190 results on '"Terrance G, Cooper"'
Search Results
2. TorC1 and nitrogen catabolite repression control of integrated GABA shunt and retrograde pathway gene expression
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Jennifer J. Tate, Rajendra Rai, and Terrance G. Cooper
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Genetics ,Bioengineering ,Applied Microbiology and Biotechnology ,Biochemistry ,Biotechnology - Published
- 2023
3. Multiple Targets on the Gln3 Transcription Activator Are Cumulatively Required for Control of Its Cytoplasmic Sequestration
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Rajendra Rai, Jennifer J. Tate, and Terrance G. Cooper
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Gln3 ,mTorC1 ,methionine sulfoximine ,nitrogen limitation ,rapamycin ,Genetics ,QH426-470 - Abstract
A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator, Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc13 sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477–493) region required for effective cytoplasmic Gln3-Myc13 sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc13 to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc13 sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477–493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration.
- Published
- 2016
- Full Text
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4. How to think about and do successful research What you probable did not learn when you first entered the laboratory
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Terrance G Cooper
- Subjects
General Medicine ,Applied Microbiology and Biotechnology ,Microbiology - Abstract
There is a logic to doing successful research, but graduate students and indeed postdoctoral fellows and young independent investigators often learn it apprentice style, by experience. The purpose of this essay is to provide the product of that experience and advice that I have found useful to young researchers as they begin their training and careers.
- Published
- 2023
5. Industry and academia—a perfect match
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Hennie J J van Vuuren and Terrance G Cooper
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General Medicine ,Applied Microbiology and Biotechnology ,Microbiology - Abstract
My career developed very differently from those of most academic researchers. After school, I worked for 6 years in industries that employed yeast to manufacture ethanol and beer. At university, I was trained as a microbiologist with very little training in molecular biology. I retrained in 1987 in molecular yeast genetics and focused on genetic engineering of industrial yeasts to minimize the production of spoilage compounds in wine and ethyl carbamate, a carcinogen, in wine. The malolactic yeast ML01 and the urea-degrading yeast were the first genetically enhanced yeasts that obtained US FDA approval for commercial applications. Apart from applied research, I was fascinated by classic molecular yeast genetic studies using sophisticated techniques such as transcriptomics, proteomics, and metabolomics. Doing research at the University of British Columbia was stimulating and exciting, we established a core microarray and metabolomics facility that was used by many scientists at UBC and hospitals in Vancouver. I also established a state-of-the-art Wine Library that was used to study aging of wines produced in British Columbia. Finally, I have been fortunate to know and collaborate with leading yeast scientists who motivated me.
- Published
- 2022
6. Effects of Abolishing Whi2 on Nitrogen Catabolite Repression‐Sensitive GATA‐Factor Localization and Protein Production
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Jennifer J. Tate, Jana Marsikova, Libuse Vachova, Zdena Palkova, and Terrance G. Cooper
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Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2022
7. From blood to stress—my life investigating cell differentiation
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Michael Breitenbach and Terrance G Cooper
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Saccharomyces cerevisiae Proteins ,Cell Cycle ,Humans ,Cell Differentiation ,Saccharomyces cerevisiae ,General Medicine ,Applied Microbiology and Biotechnology ,Microbiology ,Retrospective Studies - Abstract
This short retrospective covers more than 50 years of research. I spent most of it doing yeast genetics and genetic engineering. It has been my great privilege to be part of the international group of yeast genetics researchers. With many of them named in this retrospective, I am connected in lifelong friendships and the same is true for my students and collaborators. The question which we wanted to ask is “How does the genome of the cell and cell differentiation adapt to changing and stressful environmental conditions?” The two examples we studied were sporulation and pseudohyphal growth. Both forms of differentiation are triggered by the stress of starvation. In the pathway of regulation of pseudohyphal growth, a yeast NADPH oxidase (discovered by our group) plays a major role.
- Published
- 2022
8. Effects of abolishing Whi2 on the proteome and nitrogen catabolite repression-sensitive protein production
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Jennifer J Tate, Jana Marsikova, Libuse Vachova, Zdena Palkova, and Terrance G Cooper
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Catabolite Repression ,Saccharomyces cerevisiae Proteins ,Proteome ,Nitrogen ,Gene Expression Regulation, Fungal ,Genetics ,Molecular Biology ,GATA Transcription Factors ,Genetics (clinical) - Abstract
In yeast physiology, a commonly used reference condition for many experiments, including those involving nitrogen catabolite repression (NCR), is growth in synthetic complete (SC) medium. Four SC formulations, SCCSH,1990, SCCSH,1994, SCCSH,2005, and SCME, have been used interchangeably as the nitrogen-rich medium of choice [Cold Spring Harbor Yeast Course Manuals (SCCSH) and a formulation in the methods in enzymology (SCME)]. It has been tacitly presumed that all of these formulations support equivalent responses. However, a recent report concluded that (i) TorC1 activity is downregulated by the lower concentration of primarily leucine in SCME relative to SCCSH. (ii) The Whi2–Psr1/2 complex is responsible for this downregulation. TorC1 is a primary nitrogen-responsive regulator in yeast. Among its downstream targets is control of NCR-sensitive transcription activators Gln3 and Gat1. They in turn control production of catabolic transporters and enzymes needed to scavenge poor nitrogen sources (e.g., Proline) and activate autophagy (ATG14). One of the reporters used in Chen et al. was an NCR-sensitive DAL80-GFP promoter fusion. This intrigued us because we expected minimal if any DAL80 expression in SC medium. Therefore, we investigated the source of the Dal80-GFP production and the proteomes of wild-type and whi2Δ cells cultured in SCCSH and SCME. We found a massive and equivalent reorientation of amino acid biosynthetic proteins in both wild-type and whi2Δ cells even though both media contained high overall concentrations of amino acids. Gcn2 appears to play a significant regulatory role in this reorientation. NCR-sensitive DAL80 expression and overall NCR-sensitive protein production were only marginally affected by the whi2Δ. In contrast, the levels of 58 proteins changed by an absolute value of log2 between 3 and 8 when Whi2 was abolished relative to wild type. Surprisingly, with only two exceptions could those proteins be related in GO analyses, i.e., GO terms associated with carbohydrate metabolism and oxidative stress after shifting a whi2Δ from SCCSH to SCME for 6 h. What was conspicuously missing were proteins related by TorC1- and NCR-associated GO terms.
- Published
- 2021
9. Phosphorylated Residues of the Gln3 Ure2 Relief Sequence Abolish Nuclear Gln3 Localization and Tor1‐Gln3 Interaction
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Rajendra Rai, Terrance G. Cooper, Claudio De Virgilio, and Jennifer J. Tate
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Biochemistry ,Chemistry ,Ure2 ,Genetics ,Phosphorylation ,Molecular Biology ,Biotechnology ,Sequence (medicine) - Published
- 2021
10. N- and C-terminal Gln3–Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization
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Jennifer J. Tate, Rajendra Rai, Claudio De Virgilio, and Terrance G. Cooper
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Protein Conformation, alpha-Helical ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Saccharomyces cerevisiae ,Active Transport, Cell Nucleus ,Biology ,Serine ,Dephosphorylation ,Phosphatidylinositol 3-Kinases ,03 medical and health sciences ,Genetics ,medicine ,Threonine ,Transcription factor ,030304 developmental biology ,Cell Nucleus ,Investigation ,0303 health sciences ,Binding Sites ,030302 biochemistry & molecular biology ,Wild type ,biology.organism_classification ,Cell biology ,medicine.anatomical_structure ,Cytoplasm ,Nucleus ,Protein Binding ,Transcription Factors - Abstract
Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656–666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3–Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3–Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3–Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3–Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3–15-fold with three of them by 10–15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3–Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.
- Published
- 2021
11. Carl Singer (1945-2013) - Life on a Plate: yeast genetics meetings and micromanipulators
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Terrance G. Cooper and Harry Singer
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Male ,Narration ,Yeast genetics ,Humans ,General Medicine ,Saccharomyces cerevisiae ,Biology ,Congresses as Topic ,Applied Microbiology and Biotechnology ,Microbiology ,Tissue Dissection ,Sketch ,Visual arts - Abstract
Micromanipulators, more than any other instrument, opened the early doors to developing the powerful genetics of yeast that underlies much of the molecular work today. The ability to separate the spores of a tetrad and analyze their phenotypes generated the genetic maps and biology upon which subsequent cloning, sequencing, cutting edge molecular and cell biology depended. This work describes the development of those micromanipulators from garage to barn to factory and the developer of the sophisticated instruments we use today. For more than 30 years Carl Singer and his family were staunch and generous supporters of the International Conferences on Yeast Genetics and Molecular Biology meetings both in Europe and America. Carl Singer's displays at meetings became a traditional fixture and engaged the appetites of many students and advanced researchers to employ a technique that many perceived as too complicated or difficult, but which he made simple and easy to learn. His experiences also document a sketch of the international yeast meetings, their venues and how they developed through the years.
- Published
- 2021
12. General Amino Acid Control and 14-3-3 Proteins Bmh1/2 Are Required for Nitrogen Catabolite Repression-Sensitive Regulation of Gln3 and Gat1 Localization
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Rajendra Rai, Terrance G. Cooper, Jennifer J. Tate, and David Buford
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Catabolite Repression ,0301 basic medicine ,Protein kinase complex ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Prions ,Saccharomyces cerevisiae ,Active Transport, Cell Nucleus ,mTORC1 ,Mechanistic Target of Rapamycin Complex 1 ,Protein Serine-Threonine Kinases ,Investigations ,GATA Transcription Factors ,03 medical and health sciences ,Transcription (biology) ,Gene expression ,Genetics ,Amino Acids ,Phosphorylation ,Cell Nucleus ,chemistry.chemical_classification ,Glutathione Peroxidase ,biology ,TOR Serine-Threonine Kinases ,Ure2 ,Epistasis, Genetic ,biology.organism_classification ,Amino acid ,Basic-Leucine Zipper Transcription Factors ,030104 developmental biology ,14-3-3 Proteins ,Biochemistry ,chemistry ,Multiprotein Complexes ,Protein Processing, Post-Translational ,Transcription Factors - Abstract
Nitrogen catabolite repression (NCR), the ability of Saccharomyces cerevisiae to use good nitrogen sources in preference to poor ones, derives from nitrogen-responsive regulation of the GATA family transcription activators Gln3 and Gat1. In nitrogen-replete conditions, the GATA factors are cytoplasmic and NCR-sensitive transcription minimal. When only poor nitrogen sources are available, Gln3 is nuclear, dramatically increasing GATA factor-mediated transcription. This regulation was originally attributed to mechanistic Tor protein kinase complex 1 (mTorC1)-mediated control of Gln3. However, we recently showed that two regulatory systems act cumulatively to maintain cytoplasmic Gln3 sequestration, only one of which is mTorC1. Present experiments demonstrate that the other previously elusive component is uncharged transfer RNA-activated, Gcn2 protein kinase-mediated general amino acid control (GAAC). Gcn2 and Gcn4 are required for NCR-sensitive nuclear Gln3-Myc13 localization, and from epistasis experiments Gcn2 appears to function upstream of Ure2. Bmh1/2 are also required for nuclear Gln3-Myc13 localization and appear to function downstream of Ure2. Overall, Gln3 phosphorylation levels decrease upon loss of Gcn2, Gcn4, or Bmh1/2. Our results add a new dimension to nitrogen-responsive GATA-factor regulation and demonstrate the cumulative participation of the mTorC1 and GAAC pathways, which respond oppositely to nitrogen availability, in the nitrogen-responsive control of catabolic gene expression in yeast.
- Published
- 2017
13. Sit4 and PP2A Dephosphorylate Transcription Activator Gln3 When TorC1 Is Up‐As Well As Down‐Regulated
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Jennifer J. Tate and Terrance G. Cooper
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Chemistry ,Genetics ,Protein phosphatase 2 ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2019
14. Rapamycin‐Dependent Gln3 Transcription Factor Dephosphorylation and Nuclear Localization Depend On Distinct Regions of the Gln3 Protein
- Author
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Jennifer J. Tate and Terrance G. Cooper
- Subjects
Dephosphorylation ,Chemistry ,Genetics ,Molecular Biology ,Biochemistry ,Transcription factor ,Nuclear localization sequence ,Biotechnology ,Cell biology - Published
- 2020
15. 14‐3‐3 Protein‐Dependent GATA Transcription Factor Control in Saccharomyces cerevisiae
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David Buford, Jennifer J. Tate, Terrance G. Cooper, and Rajendra Rai
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biology ,Chemistry ,Saccharomyces cerevisiae ,Genetics ,GATA transcription factor ,biology.organism_classification ,Molecular Biology ,Biochemistry ,14-3-3 protein ,Biotechnology ,Cell biology - Published
- 2018
16. Opening the Doors for Nuclear Gln3 Entry In Saccharomyces cerevisiae
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Terrance G. Cooper, Rajendra Rai, and Jennifer J. Tate
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biology ,Chemistry ,Saccharomyces cerevisiae ,Genetics ,Doors ,biology.organism_classification ,Molecular Biology ,Biochemistry ,Biotechnology ,Cell biology - Published
- 2018
17. Premature termination ofGAT1transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production
- Author
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Isabelle, Georis, Georis, Isabelle, Jennifer J, Tate, Fabienne, Vierendeels, Terrance G, Cooper, and Evelyne, Dubois
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termination ,Gene isoform ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Blotting, Western ,Molecular Sequence Data ,Saccharomyces cerevisiae ,yeast ,Biology ,GATA Transcription Factors ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Protein biosynthesis ,Amino Acid Sequence ,RNA, Messenger ,3' Untranslated Regions ,Molecular Biology ,Gene ,Phylogeny ,Genetics ,Messenger RNA ,Base Sequence ,Models, Genetic ,Reverse Transcriptase Polymerase Chain Reaction ,Three prime untranslated region ,Alternative splicing ,Cell Biology ,Blotting, Northern ,initiation ,Alternative Splicing ,GAT1 ,Protein Biosynthesis ,Transcription Termination, Genetic ,GATA transcription factor ,Erratum ,transcription ,5' Untranslated Regions ,Research Paper - Abstract
The first step in executing the genetic program of a cell is production of mRNA. In yeast, almost every gene is transcribed as multiple distinct isoforms, differing at their 5′ and/or 3′ termini. However, the implications and functional significance of the transcriptome-wide diversity of mRNA termini remains largely unexplored. In this paper, we show that the GAT1 gene, encoding a transcriptional activator of nitrogen-responsive catabolic genes, produces a variety of mRNAs differing in their 5′ and 3′ termini. Alternative transcription initiation leads to the constitutive, low level production of 2 full length proteins differing in their N-termini, whereas premature transcriptional termination generates a short, highly nitrogen catabolite repression- (NCR-) sensitive transcript that, as far as we can determine, is not translated under the growth conditions we used, but rather likely protects the cell from excess Gat1.
- Published
- 2015
18. GATA Factor Regulation in Excess Nitrogen Occurs Independently of Gtr-Ego Complex-Dependent TorC1 Activation
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Terrance G. Cooper, Evelyne Dubois, Jennifer J. Tate, Isabelle Georis, Rajendra Rai, and Fabienne Vierendeels
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Gln3 localization ,Protein kinase complex ,Cytoplasm ,Saccharomyces cerevisiae Proteins ,Genotype ,Nitrogen ,Glutamine ,EGO complex ,tor complex one (TORC1) ,Saccharomyces cerevisiae ,Investigations ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,GATA Transcription Factors ,Nitrogen limitation ,Genes, Reporter ,Tor complex one (TORC1) ,Genetics ,medicine ,nitrogen starvation ,Kinase activity ,Molecular Biology ,Genetics (clinical) ,Monomeric GTP-Binding Proteins ,Cell Nucleus ,Nitrogen catabolite repression (NCR) ,Nitrogen starvation ,TOR Serine-Threonine Kinases ,nitrogen limitation ,Membrane Proteins ,Ego1/3 complex ,Sciences bio-médicales et agricoles ,3. Good health ,Cell biology ,Cell nucleus ,medicine.anatomical_structure ,Biochemistry ,Membrane protein ,Gtr1/2 complex ,Multiprotein Complexes ,Mutation ,nitrogen catabolite repression (NCR) ,GATA transcription factor ,Nuclear localization sequence - Abstract
The TorC1 protein kinase complex is a central component in a eukaryotic cell's response to varying nitrogen availability, with kinase activity being stimulated in nitrogen excess by increased intracellular leucine. This leucine-dependent TorC1 activation requires functional Gtr1/2 and Ego1/3 complexes. Rapamycin inhibition of TorC1 elicits nuclear localization of Gln3, a GATA-family transcription activator responsible for the expression of genes encoding proteins required to transport and degrade poor nitrogen sources, e.g. proline. In nitrogen-replete conditions, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limiting or starvation conditions, or after rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence supports the idea that TorC1 activation may not be as central to nitrogen-responsive intracellular Gln3 localization as envisioned previously. To test this idea directly, we determined whether Gtr1/2- and Ego1/3-dependent TorC1 activation also was required for cytoplasmic Gln3 sequestration and repressed GATA factor-mediated transcription by abolishing the Gtr-Ego complex proteins. We show that Gln3 is sequestered in the cytoplasm of gtr1Δ, gtr2Δ, ego1Δ, and ego3Δ strains either long term in logarithmically glutamine-grown cells or short term after refeeding glutamine to nitrogen-limited or -starved cells; GATA factor2dependent transcription also was minimal. However, in all but a gtr1Δ, nuclear Gln3 localization in response to nitrogen limitation or starvation was adversely affected. Our data demonstrate: (i) Gtr-Ego-dependent TorC1 activation is not required for cytoplasmic Gln3 sequestration in nitrogen-rich conditions; (ii) a novel Gtr-Ego-TorC1 activation-independent mechanism sequesters Gln3 in the cytoplasm; (iii) Gtr and Ego complex proteins participate in nuclear Gln3- Myc13 localization, heretofore unrecognized functions for these proteins; and (iv) the importance of searching for new mechanisms associated with TorC1 activation and/or the regulation of Gln3 localization/function in response to changes in the cells' nitrogen environment., SCOPUS: ar.j, info:eu-repo/semantics/published
- Published
- 2015
19. Nitrogen Starvation and TorC1 Inhibition Differentially Affect Nuclear Localization of the Gln3 and Gat1 Transcription Factors Through the Rare Glutamine tRNACUG in Saccharomyces cerevisiae
- Author
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Jennifer J. Tate, Terrance G. Cooper, and Rajendra Rai
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Saccharomyces cerevisiae Proteins ,Nitrogen ,Recombinant Fusion Proteins ,Mutant ,Active Transport, Cell Nucleus ,Gene Expression ,Saccharomyces cerevisiae ,Investigations ,Biology ,GATA Transcription Factors ,Genes, Reporter ,Gene Expression Regulation, Fungal ,Methionine Sulfoximine ,RNA, Transfer, Gln ,Glutamine synthetase ,Gene expression ,Genetics ,Protein biosynthesis ,Transcription factor ,Sirolimus ,Epistasis, Genetic ,Glutamine ,Protein Transport ,Phenotype ,Biochemistry ,Mutation ,GATA transcription factor ,Nuclear localization sequence ,Transcription Factors - Abstract
A leucine, leucyl-tRNA synthetase–dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of Gln3 and Gat1 to rapamycin inhibition of TorC1 and nitrogen starvation.
- Published
- 2014
20. Editorial: Saccharomyces riding the waves of technology and transition
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Terrance G. Cooper
- Subjects
0301 basic medicine ,Genetics, Microbial ,biology ,Extramural ,MEDLINE ,Historical Article ,General Medicine ,Computational biology ,Mycology ,MOLECULAR BIOLOGY METHODS ,History, 20th Century ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Microbiology ,Saccharomyces ,History, 21st Century ,03 medical and health sciences ,030104 developmental biology ,Editorial ,Humans ,Molecular Biology - Published
- 2017
21. What do the pictures say-snapshots of a career
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Terrance G. Cooper
- Subjects
0301 basic medicine ,Generosity ,media_common.quotation_subject ,education ,Retrospective ,General Medicine ,Saccharomyces cerevisiae ,Space (commercial competition) ,Biology ,History, 20th Century ,Applied Microbiology and Biotechnology ,Biochemistry ,History, 21st Century ,Microbiology ,humanities ,United States ,Visual arts ,03 medical and health sciences ,030104 developmental biology ,Isolation (psychology) ,Workforce ,Humans ,Nitrogen catabolite repression ,Castor beans ,media_common - Abstract
What follows are snapshots of my career in chicken eyes, yeast and Rhodospirillum rubrum, castor beans, Escherichia coli and finally yeast again. In contrast, only a few of the failures that realistically make up a career are included. It is a tale of the generosity and influences of those who shaped what I am and what I learned in a wonderful profession. The science described is only that which I was lucky enough to do or was performed in my laboratory by those who really deserve the credit for any success that I've enjoyed. Not mentioned for lack of space are the critical contributions of many impressive investigators in the field of nitrogen-responsive regulation for no scientific investigation occurs in isolation.
- Published
- 2017
22. A Domain in the Transcription Activator Gln3 Specifically Required for Rapamycin Responsiveness
- Author
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Rajendra Rai, Terrance G. Cooper, Martha M. Howe, Jennifer J. Tate, and Karthik Shanmuganatham
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Models, Molecular ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Catabolite repression ,Saccharomyces cerevisiae ,Biology ,medicine.disease_cause ,Biochemistry ,Serine ,Drug Resistance, Fungal ,medicine ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Transcription factor ,Sirolimus ,chemistry.chemical_classification ,Alanine ,Mutation ,Cell Biology ,Protein Structure, Tertiary ,Amino acid ,Cell biology ,Protein Transport ,Amino Acid Substitution ,chemistry ,Signal transduction ,Signal Transduction ,Transcription Factors - Abstract
Nitrogen-responsive control of Gln3 localization is implemented through TorC1-dependent (rapamycin-responsive) and TorC1-independent (nitrogen catabolite repression-sensitive and methionine sulfoximine (Msx)-responsive) regulatory pathways. We previously demonstrated amino acid substitutions in a putative Gln3 α-helix(656-666), which are required for a two-hybrid Gln3-Tor1 interaction, also abolished rapamycin responsiveness of Gln3 localization and partially abrogated cytoplasmic Gln3 sequestration in cells cultured under nitrogen-repressive conditions. Here, we demonstrate these three characteristics are not inextricably linked together. A second distinct Gln3 region (Gln3(510-589)) is specifically required for rapamycin responsiveness of Gln3 localization, but not for cytoplasmic Gln3 sequestration under repressive growth conditions or relocation to the nucleus following Msx addition. Aspartate or alanine substitution mutations throughout this region uniformly abolish rapamycin responsiveness. Contained within this region is a sequence with a predicted propensity to form an α-helix(583-591), one side of which consists of three hydrophobic amino acids flanked by serine residues. Substitution of aspartate for even one of these serines abolishes rapamycin responsiveness and increases rapamycin resistance without affecting either of the other two Gln3 localization responses. In contrast, alanine substitutions decrease rapamycin resistance. Together, these data suggest that targets in the C-terminal portion of Gln3 required for the Gln3-Tor1 interaction, cytoplasmic Gln3 sequestration, and Gln3 responsiveness to Msx addition and growth in poor nitrogen sources are distinct from those needed for rapamycin responsiveness.
- Published
- 2014
23. Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes
- Author
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Terrance G. Cooper and Jennifer J. Tate
- Subjects
Cell signaling ,Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Phosphatase ,Down-Regulation ,Saccharomyces cerevisiae ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,Biochemistry ,Gene Expression Regulation, Fungal ,TOR complex ,Protein Phosphatase 2 ,Molecular Biology ,Nitrogen cycle ,Sirolimus ,TOR Serine-Threonine Kinases ,Cell Biology ,Protein phosphatase 2 ,G1 Phase Cell Cycle Checkpoints ,Glutamine ,Multiprotein Complexes ,Leucine ,Regulatory Pathway ,Signal Transduction ,Transcription Factors - Abstract
Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc13 localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G1 arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc13 localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal. Background: Five different conditions have been employed interchangeably to down-regulate TorC1. Results: Four of the five conditions exhibit different, hierarchially related phosphatase requirements. Gln3 responses to long-term nitrogen starvation and Msx treatment exhibit the same phosphatase requirements. Conclusion: Previous conditions for down-regulating TorC1 are not physiologically equivalent. Significance: The cellular responses to varying nitrogen supplies occur via multiple, distinguishable regulatory pathways.
- Published
- 2013
24. gln3 Mutations Dissociate Responses to Nitrogen Limitation (Nitrogen Catabolite Repression) and Rapamycin Inhibition of TorC1
- Author
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Terrance G. Cooper, Rajendra Rai, David R. Nelson, and Jennifer J. Tate
- Subjects
Cytoplasm ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Glutamine ,Saccharomyces cerevisiae ,mTORC1 ,Mechanistic Target of Rapamycin Complex 1 ,Biology ,GATA Transcription Factors ,Biochemistry ,Protein Structure, Secondary ,Two-Hybrid System Techniques ,Protein Interaction Mapping ,Fluorescent Antibody Technique, Indirect ,Molecular Biology ,Transcription factor ,Sirolimus ,chemistry.chemical_classification ,Models, Genetic ,TOR Serine-Threonine Kinases ,Wild type ,Cell Biology ,Amino acid ,chemistry ,Multiprotein Complexes ,Mutation ,GATA transcription factor ,Gene Deletion ,Intracellular ,Plasmids ,Transcription Factors ,Signal Transduction - Abstract
The GATA family transcription activator, Gln3 responds to the nitrogen requirements and environmental resources of the cell. When rapidly utilized, "good" nitrogen sources, e.g., glutamine, are plentiful, Gln3 is completely sequestered in the cytoplasm, and the transcription it mediates is minimal. In contrast, during nitrogen-limiting conditions, Gln3 quickly relocates to the nucleus and activates transcription of genes required to scavenge alternative, "poor" nitrogen sources, e.g., proline. This physiological response has been designated nitrogen catabolite repression (NCR). Because rapamycin treatment also elicits nuclear Gln3 localization, TorC1 has been thought to be responsible for NCR-sensitive Gln3 regulation. However, accumulating evidence now suggests that GATA factor regulation may occur by two separate pathways, one TorC1-dependent and the other NCR-sensitive. Therefore, the present experiments were initiated to identify Gln3 amino acid substitutions capable of dissecting the individual contributions of these pathways to overall Gln3 regulation. The rationale was that different regulatory pathways might be expected to operate through distinct Gln3 sensor residues. We found that C-terminal truncations or amino acid substitutions in a 17-amino acid Gln3 peptide with a predicted propensity to fold into an α-helix partially abolished the ability of the cell to sequester Gln3 in the cytoplasm of glutamine-grown cells and eliminated the rapamycin response of Gln3 localization, but did not adversely affect its response to limiting nitrogen. However, overall wild type control of intracellular Gln3 localization requires the contributions of both individual regulatory systems. We also found that Gln3 possesses at least one Tor1-interacting site in addition to the one previously reported.
- Published
- 2013
25. Editorial: Retrospectives - lives behind the science
- Author
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Terrance G. Cooper
- Subjects
Biomedical Research ,Library science ,General Medicine ,Scientific literature ,Biology ,History, 20th Century ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Microbiology ,History, 21st Century ,Health science ,Center (algebra and category theory) ,Periodicals as Topic ,Memphis ,Editorial Policies - Abstract
Scientific investigation is an intensely and uniquely human activity. The collective record of those activities is passed with unerring accuracy from one generation to the next. It's called the scientific literature. What's almost totally missing from that literature, however, are the personal stories of those who produced it. The challenges, triumphs and disappointments of their career journeys. The laboratory lore that is … [↵][1]*Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, 858 Madison, Ave., Memphis Tennessee 38163, USA; E-mail: tcooper{at}uthsc.edu [1]: #xref-corresp-1-1
- Published
- 2016
26. Piotr P. Slonimski - The Warrior Pope: The discovery of mitochondrial (petite) mutants and split genes
- Author
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Piotr P Slonimski, Robert C Jack von Borstel, and Terrance G. Cooper
- Subjects
0301 basic medicine ,Mycology ,Biology ,Applied Microbiology and Biotechnology ,Microbiology ,DNA, Mitochondrial ,German ,03 medical and health sciences ,0302 clinical medicine ,Nothing ,Yeasts ,Potpourri ,Blitzkrieg ,World War II ,General Medicine ,History, 20th Century ,Romance ,language.human_language ,Mitochondria ,030104 developmental biology ,Genes, Mitochondrial ,Action (philosophy) ,Memoir ,Mutation ,language ,Corrigendum ,030217 neurology & neurosurgery ,Classics - Abstract
Memoirs are more often written by generals than by scientists. Although the highest rank I received during my five years in the Polish Underground Army, Armia Krajowa, did not exceed that of a corporal-chief (not a very brilliant accomplishment!), it would have been much easier, and possibly even more interesting for the readers, to write about this period. At least there was action, blood, fantastic friends and beautiful girls: a more-or-less classical potpourri of cliches that a survivor (who, in the language we are accustomed to, is nothing more than a random colony that survives on a petri plate after a drastic treatment with a highly efficient inhibitor) might use. But this is not what I’m asked to do. I have to write about yeast genetics. Strangely enough, there is a link between my interest in genetics and my ancient terrorist activities. It is an indirect, devious link, but an essential link, nevertheless. As a child, I was interested in natural sciences, and certainly, this was because of a long-standing family tradition. My father was an embryologist and histologist investigating blood formation at the University of Warsaw. Grandfathers, Chaim Zelig Slonimski and Abraham Stern, were mathematicians and astronomers. I collected beetles, grew tadpoles and paramecia in stinking water, and read ‘Microbe Hunters’ and Claude Bernard. In 1943, von Bertalanffy led me to Franz Moewus, and Franz Moewus inspired me to work on the chemical nature of the gene. And here is how that came about. Joining the Army at the age of 16 1/2 is not a good idea in peace-time, but the notion is even less romantic in war-time when there were two opposing fronts with a German blitzkrieg force at one of them and the other populated by Russian forces ready to pounce, and I as a young …
- Published
- 2016
27. Nitrogen-responsive Regulation of GATA Protein Family Activators Gln3 and Gat1 Occurs by Two Distinct Pathways, One Inhibited by Rapamycin and the Other by Methionine Sulfoximine
- Author
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Jennifer J. Tate, Isabelle Georis, Terrance G. Cooper, and Evelyne Dubois
- Subjects
Cell Nucleus ,Sirolimus ,Regulation of gene expression ,Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Glutamine ,Promoter ,Saccharomyces cerevisiae ,Cell Biology ,Protein phosphatase 2 ,Biology ,Response Elements ,GATA Transcription Factors ,Biochemistry ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Methionine Sulfoximine ,Glutamine synthetase ,Gene expression ,GATA transcription factor ,Molecular Biology ,Transcription factor ,Transcription Factors ,Signal Transduction - Abstract
Nitrogen availability regulates the transcription of genes required to degrade non-preferentially utilized nitrogen sources by governing the localization and function of transcription activators, Gln3 and Gat1. TorC1 inhibitor, rapamycin (Rap), and glutamine synthetase inhibitor, methionine sulfoximine (Msx), elicit responses grossly similar to those of limiting nitrogen, implicating both glutamine synthesis and TorC1 in the regulation of Gln3 and Gat1. To better understand this regulation, we compared Msx- versus Rap-elicited Gln3 and Gat1 localization, their DNA binding, nitrogen catabolite repression-sensitive gene expression, and the TorC1 pathway phosphatase requirements for these responses. Using this information we queried whether Rap and Msx inhibit sequential steps in a single, linear cascade connecting glutamine availability to Gln3 and Gat1 control as currently accepted or alternatively inhibit steps in two distinct parallel pathways. We find that Rap most strongly elicits nuclear Gat1 localization and expression of genes whose transcription is most Gat1-dependent. Msx, on the other hand, elicits nuclear Gln3 but not Gat1 localization and expression of genes that are most Gln3-dependent. Importantly, Rap-elicited nuclear Gln3 localization is absolutely Sit4-dependent, but that elicited by Msx is not. PP2A, although not always required for nuclear GATA factor localization, is highly required for GATA factor binding to nitrogen-responsive promoters and subsequent transcription irrespective of the gene GATA factor specificities. Collectively, our data support the existence of two different nitrogen-responsive regulatory pathways, one inhibited by Msx and the other by rapamycin.
- Published
- 2011
28. Rapamycin-induced Gln3 Dephosphorylation Is Insufficient for Nuclear Localization
- Author
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Isabelle Georis, André Feller, Jennifer J. Tate, Terrance G. Cooper, and Evelyne Dubois
- Subjects
Activator (genetics) ,Phosphatase ,Cell Biology ,Protein phosphatase 2 ,Biology ,Biochemistry ,Dephosphorylation ,Cell nucleus ,medicine.anatomical_structure ,medicine ,Phosphorylation ,Molecular Biology ,Cellular compartment ,Nuclear localization sequence - Abstract
Gln3, the major activator of nitrogen catabolite repression (NCR)-sensitive transcription, is often used as an assay of Tor pathway regulation in Saccharomyces cerevisiae. Gln3 is cytoplasmic in cells cultured with repressive nitrogen sources (Gln) and nuclear with derepressive ones (Pro) or after treating Gln-grown cells with the Tor inhibitor, rapamycin (Rap). In Raptreated or Pro-grown cells, Sit4 is posited to dephosphorylate Gln3, which then dissociates from a Gln3-Ure2 complex and enters the nucleus. However, in contrast with this view, Sit4-dependent Gln3 dephosphorylation is greater in Gln than Pro. Investigating this paradox, we show that PP2A (another Tor pathway phosphatase)-dependent Gln3 dephosphorylation is regulated oppositely to that of Sit4, being greatest in Pro- and least in Gln-grown cells. It thus parallels nuclear Gln3 localization and NCR-sensitive transcription. However, because PP2A is not required for nuclear Gln3 localization in Pro, PP2A-dependent Gln3 dephosphorylation and nuclear localization are likely parallel responses to derepressive nitrogen sources. In contrast, Rap-induced nuclear Gln3 localization absolutely requires all four PP2A components (Pph21/22, Tpd3, Cdc55, and Rts1). In pph21Δ22Δ, tpd3Δ, or cdc55Δ cells, however, Gln3 is dephosphorylated to the same level as in Rap-treated wild-type cells, indicating Rap-induced Gln3 dephosphorylation is insufficient to achieve nuclear localization. Finally, PP2A-dependent Gln3 dephosphorylation parallels conditions where Gln3 is mostly nuclear, while Sit4-dependent and Rap-induced dephosphorylation parallels those where Gln3 is mostly cytoplasmic, suggesting the effects of these phosphatases on Gln3 may occur in different cellular compartments.
- Published
- 2009
29. Formalin can alter the intracellular localization of some transcription factors inSaccharomyces cerevisiae
- Author
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Terrance G. Cooper and Jennifer J. Tate
- Subjects
Cytoplasm ,Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Osmotic shock ,Nitrogen ,Saccharomyces cerevisiae ,Biology ,GATA Transcription Factors ,Applied Microbiology and Biotechnology ,Microbiology ,Article ,Fixatives ,Osmotic Pressure ,Transcription (biology) ,Formaldehyde ,Gene Expression Regulation, Fungal ,medicine ,Transcription factor ,Cell Nucleus ,Sirolimus ,Osmotic concentration ,General Medicine ,biology.organism_classification ,Molecular biology ,Repressor Proteins ,Cell nucleus ,medicine.anatomical_structure ,Microscopy, Fluorescence ,GATA transcription factor ,Transcription Factors - Abstract
Indirect immunofluorescence (IF) microscopy is one of the most frequently employed methods to determine intracellular protein localization in yeast. It is especially useful for low abundance proteins, eg., the GATA-factors (Gln3, Gat1) which activate NCR-sensitive transcription. Limiting the nitrogen supply or treating cells with the Tor pathway inhibitor, rapamycin, elicits nuclear GATA-factor localization and increased NCR-sensitive transcription, whereas excess nitrogen restricts these proteins to the cytoplasm and decreases transcription. The initial step of the IF procedure is formalin-fixation that quenches cellular activity and fixes protein locations via cross-linking. Indeed, it is on the success of this immobilization step that the assay depends. We have found that under some conditions, formalin itself can influence GATA-factor localization. With low concentrations of formalin (1.6%), Gat1-Myc13 became more nuclear, and with higher concentrations (5.6%), it became more cytoplasmic. Gln3-Myc13 localization, on the other hand, did not respond to low formalin, but became more cytoplasmic at the higher concentration. Interestingly, the high concentration of formalin had no demonstrable effect when the GATA-factors were completely nuclear, i.e., after rapamycin-(Gat1-Myc13) or Msx-(Gln3-Myc13) treatment. Our data indicate that these effects are most likely elicited by methylene and polyoxymethylene glycols, which account for more than 99% of the formaldehyde in formalin. These compounds greatly increased the osmolarity of the medium (0.5–2) and leads us to suggest that varying degrees of osmotic stress, to which both Gln3 and Gat1 are known to respond, and protein movement in response to it can occur after the beginning of fixation but before proteins become immobilized. Therefore, precautions are required to minimize the problem if possible or to account for it during interpretation of IF or other experimental data derived from cells treated with formalin when it cannot be avoided.
- Published
- 2008
30. Tor Pathway Control of the Nitrogen-responsive DAL5 Gene Bifurcates at the Level of Gln3 and Gat1 Regulation in Saccharomyces cerevisiae
- Author
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Isabelle Georis, Evelyne Dubois, Terrance G. Cooper, and Jennifer J. Tate
- Subjects
Cytoplasm ,Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Nitrogen ,Prions ,Glutamine ,Phosphatase ,Active Transport, Cell Nucleus ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,Biology ,GATA Transcription Factors ,Biochemistry ,Phosphatidylinositol 3-Kinases ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Gene expression ,Protein Phosphatase 2 ,Molecular Biology ,Gene ,Transcription factor ,Cell Nucleus ,Sirolimus ,Glutathione Peroxidase ,Kinase ,Mechanisms of Signal Transduction ,Ure2 ,Membrane Transport Proteins ,Cell Biology ,Repressor Proteins ,Phosphotransferases (Alcohol Group Acceptor) ,Transcription Factors - Abstract
The Tor1,2 protein kinases globally influence many cellular processes including nitrogen-responsive gene expression that correlates with intracellular localization of GATA transcription activators Gln3 and Gat1/Nil1. Gln3-Myc13 and Gat1-Myc13 are restricted to the cytoplasm of cells provided with good nitrogen sources, e.g. glutamine. Following the addition of the Tor1,2 inhibitor, rapamycin, both transcription factors relocate to the nucleus. Gln3-Myc13 localization is highly dependent upon Ure2 and type 2A-related phosphatase, Sit4. Ure2 is required for Gln3 to be restricted to the cytoplasm of cells provided with good nitrogen sources, and Sit4 is required for its location to the nucleus following rapamycin treatment. The paucity of analogous information concerning Gat1 regulation prompted us to investigate the effects of deleting SIT4 and URE2 on Gat1-Myc13 localization, DNA binding, and NCR-sensitive transcription. Our data demonstrate that Tor pathway control of NCR-responsive transcription bifurcates at the regulation of Gln3 and Gat1. Gat1-Myc13 localization is not strongly influenced by deleting URE2, nor is its nuclear targeting following rapamycin treatment strongly dependent on Sit4. ChIP experiments demonstrated that Gat1-Myc13 can bind to the DAL5 promoter in the absence of Gln3. Gln3-Myc13, on the other hand, cannot bind to DAL5 in the absence of Gat1. We conclude that: (i) Tor pathway regulation of Gat1 differs markedly from that of Gln3, (ii) nuclear targeting of Gln3-Myc13 is alone insufficient for its recruitment to the DAL5 promoter, and (iii) the Tor pathway continues to play an important regulatory role in NCR-sensitive transcription even after Gln3-Myc13 is localized to the nucleus.
- Published
- 2008
31. Nuclear Gln3 Import Is Regulated by Nitrogen Catabolite Repression Whereas Export Is Specifically Regulated by Glutamine
- Author
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Terrance G. Cooper, Martha M. Howe, Karthik Shanmuganatham, David R. Nelson, Rajendra Rai, and Jennifer J. Tate
- Subjects
Catabolite Repression ,Cytoplasm ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Glutamine ,Catabolite repression ,Active Transport, Cell Nucleus ,Saccharomyces cerevisiae ,Biology ,Investigations ,Transcription (biology) ,Genetics ,medicine ,Nuclear export signal ,Transcription factor ,Cell Nucleus ,Binding Sites ,Cell nucleus ,Regulon ,medicine.anatomical_structure ,Biochemistry ,Amino Acid Substitution ,Mutation ,Nuclear transport ,Transcription Factors - Abstract
Gln3, a transcription activator mediating nitrogen-responsive gene expression in Saccharomyces cerevisiae, is sequestered in the cytoplasm, thereby minimizing nitrogen catabolite repression (NCR)-sensitive transcription when cells are grown in nitrogen-rich environments. In the face of adverse nitrogen supplies, Gln3 relocates to the nucleus and activates transcription of the NCR-sensitive regulon whose products transport and degrade a variety of poorly used nitrogen sources, thus expanding the cell’s nitrogen-acquisition capability. Rapamycin also elicits nuclear Gln3 localization, implicating Target-of-rapamycin Complex 1 (TorC1) in nitrogen-responsive Gln3 regulation. However, we long ago established that TorC1 was not the sole regulatory system through which nitrogen-responsive regulation is achieved. Here we demonstrate two different ways in which intracellular Gln3 localization is regulated. Nuclear Gln3 entry is regulated by the cell’s overall nitrogen supply, i.e., by NCR, as long accepted. However, once within the nucleus, Gln3 can follow one of two courses depending on the glutamine levels themselves or a metabolite directly related to glutamine. When glutamine levels are high, e.g., glutamine or ammonia as the sole nitrogen source or addition of glutamine analogues, Gln3 can exit from the nucleus without binding to DNA. In contrast, when glutamine levels are lowered, e.g., adding additional nitrogen sources to glutamine-grown cells or providing repressive nonglutamine nitrogen sources, Gln3 export does not occur in the absence of DNA binding. We also demonstrate that Gln3 residues 64–73 are required for nuclear Gln3 export.
- Published
- 2015
32. The Gln3 Response To Nitrogen‐Rich Environments Is Independent of Vam6‐, Gtr1/2‐, Ego1/3‐Dependent TorC1 Activation
- Author
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Fabienne Vierendeels, Isabelle Georis, Terrance G. Cooper, Evelyne Dubois, Jennifer J. Tate, and Rajendra Rai
- Subjects
Chemistry ,Kinase ,Multiple modes ,Biochemistry ,Cell biology ,Nitrogen rich ,Cytoplasm ,Transcription (biology) ,Rapamycin treatment ,Genetics ,Molecular Biology ,Nuclear localization sequence ,Intracellular ,Biotechnology - Abstract
Vam6, Gtr1/2, and Ego1/3 are required for leucine-dependent TorC1 kinase activation which is central to nitrogen-responsive regulation. However, Gln3, a nitrogen-responsive transcription activator, does not respond to leucine-dependent TorC1 activation. In nitrogen excess, Gln3 is cytoplasmic and Gln3-mediated transcription minimal, whereas in nitrogen limitation, starvation, or following rapamycin treatment, Gln3 is nuclear and transcription greatly increased. Increasing evidence demonstrates nitrogen-responsive intracellular Gln3 localization is subject to multiple modes of regulation. To ascertain whether the Vam6, Gtr-Ego complexes participate in the regulation of Gln3, we determined the requirements of the above proteins for nuclear localization and cytoplasmic sequestration of Gln3 in response to nitrogen excess, starvation or limitation. We show that Gln3 is sequestered in the cytoplasm of vam6Δ, gtr1Δ, gtr2Δ, ego1Δ and ego3Δ either long-term in logarithmically glutamine-grown cells or short-term a...
- Published
- 2015
33. Glutamine tRNA CUG ‐Dependent, Nitrogen‐Responsive Nuclear Gln3 and Gat1 Localization
- Author
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Terrance G. Cooper, Rajendra Rai, and Jennifer J. Tate
- Subjects
Chemistry ,Kinase ,Stimulation ,Biochemistry ,Cell biology ,Glutamine ,medicine.anatomical_structure ,Downregulation and upregulation ,Cytoplasm ,Transcription (biology) ,Genetics ,Protein biosynthesis ,medicine ,Molecular Biology ,Nucleus ,Biotechnology - Abstract
A leucine-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis. However, Gln3 and Gat1, nitrogen-responsive transcription activators, don't respond to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down regulation of Gln3 occurs via a second mechanism. A major site of Gln3/Gat1 control occurs at their access to the nucleus. In excess nitrogen, Gln3/Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear when nitrogen or glutamine is limiting. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis by methionine sulfoximine (Msx) prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on Gln3/Gat1 regulation. Contrary to prediction, we show nuclear Gln3 localization - elicited by short- and long-term nitrogen starvation, growth in proline, during Msx or rapamycin treatment or in a ure2Δ - is abolished by alteration of glutamine tRNACUG. In ...
- Published
- 2015
34. Differing responses of Gat1 and Gln3 phosphorylation and localization to rapamycin and methionine sulfoximine treatment in Saccharomyces cerevisiae
- Author
-
Ajit Kulkarni, Rajendra Rai, Terrance G. Cooper, and Thomas D. Buford
- Subjects
Catabolite repression ,Hyperphosphorylation ,General Medicine ,Biology ,Applied Microbiology and Biotechnology ,Microbiology ,Cell nucleus ,medicine.anatomical_structure ,Biochemistry ,Transcription (biology) ,Glutamine synthetase ,medicine ,GATA transcription factor ,Phosphorylation ,Transcription factor - Abstract
Gln3 and Gat1/Nil1 are GATA-family transcription factors responsible for transcription of nitrogen-catabolic genes in Saccharomyces cerevisiae. Intracellular Gln3 localization and Gln3-dependent transcription respond in parallel to the nutritional environment and inhibitors of Tor1/2 (rapamycin) and glutamine synthetase (L-methionine sulfoximine, MSX). However, detectable Gln3 phosphorylation, though influenced by nutrients and inhibitors, correlates neither with Gln3 localization nor nitrogen catabolite repression-sensitive transcription in a consistent way. To establish relationships between Gln3 and Gat1 regulation, we performed experiments parallel to those we previously reported for Gln3. Gat1 and Gln3 localization are similar during steady-state growth, being cytoplasmic and nuclear with good and poor nitrogen sources, respectively. Localization correlates with Gat1- and Gln3-mediated transcription. In contrast, three characteristics of Gat1 and Gln3 differ significantly: (i) the kinetics of their localization in response to nutritional transitions and rapamycin-treatment; (ii) their opposite responses to MSX-treatment, i.e. that cytoplasmic Gln3 becomes nuclear following MSX addition, whereas nuclear Gat1 becomes cytoplasmic; and (iii) their phosphorylation levels in the above situations. In instances where Gln3 phosphorylation can be straightforwardly demonstrated to change, Gat1 phosphorylation (in the same samples) appears invariant. The only exception was following carbon starvation, where Gat1, like Gln3, is hyperphosphorylated in a Snf1-dependent manner. However, neither carbon starvation nor MSX treatment elicits Snf1-independent Gat1 hyperphosphorylation, as observed for Gln3.
- Published
- 2006
35. Synergistic operation of four -acting elements mediate high level transcription in
- Author
-
Thomas D. Buford, Jennifer J. Tate, Terrance G. Cooper, Rajendra Rai, and Jon R. Daugherty
- Subjects
Saccharomyces cerevisiae ,Catabolite repression ,General Medicine ,Biology ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Microbiology ,Biochemistry ,Transcription (biology) ,Transcription Coactivator ,Gene expression ,Electrophoretic mobility shift assay ,Binding site ,Gene - Abstract
The Saccharomyces cerevisiae allantoate/ureidosuccinate permease gene (DAL5) is often used as a reporter in studies of the Tor1/2 protein kinases which are specifically inhibited by the clinically important immunosuppressant and anti-neoplastic drug, rapamycin. To date, only a single type of cis-acting element has been shown to be required for DAL5 expression, two copies of the GATAA-containing UASNTR element that mediates nitrogen catabolite repression-sensitive transcription. UASNTR is the binding site for the transcriptional activator, Gln3 whose intracellular localization responds to the nitrogen supply, accumulating in the nuclei of cells provided with poor nitrogen sources and in the cytoplasm when excess nitrogen is available. Recent data raised the possibility that DAL5 might also be regulated by the retrograde system responsible for control of early TCA cycle gene expression, prompting us to investigate the structure of the DAL5 promoter in more detail. Here, we show that clearly one (UASB), and possibly two (UASA), additional cis-acting elements are required for full DAL5 expression. One of these elements (UASB) is in a region that is heavily protected from DNaseI digestion and functions in a highly synergistic manner with the two UASNTR elements. Cis-acting elements UASNTR–UASA and UASNTR–UASB are situated on the same face of the DNA two and one turn apart, respectively. We also found that decreased DAL5 expression in glutamate-grown cells, a characteristic shared with retrograde regulation, likely derives from decreased nuclear Gln3 levels that occur under these growth conditions rather than direct retrograde system control.
- Published
- 2004
36. Actin Cytoskeleton Is Required For Nuclear Accumulation of Gln3 in Response to Nitrogen Limitation but Not Rapamycin Treatment in Saccharomyces cerevisiae
- Author
-
Terrance G. Cooper, Kathleen H. Cox, and Jennifer J. Tate
- Subjects
Saccharomyces cerevisiae Proteins ,Nitrogen ,Active Transport, Cell Nucleus ,Arp2/3 complex ,Cell Cycle Proteins ,Saccharomyces cerevisiae ,macromolecular substances ,Biology ,Biochemistry ,Article ,Phosphatidylinositol 3-Kinases ,Cytoskeleton ,Molecular Biology ,Transcription factor ,Actin ,Phosphoinositide-3 Kinase Inhibitors ,Sirolimus ,Cell Biology ,Bridged Bicyclo Compounds, Heterocyclic ,Actin cytoskeleton ,Actins ,Cell biology ,Repressor Proteins ,Phosphotransferases (Alcohol Group Acceptor) ,Protein Transport ,Thiazoles ,Profilin ,Cytoplasm ,biology.protein ,Thiazolidines ,Latrunculin ,Transcription Factors - Abstract
Saccharomyces cerevisiae selectively utilizes good nitrogen sources in preference to poor ones by down-regulating transcription of genes encoding proteins that transport and degrade poor nitrogen sources when excess nitrogen is available. This regulation is designated nitrogen catabolite repression (NCR). When cells are transferred from a good to a poor nitrogen source (glutamine to proline) or treated with rapamycin, an inhibitor of the protein kinases Tor1/2, Gln3 (NCR-sensitive transcription activator) moves from the cytoplasm into the nucleus. Gln3 re-accumulates in the cytoplasm when cells are returned to a good nitrogen source. However, Gln3 is not uniformly distributed in the cytoplasm. Such non-uniform distribution could result from a variety of interactions including association with a cytoplasmic vesicular system or components of the cytoskeleton. We used latrunculin, a drug that disrupts the actin cytoskeleton by inhibiting actin polymerization, to determine whether the actin cytoskeleton participates in intracellular Gln3 movement. Latrunculin-treatment prevents nuclear accumulation of Gln3 and NCR-sensitive transcription in cells transferred from ammonia to proline medium but does not prevent its accumulation in the cytoplasm of cells transferred from proline to glutamine medium. In contrast, rapamycin-induced nuclear accumulation of Gln3 is not demonstrably affected by latrunculin treatment. These data indicate the actin cytoskeleton is required for nuclear localization of Gln3 in response to limiting nitrogen but not rapamycin-treatment. Therefore, the actin cytoskeleton either participates in the response of Gln3 intracellular localization to nitrogen limitation before Tor1/2, or Tor1/2 inhibition only mimics the outcome of nitrogen limitation rather than directly regulating it.
- Published
- 2004
37. Tor1/2 Regulation of Retrograde Gene Expression in Saccharomyces cerevisiae Derives Indirectly as a Consequence of Alterations in Ammonia Metabolism
- Author
-
Terrance G. Cooper and Jennifer J. Tate
- Subjects
Transcriptional Activation ,Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Time Factors ,Nitrogen ,viruses ,Saccharomyces cerevisiae ,Down-Regulation ,Glutamic Acid ,Cell Cycle Proteins ,Models, Biological ,Biochemistry ,Article ,Histones ,Phosphatidylinositol 3-Kinases ,Ammonia ,Gene Expression Regulation, Fungal ,Gene expression ,Animals ,Molecular Biology ,Gene ,Sirolimus ,Regulation of gene expression ,biology ,Catabolism ,Biological Transport ,Cell Biology ,Metabolism ,Blotting, Northern ,biology.organism_classification ,Glutamine ,Phosphotransferases (Alcohol Group Acceptor) ,Glucose ,Regulon ,Gene Expression Regulation ,Liver ,Fermentation ,Ketoglutaric Acids ,Cattle - Abstract
Retrograde genes of Saccharomyces cerevisiae encode the enzymes needed to synthesize alpha-ketoglutarate, required for ammonia assimilation, when mitochondria are damaged or non-functional because of glucose fermentation. Therefore, it is not surprising that a close association exists between control of the retrograde regulon and expression of nitrogen catabolic genes. Expression of these latter genes is nitrogen catabolite repression (NCR)-sensitive, i.e. expression is low with good nitrogen sources (e.g. glutamine) and high when only poor (e.g. proline) or limiting nitrogen sources are available. It has been reported recently that both NCR-sensitive and retrograde gene expression is negatively regulated by glutamine and induced by treating cells with the Tor1/2 inhibitor, rapamycin. These conclusions predict that NCR-sensitive and retrograde gene expression should respond in parallel to nitrogen sources, ranging from those that highly repress NCR-sensitive transcription to those that elicit minimal NCR. Because this prediction did not accommodate earlier observations that CIT2 (a retrograde gene) expression is higher in glutamine than proline containing medium, we investigated retrograde regulation further. We show that (i) retrograde gene expression correlates with intracellular ammonia and alpha-ketoglutarate generated by a nitrogen source rather than the severity of NCR it elicits, and (ii) in addition to its known regulation by NCR, NAD-glutamate dehydrogenase (GDH2) gene expression is down-regulated by ammonia under conditions where NCR is minimal. Therefore, intracellular ammonia plays a pivotal dual role, regulating the interface of nitrogen and carbon metabolism at the level of ammonia assimilation and production. Our results also indicate the effects of rapamycin treatment on CIT2 transcription, and hence Tor1/2 regulation of retrograde gene expression occur indirectly as a consequence of alterations in ammonia and glutamate metabolism.
- Published
- 2003
38. Ure2, a Prion Precursor with Homology to Glutathione S-Transferase, Protects Saccharomyces cerevisiae Cells from Heavy Metal Ion and Oxidant Toxicity
- Author
-
Terrance G. Cooper, Rajendra Rai, and Jennifer J. Tate
- Subjects
Saccharomyces cerevisiae Proteins ,Prions ,Saccharomyces cerevisiae ,Mutant ,Glutamic Acid ,Biochemistry ,Article ,chemistry.chemical_compound ,Ammonia ,Metals, Heavy ,RNA, Messenger ,Molecular Biology ,Gene ,Glutathione Transferase ,chemistry.chemical_classification ,Glutathione Peroxidase ,biology ,Glutathione peroxidase ,Genetic Complementation Test ,Ure2 ,Cell Biology ,Glutathione ,Oxidants ,biology.organism_classification ,Culture Media ,Kinetics ,Glutathione S-transferase ,chemistry ,biology.protein ,Schizosaccharomyces ,Plasmids - Abstract
Ure2, the protein that negatively regulates GATA factor (Gln3, Gat1)-mediated transcription in Saccharomyces cerevisiae, possesses prion-like characteristics. Identification of metabolic and environmental factors that influence prion formation as well as any activities that prions or prion precursors may possess are important to understanding them and developing treatment strategies for the diseases in which they participate. Ure2 exhibits primary sequence and three-dimensional homologies to known glutathione S-transferases. However, multiple attempts over nearly 2 decades to demonstrate Ure2-mediated S-transferase activity have been unsuccessful, leading to the possibility that Ure2 may well not participate in glutathionation reactions. Here we show that Ure2 is required for detoxification of glutathione S-transferase substrates and cellular oxidants. ure2 Delta mutants are hypersensitive to cadmium and nickel ions and hydrogen peroxide. They are only slightly hypersensitive to diamide, which is nitrogen source-dependent, and minimally if at all hypersensitive to 1-chloro-2,4-dinitrobenzene, the most commonly used substrate for glutathione S-transferase enzyme assays. Therefore, Ure2 shares not only structural homology with various glutathione S-transferases, but ure2 mutations possess the same phenotypes as mutations in known S. cerevisiae and Schizosaccharomyces pombe glutathione S-transferase genes. These findings are consistent with Ure2 serving as a glutathione S-transferase in S. cerevisiae.
- Published
- 2003
39. Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite Repression and the Mechanism of Its Nuclear Localization during Carbon Starvation in Saccharomyces cerevisiae
- Author
-
Jennifer J. Tate, Terrance G. Cooper, and Kathleen H. Cox
- Subjects
Cytoplasm ,Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Nitrogen ,Glutamine ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,Article ,Fungal Proteins ,Gene Expression Regulation, Fungal ,Gene expression ,medicine ,Molecular Biology ,Transcription factor ,Cell Nucleus ,Fungal protein ,Cell Biology ,biology.organism_classification ,Carbon ,Culture Media ,DNA-Binding Proteins ,Repressor Proteins ,Protein Transport ,Cytosol ,Cell nucleus ,medicine.anatomical_structure ,Nuclear localization sequence ,Subcellular Fractions ,Transcription Factors - Abstract
Regulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g. glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus.
- Published
- 2002
40. Transmitting the signal of excess nitrogen inSaccharomyces cerevisiaefrom the Tor proteins to the GATA factors: connecting the dots
- Author
-
Terrance G. Cooper
- Subjects
Antifungal Agents ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Prions ,Regulator ,Saccharomyces cerevisiae ,Biology ,Ribosomal Protein S6 Kinases, 90-kDa ,Microbiology ,Article ,Fungal Proteins ,Gene Expression Regulation, Fungal ,Gene expression ,Transcription factor ,Gene ,Sirolimus ,Regulation of gene expression ,Glutathione Peroxidase ,Ure2 ,Pyrrolidonecarboxylic Acid ,DNA-Binding Proteins ,Repressor Proteins ,Infectious Diseases ,Biochemistry ,Signal transduction ,Regulatory Pathway ,Oligopeptides ,Signal Transduction ,Transcription Factors - Abstract
Major advances have recently occurred in our understanding of GATA factor-mediated, nitrogen catabolite repression (NCR)-sensitive gene expression in Saccharomyces cerevisiae. Under nitrogen-rich conditions, the GATA family transcriptional activators, Gln3 and Gat1, form complexes with Ure2, and are localized to the cytoplasm, which decreases NCR-sensitive expression. Under nitrogen-limiting conditions, Gln3 and Gat1 are dephosphorylated, move from the cytoplasm to the nucleus, in wild-type but not rna1 and srp1 mutants, and increase expression of NCR-sensitive genes. 'Induction' of NCR-sensitive gene expression and dephosphorylation of Gln3 (and Ure2 in some laboratories) when cells are treated with rapamycin implicates the Tor1/2 signal transduction pathway in this regulation. Mks1 is posited to be a negative regulator of Ure2, positive regulator of retrograde gene expression and to be itself negatively regulated by Tap42. In addition to Tap42, phosphatases Sit4 and Pph3 are also argued by some to participate in the regulatory pathway. Although a treasure trove of information has recently become available, much remains unknown (and sometimes controversial) with respect to the precise biochemical functions and regulatory pathway connections of Tap42, Sit4, Pph3, Mks1 and Ure2, and how precisely Gln3 and Gat1 are prevented from entering the nucleus. The purpose of this review is to provide background information needed by students and investigators outside of the field to follow and evaluate the rapidly evolving literature in this exciting field.
- Published
- 2002
41. Gtr1‐Gtr2, Ego1‐Ego3 and Vam6‐independent cytoplasmic Gln3 sequestration in conditions of nitrogen excess (609.17)
- Author
-
Rajendra Rai, Isabelle Georis, Terrance G. Cooper, Evelyne Dubois, and Jennifer J. Tate
- Subjects
Turn (biochemistry) ,chemistry ,Cytoplasm ,Kinase ,Genetics ,Biophysics ,chemistry.chemical_element ,Molecular Biology ,Biochemistry ,Nitrogen ,Biotechnology - Abstract
The Gtr1/2, Ego1/3 complexes and Vam6 have been reported to be required for TorC1 kinase activation. In turn, activated TorC1 is accepted to be required for cytoplasmic Gln3 sequestration based on ...
- Published
- 2014
42. Rapamycin‐responsiveness and Gln3‐Tor1 interaction are associated with different target sites on the Gln3 protein (609.13)
- Author
-
Jennifer J. Tate, Rajendra Rai, and Terrance G. Cooper
- Subjects
Chemistry ,Genetics ,Molecular Biology ,Biochemistry ,Biotechnology - Published
- 2014
43. Constitutive and nitrogen catabolite repression-sensitive production of Gat1 isoforms
- Author
-
Jennifer J. Tate, Isabelle Georis, Terrance G. Cooper, Rajendra Rai, and Evelyne Dubois
- Subjects
Gene isoform ,Saccharomyces cerevisiae Proteins ,Nitrogen ,Glutamine ,Biochimie ,Response element ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,GATA Transcription Factors ,Sp3 transcription factor ,Isomerism ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Amino Acid Sequence ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Transcription Initiation, Genetic ,General transcription factor ,Wild type ,Biologie moléculaire ,Cell Biology ,Mutagenesis ,GATA transcription factor ,Biologie cellulaire ,Protein Processing, Post-Translational ,Signal Transduction ,Transcription Factors - Abstract
Nitrogen catabolite repression (NCR)-sensitive transcription is activatedby Gln3 and Gat1. Innitrogen excess, Gln3 and Gat1 are cytoplasmic, and transcription is minimal. In poor nitrogen, Gln3 and Gat1 become nuclear and activate transcription. A long standing paradox has surrounded Gat1 production. Gat1 was first reported as an NCR-regulated activity mediating NCR-sensitive transcription in gln3 deletion strains. Upon cloning, GAT1 transcription was, as predicted, NCR-sensitive and Gln3- and Gat1-activated. In contrast, Western blots of Gat1-Myc13 exhibited two constitutively produced species. Investigating this paradox, wedemonstrate that wild type Gat1 isoforms (IsoA and IsoB) are initiated at Gat1 methionines 40, 95, and/or 102, but not at methionine 1. Their low level production is the same in rich and poor nitrogen conditions. When the Myc13 tag is placed after Gat1 Ser-233, four N-terminal Gat1 isoforms (IsoC-F) are also initiated at methionines 40, 95, and/or 102. However, their production is highly NCR-sensitive, being greater in proline than glutamine medium. Surprisingly, all Gat1 isoforms produced in sufficient quantities to be confidently analyzed (IsoA, IsoC, and IsoD) require Gln3 and UASGATA promoter elements, both requirements typical of NCR-sensitive transcription. These data demonstrate that regulated Gat1 production is more complex than previously recognized, with wild type versus truncated Gat1 proteins failing to be regulated in parallel. This is the first reported instance of Gln3 UAS GATA-dependent protein production failing to derepress in nitrogen poor conditions. A Gat1-lacZ ORF swap experiment indicated sequence(s) responsible for the nonparallel production are downstream of Gat1 leucine 61. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc., SCOPUS: ar.j, info:eu-repo/semantics/published
- Published
- 2014
44. Components of Golgi-to-vacuole trafficking are required for nitrogen- and TORC1-responsive regulation of the yeast GATA factors
- Author
-
Isabelle Georis, Evelyne Dubois, Mohammad Fayyad-Kazan, Terrance G. Cooper, Jennifer J. Tate, and Fabienne Vierendeels
- Subjects
Saccharomyces cerevisiae Proteins ,Nitrogen ,Saccharomyces cerevisiae ,Biotechnologie ,Golgi Apparatus ,nitrogen availability ,Vacuole ,Nitrogen availability ,Biology ,yeast ,Microbiology ,GATA Transcription Factors ,03 medical and health sciences ,symbols.namesake ,Transcription (biology) ,Gene Expression Regulation, Fungal ,GATA factor ,Rapamycin ,Transcription factor ,030304 developmental biology ,Original Research ,0303 health sciences ,rapamycin ,030302 biochemistry & molecular biology ,Ure2 ,Golgi apparatus ,biology.organism_classification ,Yeast ,3. Good health ,Culture Media ,Biochemistry ,Vacuoles ,symbols ,GATA transcription factor ,Vesicular trafficking ,vesicular trafficking ,Transcription Factors - Abstract
Nitrogen catabolite repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae responds to the available nitrogen status and selectively utilizes rich nitrogen sources in preference to poor ones. Expression of NCR-sensitive genes is mediated by two transcription activators, Gln3 and Gat1, in response to provision of a poorly used nitrogen source or following treatment with the TORC1 inhibitor, rapamycin. During nitrogen excess, the transcription activators are sequestered in the cytoplasm in a Ure2-dependent fashion. Here, we show that Vps components are required for Gln3 localization and function in response to rapamycin treatment when cells are grown in defined yeast nitrogen base but not in complex yeast peptone dextrose medium. On the other hand, Gat1 function was altered in vps mutants in all conditions tested. A significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. Further, our results are consistent with the possibility that Ure2 might function downstream of the Vps components during the control of GATA factor-mediated gene expression. These observations demonstrate distinct media-dependent requirements of vesicular trafficking components for wild-type responses of GATA factor localization and function. As a result, the current model describing participation of Vps system components in events associated with translocation of Gln3 into the nucleus following rapamycin treatment or growth in nitrogen-poor medium requires modification. © 2014 The Authors., SCOPUS: ar.j, info:eu-repo/semantics/published
- Published
- 2014
45. The Level of DAL80 Expression Down-Regulates GATA Factor-Mediated Transcription in Saccharomyces cerevisiae
- Author
-
Thomas S. Cunningham, Terrance G. Cooper, and Rajendra Rai
- Subjects
Saccharomyces cerevisiae Proteins ,Transcription, Genetic ,Nitrogen ,Recombinant Fusion Proteins ,Genes, Fungal ,Response element ,Down-Regulation ,Saccharomyces cerevisiae ,Biology ,GATA Transcription Factors ,Microbiology ,Fungal Proteins ,Transcription (biology) ,Gene Expression Regulation, Fungal ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Regulation of gene expression ,Genetics ,GATA4 ,GATA2 ,Promoter ,Culture Media ,DNA-Binding Proteins ,Repressor Proteins ,Eukaryotic Cells ,GATA transcription factor ,Transcription Factors - Abstract
Nitrogen-catabolic gene expression in Saccharomyces cerevisiae is regulated by the action of four GATA family transcription factors: Gln3p and Gat1p/Nil1p are transcriptional activators, and Dal80 and Deh1p/Gzf3p are repressors. In addition to the GATA sequences situated upstream of all nitrogen catabolite repression-sensitive genes that encode enzyme and transport proteins, the promoters of the GAT1 , DAL80 , and DEH1 genes all contain multiple GATA sequences as well. These GATA sequences are the binding sites of the GATA family transcription factors and are hypothesized to mediate their autogenous and cross regulation. Here we show, using DAL80 fused to the carbon-regulated GAL1 , 10 or copper-regulated CUP1 promoter, that GAT1 expression is inversely regulated by the level of DAL80 expression, i.e., as DAL80 expression increases, GAT1 expression decreases. The amount of DAL80 expression also dictates the level at which DAL3 , a gene activated almost exclusively by Gln3p, is transcribed. Gat1p was found to partially substitute for Gln3p in transcription. These data support the contention that regulation of GATA-factor gene expression is tightly and dynamically coupled. Finally, we suggest that the complicated regulatory circuit in which the GATA family transcription factors participate is probably most beneficial as cells make the transition from excess to limited nitrogen availability.
- Published
- 2000
46. Functional Domain Mapping and Subcellular Distribution of Dal82p in Saccharomyces cerevisiae
- Author
-
Rosemary A. Dorrington, Stephanie Scott, Alexander E. Beeser, Mackenzie Distler, Vladimir Svetlov, and Terrance G. Cooper
- Subjects
Transcriptional Activation ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Saccharomyces cerevisiae ,Peptide ,Biochemistry ,Article ,Fungal Proteins ,chemistry.chemical_compound ,Gene expression ,Binding site ,Molecular Biology ,chemistry.chemical_classification ,Fungal protein ,Binding Sites ,Base Sequence ,biology ,C-terminus ,Membrane Transport Proteins ,DNA ,Cell Biology ,biology.organism_classification ,Molecular biology ,Amino acid ,Cell biology ,chemistry - Abstract
Previous studies have shown that (i) Dal81p and Dal82p are required for allophanate-induced gene expression inSaccharomyces cerevisiae; (ii) the cis-acting element mediating the induced transcriptional response to allophanate is a dodecanucleotide, UIS ALL; and (iii) Dal82p binds specifically to UIS ALL. Here we show that Dal82p is localized to the nucleus and parallels movement of the DNA through the cell cycle. Deletion analysis of DAL82identified and localized three functional domains. Electrophoretic mobility shift assays identified a peptide (consisting of Dal82p amino acids 1–85) that is sufficient to bind a DNA fragment containingUIS ALL. LexA-tethering experiments demonstrated that Dal82p is capable of mediating transcriptional activation. The activation domain consists of two parts: (i) an absolutely required core region (amino acids 66–99) and (ii) less well defined regions flanking residues 66–99 that are required for full wild-type levels of activation. The Dal82p C terminus contains a predicted coiled-coil motif that down-regulates Dal82p-mediated transcriptional activation.
- Published
- 2000
47. Synergistic Operation of the CAR2 (Ornithine Transaminase) Promoter Elements in Saccharomyces cerevisiae
- Author
-
Stephanie Scott, Rajendra Rai, Rosemary A. Dorrington, Heui-Dong Park, and Terrance G. Cooper
- Subjects
Transcriptional Activation ,Saccharomyces cerevisiae Proteins ,Molecular Sequence Data ,Telomere-Binding Proteins ,Catabolite repression ,Saccharomyces cerevisiae ,Biology ,Microbiology ,DNA-binding protein ,Shelterin Complex ,Fungal Proteins ,Transformation, Genetic ,Transcription (biology) ,Gene expression ,Ornithine-Oxo-Acid Transaminase ,Inducer ,Promoter Regions, Genetic ,Molecular Biology ,Transcription factor ,Gene ,Binding Sites ,Base Sequence ,beta-Galactosidase ,DNA-Binding Proteins ,Eukaryotic Cells ,Biochemistry ,Trans-Activators ,5' Untranslated Regions ,Gene Deletion ,Plasmids ,Transcription Factors - Abstract
Dal82p binds to the UIS ALL sites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression. CAR2 , which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer. The dominant activators of CAR2 transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observations prompted us to investigate the structure of the CAR2 promoter with the objectives of determining whether other transcription factors were required for CAR2 expression and, if so, of ascertaining their relative contributions to CAR2 ’s expression and control. We show that Rap1p binds upstream of CAR2 and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer. The observations reported suggest that CAR2 gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is then tipped in the direction of expression by the presence of the inducer. The formal structure of the CAR2 promoter and its operation closely follow the model proposed for CAR1 .
- Published
- 1999
48. The Dual-Specificity Protein Phosphatase Yvh1p Acts Upstream of the Protein Kinase Mck1p in Promoting Spore Development in Saccharomyces cerevisiae
- Author
-
Alexander E. Beeser and Terrance G. Cooper
- Subjects
Saccharomyces cerevisiae Proteins ,Saccharomyces cerevisiae ,Protein tyrosine phosphatase ,Microbiology ,Substrate Specificity ,Fungal Proteins ,Glycogen Synthase Kinase 3 ,Dual-specificity phosphatase ,Nuclear protein ,Protein kinase A ,Molecular Biology ,Fluorescent Dyes ,Fungal protein ,biology ,fungi ,Nuclear Proteins ,Protein-Tyrosine Kinases ,Spores, Fungal ,biology.organism_classification ,Phenotype ,Eukaryotic Cells ,Biochemistry ,biology.protein ,Dual-Specificity Phosphatases ,Protein Tyrosine Phosphatases ,Signal transduction ,Signal Transduction ,Transcription Factors - Abstract
Diploid Saccharomyces cerevisiae cells induce YVH1 expression and enter the developmental pathway, leading to sporulation when starved for nitrogen. We show that yvh1 disruption causes a defect in spore maturation; overexpression of MCK1 or IME1 suppresses this yvh1 phenotype. While mck1 mutations are epistatic to those in yvh1 relative to spore maturation, overexpression of MCK1 does not suppress the yvh1 slow-vegetative-growth phenotype. We conclude that (i) Yvh1p functions earlier than Mck1p and Ime1p in the signal transduction cascade that regulates sporulation and is triggered by nitrogen starvation and (ii) the role of Yvh1p in gametogenesis can be genetically distinguished from its role in vegetative growth.
- Published
- 1999
49. Genome-wide transcriptional analysis inS. cerevisiae by mini-array membrane hybridization
- Author
-
Anna B. Pinchak, Kathleen H. Cox, and Terrance G. Cooper
- Subjects
Genetics ,Bioengineering ,Transcriptional analysis ,Instrumentation (computer programming) ,Biology ,Applied Microbiology and Biotechnology ,Biochemistry ,Gene ,Genome ,Biotechnology - Abstract
Access to the powerful micro-array analytical methods used for genome-wide transcriptional analysis has so far been restricted by the high cost and/or lack of availability of the sophisticated instrumentation and materials needed to perform it. Mini-array membrane hybridization provides a less expensive alternative. The reliability of this technique, however, is not well documented and its reported use has, up to this point, been very limited. Our objective was to test whether or not mini-array membrane hybridization would reliably identify genes whose expression was controlled by a specific set of genetic and/or physiological signals. Our results demonstrate that mini-array hybridization can correctly identify genes whose expression is known to be controlled by the GATA-factor regulatory network in S. cerevisiae and in addition can reliably identify genes not previously reported to be associated with this nitrogen control system. Copyright © 1999 John Wiley & Sons, Ltd.
- Published
- 1999
50. Premature termination of GAT1 transcription explains paradoxical negative correlation between nitrogen-responsive mRNA, but constitutive low-level protein production
- Author
-
Georis Isabelle, Jennifer J Tate, Fabienne Vierendeels, Terrance G Cooper, Evelyne Dubois, Georis Isabelle, Jennifer J Tate, Fabienne Vierendeels, Terrance G Cooper, and Evelyne Dubois
- Published
- 2015
- Full Text
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