Text
- View/download PDF
40. Closed-loop cycles of experiment design, execution, and learning accelerate systems biology model development in yeast
- Author
-
Ross D. King, Jacek Grzebyta, Martin Carpenter, Jan Ramon, Henry Soldano, Céline Rouveirol, Guillaume Santini, Anthony Coutant, Katherine Roper, Larisa N. Soldatova, Daniel Trejo-Banos, Dominique Bouthinon, Mohamed Elati, Laboratoire d'Informatique de Paris-Nord (LIPN), Université Paris 13 (UP13)-Institut Galilée-Université Sorbonne Paris Cité (USPC)-Centre National de la Recherche Scientifique (CNRS), University of Manchester [Manchester], Génomique métabolique (UMR 8030), Genoscope - Centre national de séquençage [Evry] (GENOSCOPE), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Brunel University London [Uxbridge], Institut de Systématique, Evolution, Biodiversité (ISYEB ), Muséum national d'Histoire naturelle (MNHN)-École Pratique des Hautes Études (EPHE), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université des Antilles (UA), Programme d'Épigénomique, Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS), Machine Learning in Information Networks (MAGNET), Inria Lille - Nord Europe, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Centre de Recherche en Informatique, Signal et Automatique de Lille - UMR 9189 (CRIStAL), Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Centrale Lille-Université de Lille-Centre National de la Recherche Scientifique (CNRS), University of London [London], The Alan Turing Institute, National Institute of Advanced Industrial Science and Technology (AIST), Université Sorbonne Paris Cité (USPC)-Institut Galilée-Université Paris 13 (UP13)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), and Muséum national d'Histoire naturelle (MNHN)-École pratique des hautes études (EPHE)
- Subjects
0301 basic medicine ,Computer science ,Systems biology ,Distributed computing ,0206 medical engineering ,Cloud computing ,Saccharomyces cerevisiae ,02 engineering and technology ,Reuse ,[INFO.INFO-AI]Computer Science [cs]/Artificial Intelligence [cs.AI] ,03 medical and health sciences ,Software ,Gene Expression Regulation, Fungal ,Semantic Web ,[SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology ,Multidisciplinary ,business.industry ,Systems Biology ,Computational Biology ,diauxic shift ,Robotics ,Biological Sciences ,artificial intelligence ,Biophysics and Computational Biology ,ComputingMethodologies_PATTERNRECOGNITION ,030104 developmental biology ,Laboratory robotics ,machine learning ,Physical Sciences ,Laboratory automation ,Robot ,[INFO.INFO-BI]Computer Science [cs]/Bioinformatics [q-bio.QM] ,business ,020602 bioinformatics - Abstract
Significance Systems biology involves the development of large computational models of biological systems. The radical improvement of systems biology models will necessarily involve the automation of model improvement cycles. We present here a general approach to automating systems biology model improvement. Humans are eukaryotic organisms, and the yeast Saccharomyces cerevisiae is widely used in biology as a “model” for eukaryotic cells. The yeast diauxic shift is the most studied cellular transformation. We combined multiple software tools with integrated laboratory robotics to execute three semiautomated cycles of diauxic shift model improvement. All the experiments were formalized and communicated to a cloud laboratory automation system (Eve) for execution. The resulting improved model is relevant to understanding cancer, the immune system, and aging., One of the most challenging tasks in modern science is the development of systems biology models: Existing models are often very complex but generally have low predictive performance. The construction of high-fidelity models will require hundreds/thousands of cycles of model improvement, yet few current systems biology research studies complete even a single cycle. We combined multiple software tools with integrated laboratory robotics to execute three cycles of model improvement of the prototypical eukaryotic cellular transformation, the yeast (Saccharomyces cerevisiae) diauxic shift. In the first cycle, a model outperforming the best previous diauxic shift model was developed using bioinformatic and systems biology tools. In the second cycle, the model was further improved using automatically planned experiments. In the third cycle, hypothesis-led experiments improved the model to a greater extent than achieved using high-throughput experiments. All of the experiments were formalized and communicated to a cloud laboratory automation system (Eve) for automatic execution, and the results stored on the semantic web for reuse. The final model adds a substantial amount of knowledge about the yeast diauxic shift: 92 genes (+45%), and 1,048 interactions (+147%). This knowledge is also relevant to understanding cancer, the immune system, and aging. We conclude that systems biology software tools can be combined and integrated with laboratory robots in closed-loop cycles.
- Published
- 2019
41. The proteasome lid triggers COP9 signalosome activity during the transition of Saccharomyces cerevisiae cells into quiescence
- Author
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Lylan, Bramasole, Abhishek, Sinha, Cirigliano, Angela, Sylvia, Gurevich, Zanlin, Yu, Dana, Harshuk, Glickman, Michael H., Rinaldi, Teresa, and Elah, Pick
- Subjects
Rpn11 ,F-box containing complex) ,NEDD8 (neural precursor cell expressed developmentally down-regulated 8) ,Rub1 (Related ubiquitin 1) ,CSN (COP9 signalosome) ,diauxic shift ,Saccharomyces cerevisiae ,Cullin ,26S proteasome ,proteasome lid ,Cdc53 ,SCF (Skp, Cullin, F-box containing complex) ,budding yeast ,SCF (Skp - Published
- 2019
42. 出芽酵母のDiauxic shift時におけるunfolded protein response経路の活性化と、それに伴うミトコンドリア伸展
- Author
-
Tran, Minh Duc
- Subjects
Unfolded protein response ,Diauxic shift ,Mitochondria enlargement ,ER stress ,Reactive oxygen species - Published
- 2018
43. Categorization of Phosphorylation Site Behavior during the Diauxic Shift in Saccharomyces cerevisiae .
- Author
-
Gassaway BM, Paulo JA, and Gygi SP
- Subjects
- Fermentation, Gene Expression Regulation, Fungal, Phosphorylation, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism
- Abstract
Protein phosphorylation has long been recognized as an essential regulator of protein activity, structure, complex formation, and subcellular localization among other cellular mechanisms. However, interpretation of the changes in protein phosphorylation is difficult. To address this difficulty, we measured protein and phosphorylation site changes across 11 points of a time course and developed a method for categorizing phosphorylation site behavior relative to protein level changes using the diauxic shift in yeast as a model and TMT11 sample multiplexing. We classified quantified proteins into behavioral categories that reflected differences in kinase activity, protein complex structure, and growth and metabolic pathway regulation across different phases of the diauxic shift. These data also provide a valuable resource for the study of fermentative versus respiratory growth and set a new benchmark for temporal quantitative proteomics and phosphoproteomics for the diauxic shift in Saccharomyces cerevisiae . Data are available via ProteomeXchange with identifier PXD022741.
- Published
- 2021
- Full Text
- View/download PDF
44. The Proteasome Lid Triggers COP9 Signalosome Activity during the Transition of Saccharomyces cerevisiae Cells into Quiescence
- Author
-
Michael H. Glickman, Abhishek Sinha, Elah Pick, Laylan Bramasole, Rinat Lift Carmeli, Dana Harshuk, Sylvia Gurevich, Zanlin Yu, Angela Cirigliano, and Teresa Rinaldi
- Subjects
Proteolysis ,lcsh:QR1-502 ,CSN (COP9 signalosome) ,Biochemistry ,NEDD8 ,lcsh:Microbiology ,Cullin ,Deubiquitinating enzyme ,Saccharomyces cerevisiae ,03 medical and health sciences ,0302 clinical medicine ,Ubiquitin ,medicine ,budding yeast ,COP9 signalosome ,26S proteasome ,Molecular Biology ,030304 developmental biology ,Rpn11 ,NEDD8 (neural precursor cell expressed developmentally down-regulated 8) ,Rub1 (Related ubiquitin 1) ,0303 health sciences ,biology ,medicine.diagnostic_test ,Chemistry ,diauxic shift ,SCF (Skp, Cullin, F-box containing complex) ,Cell biology ,Proteasome ,biology.protein ,proteasome lid ,Cdc53 ,030217 neurology & neurosurgery ,Deubiquitination - Abstract
The class of Cullin−RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8−Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1−CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53−Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes.
- Published
- 2019
45. Fluorescence Detection of Increased Reactive Oxygen Species Levels in Saccharomyces cerevisiae at the Diauxic Shift.
- Author
-
Sinha A and Pick E
- Subjects
- Carbon metabolism, Cell Respiration physiology, Ethanol metabolism, Fermentation, Fluorescence, Gene Expression Regulation, Fungal genetics, Glucose metabolism, Mitochondria metabolism, Oxidative Phosphorylation, Reactive Oxygen Species metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Microscopy, Fluorescence methods, Reactive Oxygen Species analysis
- Abstract
The budding yeast Saccharomyces cerevisiae is a facultative organism that is able to utilize both anaerobic and aerobic metabolism, depending on the composition of carbon source in the growth medium. When glucose is abundant, yeast catabolizes it to ethanol and other by-products by anaerobic fermentation through the glycolysis pathway. Following glucose exhaustion, cells switch to oxygenic respiration (a.k.a. "diauxic shift"), which allows catabolizing ethanol and the other carbon compounds via the TCA cycle and oxidative phosphorylation in the mitochondria. The diauxic shift is accompanied by elevated reactive oxygen species (ROS) levels and is characterized by activation of ROS defense mechanisms. Traditional measurement of the diauxic shift is done through measuring optical density of cultures grown in a batch at intermediate time points and generating a typical growth curve or by estimating the reduction of glucose and accumulation of ethanol in growth media over time. In this manuscript, we describe a method for determining changes in ROS levels upon yeast growth, using carboxy-H(2)-dichloro-dihydrofluorescein diacetate (carboxy-H(2)-DCFDA). H
2 -DCFDA is a widely used fluorescent dye for measuring intracellular ROS levels. H2 -DCFDA enables a direct measurement of ROS in yeast cells at intermediate time points. The outcome of H2 -DCFDA fluorescent readout measurements correlates with the growth curve information, hence providing a clear understanding of the diauxic shift.- Published
- 2021
- Full Text
- View/download PDF
46. Discovery and characterization of a new methionyl-tRNA synthetase in Saccharomyces cerevisiae
- Author
-
Laporte, Daphné and STAR, ABES
- Subjects
TRNA ,Aminoacyl-ARNt synthétase ,Diauxic shift ,Transition diauxique ,Saccharomyces cerevisiae ,[SDV.MP.MYC] Life Sciences [q-bio]/Microbiology and Parasitology/Mycology ,Nucleus ,Noyau ,ARNt ,Aminoacyl-tRNA synthetase ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Transcription ,Clivage ,Cleavage - Abstract
Methionyl-tRNA synthetase (MetRS) is the enzyme in charge of aminocylation of tRNA methionine initiator and elongator (tRNAiMet et tRNAeMet), but also displays atypical functions in Saccharomyces cerevisiae. In the present work, we showed that MetRS is imported to the nucleus during the diauxic shift in order to regulate transcription of genes coding for the complexes III and V subunits of the mitochondrial respiratory chain. To do so, MetRS harbors at least two nuclear localization signals (NLS), located within the 55 first aminoacids (NLS1) and beyond the N-terminal part (NLS2). The N- terminal part is responsible for the recruitment of RNA pol II subunits Rpb4 and Rpb7. We also showed that MetRS is cleaved through the 114th and the 132nd aminoacid during fermentation and that the proteolysed form is essential for the viability of the cell, since a mutant of MetRS which is not cleaved (MetRSK11A) did not allows the growth. We showed that an overproduced and purified a mutant representative of the cleaved form (MetRSΔ142) is more efficient for tRNAiMet aminoacylation than the full length MetRS. Thus, our study suggests that in S. cerevisiae, the cytoplasmic full length MetRS aminoacylates tRNAeMet, the nuclear full length MetRS regulates genes transcription, and the cytoplasmic and nuclear cleaved MetRS aminoacylates the tRNAiMet., La methionyl-ARNt synthétase (MetRS) de Saccharomyces cerevisiae aminoacyle les ARNt méthionine initiateur et élongateur (ARNtiMet et ARNteMet), mais possède également des fonctions atypiques. Nous avons montré que la MetRS rejoint le noyau durant la transition diauxique afin de réguler la transcription des gènes nucléaires des complexes III et V de la chaîne respiratoire mitochondriale. Pour ce faire, la MetRS possède au moins deux signaux de localisation nucléaire (NLS) dans sa séquence, l’un se situant dans les 55 premiers acides aminés (aa) et le second, au delà de la partie N-terminale lui permettant de recruter les sous-unités Rpb4 et Rpb7 de l’ARN pol II. Nous avons montré qu’en fermentation, la MetRS est clivée entre le 114ème et le 132ème aa et que cette forme clivée est essentielle à la viabilité des cellules, puisqu’un variant non clivé (MetRSK11A) ne permet pas la croissance. Nous avons surproduit et purifié un mutant de la MetRS clivée (MetRSΔ142) et montré que ce variant est plus efficace pour l’aminoacylation de l’ARNtiMet que la forme entière de MetRS. Ainsi, notre étude suggère que chez S. cerevisiae, la forme longue de MetRS cytoplasmique permet l’aminoacylation de l’ARNteMet, la forme longue de MetRS nucléaire régule la transcription, et la forme clivée de MetRS nucléaire et cytoplasmique permet l’aminoacylation de l’ARNtiMet
- Published
- 2016
47. The Proteasome Lid Triggers COP9 Signalosome Activity during the Transition of Sachharomyces cerevisiae Cells into Quiescence.
- Author
-
Bramasole, Laylan, Sinha, Abhishek, Harshuk, Dana, Cirigliano, Angela, Sylvia, Gurevich, Yu, Zanlin, Carmeli, Rinat Lift, Glickman, Michael H., Rinaldi, Teresa, and Pick, Elah
- Subjects
- *
PROTEASOMES , *UBIQUITIN ligases , *SACCHAROMYCES cerevisiae , *LIGASES , *CAPS & closures , *ENZYMES , *CELLS - Abstract
The class of Cullin–RING E3 ligases (CRLs) selectively ubiquitinate a large portion of proteins targeted for proteolysis by the 26S proteasome. Before degradation, ubiquitin molecules are removed from their conjugated proteins by deubiquitinating enzymes, a handful of which are associated with the proteasome. The CRL activity is triggered by modification of the Cullin subunit with the ubiquitin-like protein, NEDD8 (also known as Rub1 in Saccharomyces cerevisiae). Cullin modification is then reversed by hydrolytic action of the COP9 signalosome (CSN). As the NEDD8–Rub1 catalytic cycle is not essential for the viability of S. cerevisiae, this organism is a useful model system to study the alteration of Rub1–CRL conjugation patterns. In this study, we describe two distinct mutants of Rpn11, a proteasome-associated deubiquitinating enzyme, both of which exhibit a biochemical phenotype characterized by high accumulation of Rub1-modified Cdc53–Cullin1 (yCul1) upon entry into quiescence in S. cerevisiae. Further characterization revealed proteasome 19S-lid-associated deubiquitination activity that authorizes the hydrolysis of Rub1 from yCul1 by the CSN complex. Thus, our results suggest a negative feedback mechanism via proteasome capacity on upstream ubiquitinating enzymes. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
48. Dynamic regulation of gene expression using sucrose responsive promoters and RNA interference in Saccharomyces cerevisiae
- Author
-
Thomas C. Williams, Monica I. Espinosa, Lars K. Nielsen, and Claudia E. Vickers
- Subjects
Sucrose ,Saccharomyces cerevisiae Proteins ,SUC2 ,Saccharomyces cerevisiae ,Gene Expression ,Bioengineering ,Computational biology ,GFP ,Applied Microbiology and Biotechnology ,Metabolic engineering ,Synthetic biology ,RNA interference ,Gene expression ,Promoter Regions, Genetic ,TEF1 ,Regulation of gene expression ,Genetics ,biology ,Promoter ,Diauxic shift ,biology.organism_classification ,Yeast ,Metabolic Engineering ,RNA Interference ,Technical Notes ,Biotechnology - Abstract
Background Engineering dynamic, environmentally- and temporally-responsive control of gene expression is one of the principle objectives in the field of synthetic biology. Dynamic regulation is desirable because many engineered functions conflict with endogenous processes which have evolved to facilitate growth and survival, and minimising conflict between growth and production phases can improve product titres in microbial cell factories. There are a limited number of mechanisms that enable dynamic regulation in yeast, and fewer still that are appropriate for application in an industrial setting. Results To address this problem we have identified promoters that are repressed during growth on glucose, and activated during growth on sucrose. Catabolite repression and preferential glucose utilisation allows active growth on glucose before switching to production on sucrose. Using sucrose as an activator of gene expression circumvents the need for expensive inducer compounds and enables gene expression to be triggered during growth on a fermentable, high energy-yield carbon source. The ability to fine-tune the timing and population density at which gene expression is activated from the SUC2 promoter was demonstrated by varying the ratio of glucose to sucrose in the growth medium. Finally, we demonstrated that the system could also be used to repress gene expression (a process also required for many engineering projects). We used the glucose/sucrose system to control a heterologous RNA interference module and dynamically repress the expression of a constitutively regulated GFP gene. Conclusions The low noise levels and high dynamic range of the SUC2 promoter make it a promising option for implementing dynamic regulation in yeast. The capacity to repress gene expression using RNA interference makes the system highly versatile, with great potential for metabolic engineering applications. Electronic supplementary material The online version of this article (doi:10.1186/s12934-015-0223-7) contains supplementary material, which is available to authorized users.
- Published
- 2015
49. Novel method for the quantification of inorganic polyphosphate (iPoP) in Saccharomyces cerevisiae shows dependence of iPoP content on the growth phase
- Author
-
Thomas P. Werner, Nikolaus Amrhein, and Florian M. Freimoser
- Subjects
Specific growth ,Growth phase ,Saccharomyces cerevisiae ,Biochemistry ,Microbiology ,Phosphates ,Inorganic polyphosphate ,Polyphosphate quantification method ,chemistry.chemical_compound ,Polyphosphates ,Genetics ,PolyP ,Yeast ,"Overplus" ,Diauxic shift ,Molecular Biology ,biology ,Polyphosphate ,General Medicine ,Limiting ,biology.organism_classification ,Phosphate ,Culture Media ,Glucose ,chemistry - Abstract
Archives of Microbiology, 184 (2), ISSN:0302-8933, ISSN:1432-072X
- Published
- 2005
50. The multisynthetasic AME complex in yeast : dynamics of the complex and non canonical roles of its components
- Author
-
Enkler, Ludovic and STAR, ABES
- Subjects
Translation ,TRNA ,Aminoacyl-ARNt synthétase ,Diauxic shift ,Transition diauxique ,Saccharomyces cerevisiae ,Mitochondrie ,Mitochondria ,ARNt ,Traduction ,Aminoacyl-tRNA synthetase ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Transcription ,Multisynthetase complex ,Complexe multisynthétasique - Abstract
Multisynthetase complexes (MSC) are complexes made of several proteins and were identified in a wide variety of organisms from pro- to eukaryotes. They are usually made of assembly factors and aminoacyl-tRNA synthetases (aaRSs), which are responsible for the aminoacylation of their corresponding tRNAs during translation. Depending on the organisms, size and composition of these complexes differ greatly and their role is not fully understood yet. Although it seems that in eukaryotes, accretions of aaRSs into MSC prevent aaRSs to perform their additional functions. In the yeast Saccharomyces cerevisiae, we show that the dynamic of the AME complex, made of the méthionyl- and glutamyl-tRNA synthetases (MRS and ERS) and the assembly protein Arc1p is linkedto yeast metabolism. In respiration, MRS is imported in the nucleus to act as a transcription factor and regulates the expression of nuclear genes belonging to complex III and V of the respiratory chain, while ERS is imported in mitochondria to activate translation. This study shows that synchronous relocation of both aaRSs is crucial for yeast cells to adapt to respiratory metabolism., Les complexes multisynthétasiques (MSC) sont des complexes multi-protéiques identifiés dans un grand nombre d’organismes pro- et eucaryotes. Ils impliquent des protéines d’assemblages et des aminoacyl-ARNt synthétases (aaRSs), responsables de l’aminoacylation de leurs ARNts homologues au cours de la traduction. La taille et la composition des MSC varient selon les organismes, et le rôle de ces complexes n’est pas encore totalement compris. Il semblerait néanmoins que chez les eucaryotes, l’accrétion en complexe soit une stratégie mise en oeuvre par les cellules pour empêcher les aaRSs d’assurer des fonctions additionnelles. Chez S.cerevisiae,nous montrons que la dynamique du complexe AME, composé de la méthionyl- et de la glutamyl-ARNt synthétase (MRS et ERS) ainsi que de la protéine d’ancrage Arc1p, est dépendante du métabolisme de la levure. En respiration la MRS joue le rôle de facteur de transcription et régule l’expression des gènes nucléaires du complexe III et V de la chaîne respiratoire, tandis que l’ERS active la traduction mitochondriale. Cette étude montre que la relocalisation synchrone est primordiale pour l’adaptation des cellules au métabolisme respiratoire.
- Published
- 2014
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