scholarly journals Effects of Abolishing Whi2 on the Proteome and Nitrogen Catabolite Repression-Sensitive Protein Production

2021 ◽  
Author(s):  
Jennifer J Tate ◽  
Jana Marsikova ◽  
Libuse Vachova ◽  
Zdena Palkova ◽  
Terrance G Cooper
Keyword(s):  
Catabolite Repression ◽  
Protein Production ◽  
Nitrogen Sources ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Synthetic Complete ◽  
Promoter Fusion ◽  
Control Production ◽  
Transcription Activators ◽  
Go Terms

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, Chen et al. (2018) concluded that: (i) TorC1 activity is down regulated by the lower concentration of primarily leucine in SCME relative to SCCSH. (ii) The Whi2-Psr1/2 complex is responsible for this down regulation. 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 hours. What was conspicuously missing were proteins related by TorC1- and NCR-associated GO terms.

scholarly journals Genes of Different Catabolic Pathways Are Coordinately Regulated by Dal81 in Saccharomyces cerevisiae

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Marcos D. Palavecino ◽  
Susana R. Correa-García ◽  
Mariana Bermúdez-Moretti
Keyword(s):  
Catabolite Repression ◽  
Transcriptional Control ◽  
Nitrogen Limitation ◽  
Temporal Order ◽  
Nitrogen Sources ◽  
Nitrogen Utilization ◽  
Aminobutyric Acid ◽  
General Mechanism ◽  
Nitrogen Catabolite Repression ◽  
Catabolic Enzymes

Yeast can use a wide variety of nitrogen compounds. However, the ability to synthesize enzymes and permeases for catabolism of poor nitrogen sources is limited in the presence of a rich one. This general mechanism of transcriptional control is called nitrogen catabolite repression. Poor nitrogen sources, such as leucine, γ-aminobutyric acid (GABA), and allantoin, enable growth after the synthesis of pathway-specific catabolic enzymes and permeases. This synthesis occurs only under conditions of nitrogen limitation and in the presence of a pathway-specific signal. In this work we studied the temporal order in the induction of AGP1, BAP2, UGA4, and DAL7, genes that are involved in the catabolism and use of leucine, GABA, and allantoin, three poor nitrogen sources. We found that when these amino acids are available, cells will express AGP1 and BAP2 in the first place, then DAL7, and at last UGA4. Dal81, a general positive regulator of genes involved in nitrogen utilization related to the metabolisms of GABA, leucine, and allantoin, plays a central role in this coordinated regulation.

scholarly journals Global Expression Analysis of the Yeast Lachancea (Saccharomyces) kluyveri Reveals NewURCGenes Involved in Pyrimidine Catabolism

2013 ◽  
Vol 13 (1) ◽  
pp. 31-42 ◽  
Author(s):  
Anna Andersson Rasmussen ◽  
Dineshkumar Kandasamy ◽  
Halfdan Beck ◽  
Seth D. Crosby ◽  
Olof Björnberg ◽  
...  
Keyword(s):  
Expression Pattern ◽  
Catabolite Repression ◽  
Nitrogen Sources ◽  
Global Changes ◽  
Nitrogen Catabolite Repression ◽  
Content Type ◽  
Double Deletion ◽  
Pyrimidine Catabolism ◽  
Saccharomyces Kluyveri ◽  
Similar Expression Pattern

ABSTRACTPyrimidines are important nucleic acid precursors which are constantly synthesized, degraded, and rebuilt in the cell. Four degradation pathways, two of which are found in eukaryotes, have been described. One of them, theURCpathway, has been initially discovered in our laboratory in the yeastLachancea kluyveri. Here, we present the global changes in gene expression inL. kluyveriin response to different nitrogen sources, including uracil, uridine, dihydrouracil, and ammonia. The expression pattern of the knownURCgenes,URC1-6, helped to identify nine putative novelURCgenes with a similar expression pattern. The microarray analysis provided evidence that both theURCandPYDgenes are under nitrogen catabolite repression inL. kluyveriand are induced by uracil or dihydrouracil, respectively. We determined the function ofURC8, which was found to catalyze the reduction of malonate semialdehyde to 3-hydroxypropionate, the final degradation product of the pathway. The other eight genes studied were all putative permeases. Our analysis of double deletion strains showed that theL. kluyveriFui1p protein transported uridine, just like its homolog inSaccharomyces cerevisiae, but we demonstrated that is was not the only uridine transporter inL. kluyveri. We also showed that theL. kluyverihomologs ofDUR3andFUR4do not have the same function that they have inS. cerevisiae, where they transport urea and uracil, respectively. InL. kluyveri, both of these deletion strains grew normally on uracil and urea.

scholarly journals The Yeast GATA Factor Gat1 Occupies a Central Position in Nitrogen Catabolite Repression-Sensitive Gene Activation

2009 ◽  
Vol 29 (13) ◽  
pp. 3803-3815 ◽  
Author(s):  
Isabelle Georis ◽  
André Feller ◽  
Fabienne Vierendeels ◽  
Evelyne Dubois
Keyword(s):  
Gene Expression ◽  
Catabolite Repression ◽  
Nitrogen Availability ◽  
Gene Activation ◽  
Nitrogen Sources ◽  
Central Position ◽  
Limiting Factor ◽  
Nitrogen Catabolite Repression ◽  
Gata Factor ◽  
Gata Factors

ABSTRACT Saccharomyces cerevisiae cells are able to adapt their metabolism according to the quality of the nitrogen sources available in the environment. Nitrogen catabolite repression (NCR) restrains the yeast's capacity to use poor nitrogen sources when rich ones are available. NCR-sensitive expression is modulated by the synchronized action of four DNA-binding GATA factors. Although the first identified GATA factor, Gln3, was considered the major activator of NCR-sensitive gene expression, our work positions Gat1 as a key factor for the integrated control of NCR in yeast for the following reasons: (i) Gat1 appeared to be the limiting factor for NCR gene expression, (ii) GAT1 expression was regulated by the four GATA factors in response to nitrogen availability, (iii) the two negative GATA factors Dal80 and Gzf3 interfered with Gat1 binding to DNA, and (iv) Gln3 binding to some NCR promoters required Gat1. Our study also provides mechanistic insights into the mode of action of the two negative GATA factors. Gzf3 interfered with Gat1 by nuclear sequestration and by competition at its own promoter. Dal80-dependent repression of NCR-sensitive gene expression occurred at three possible levels: Dal80 represses GAT1 expression, it competes with Gat1 for binding, and it directly represses NCR gene transcription.

scholarly journals Gat1p, a GATA family protein whose production is sensitive to nitrogen catabolite repression, participates in transcriptional activation of nitrogen-catabolic genes in Saccharomyces cerevisiae.

1996 ◽  
Vol 16 (3) ◽  
pp. 847-858 ◽  
Author(s):  
J A Coffman ◽  
R Rai ◽  
T Cunningham ◽  
V Svetlov ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Catabolite Repression ◽  
Nitrogen Sources ◽  
Nitrogen Catabolite Repression ◽  
Triple Mutant ◽  
Regulatory Circuit ◽  
Catabolic Genes ◽  
Family Protein ◽  
Sequence Elements

Saccharomyces cerevisiae cells selectively use nitrogen sources in their environment. Nitrogen catabolite repression (NCR) is the basis of this selectivity. Until recently NCR was thought to be accomplished exclusively through the negative regulation of Gln3p function by Ure2p. The demonstration that NCR-sensitive expression of multiple nitrogen-catabolic genes occurs in a gln3 delta ure2 delta dal80::hisG triple mutant indicated that the prevailing view of the nitrogen regulatory circuit was in need of revision; additional components clearly existed. Here we demonstrate that another positive regulator, designated Gat1p, participates in the transcription of NCR-sensitive genes and is able to weakly activate transcription when tethered upstream of a reporter gene devoid of upstream activation sequence elements. Expression of GAT1 is shown to be NCR sensitive, partially Gln3p dependent, and Dal80p regulated. In agreement with this pattern of regulation, we also demonstrate the existence of Gln3p and Dal80p binding sites upstream of GAT1.

scholarly journals Control of Nitrogen Catabolite Repression Is Not Affected by the tRNAGln-CUU Mutation, Which Results in Constitutive Pseudohyphal Growth of Saccharomyces cerevisiae

1999 ◽  
Vol 181 (8) ◽  
pp. 2472-2476 ◽  
Author(s):  
Alexander E. Beeser ◽  
Terrance G. Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Catabolite Repression ◽  
Doubling Time ◽  
Nitrogen Availability ◽  
Nitrogen Starvation ◽  
Nitrogen Sources ◽  
Nitrogen Catabolite Repression ◽  
Pseudohyphal Growth ◽  
Specific Allele

ABSTRACT Saccharomyces cerevisiae responds to nitrogen availability in several ways. (i) The cell is able to distinguish good nitrogen sources from poor ones through a process designated nitrogen catabolite repression (NCR). Good and poor nitrogen sources do not demonstrably affect the cell cycle other than to influence the cell’s doubling time. (ii) Nitrogen starvation promotes the initiation of sporulation and pseudohyphal growth. (iii) Nitrogen starvation strongly affects the cell cycle; nitrogen-starved cells arrest in G1. A specific allele of the SUP70/CDC65tRNAGln gene (sup70-65) has been reported to be defective in nitrogen signaling associated with pseudohyphal formation, sporulation, and NCR. Our data confirm that pseudohyphal growth occurs gratuitously in sup70-65 mutants cultured in nitrogen-rich medium at 30°C. However, we find neither any defect in NCR in thesup70-65 mutant nor any alteration in the control ofYVH1 expression, which has been previously shown to be specifically induced by nitrogen starvation.

scholarly journals Synergistic Operation of the CAR2(Ornithine Transaminase) Promoter Elements in Saccharomyces cerevisiae

1999 ◽  
Vol 181 (22) ◽  
pp. 7052-7064 ◽  
Author(s):  
Heui-Dong Park ◽  
Stephanie Scott ◽  
Rajendra Rai ◽  
Rosemary Dorrington ◽  
Terrance G. Cooper
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Catabolite Repression ◽  
Early Report ◽  
Formal Structure ◽  
Nitrogen Catabolite Repression ◽  
Ornithine Transaminase ◽  
Transcription Activators ◽  
And Control ◽  
Gata Family

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 ofCAR2 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 theCAR2 promoter with the objectives of determining whether other transcription factors were required for CAR2expression and, if so, of ascertaining their relative contributions toCAR2’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 theCAR2 promoter and its operation closely follow the model proposed for CAR1.

scholarly journals Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae.

1986 ◽  
Vol 6 (11) ◽  
pp. 3954-3964 ◽  
Author(s):  
F S Genbauffe ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Normal Control ◽  
Catabolite Repression ◽  
Gene Products ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Recombinant Plasmids ◽  
Control Pattern ◽  
Steady State Levels

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

Induction and repression of the urea amidolyase gene in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (11) ◽  
pp. 3954-3964
Author(s):  
F S Genbauffe ◽  
T G Cooper
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Steady State ◽  
Normal Control ◽  
Catabolite Repression ◽  
Gene Products ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Recombinant Plasmids ◽  
Control Pattern ◽  
Steady State Levels

The DUR1,2 gene from Saccharomyces cerevisiae has been isolated on recombinant plasmids along with all DNA between the DUR1,2 and MET8 loci. DUR1,2 was found to encode a 5.7-kilobase transcript, which is consistent with our earlier suggestion that the DUR1 and DUR2 loci are two domains of a single multifunctional gene. Steady-state levels of the DUR1,2 transcript responded to induction and nitrogen catabolite repression in the same way as urea amidolyase activity. dal81 mutants (grown with inducer) contained barely detectable amounts of DUR1,2 RNA, whereas dal80 mutants (grown without inducer) contained the same amount as a wild-type induced culture. These observations support our earlier hypothesis that DUR1,2 is transcriptionally regulated, with control being mediated by the DAL80 and DAL81 gene products. We cloned the DUR1,2-Oh mutation and found it to be a Ty insertion near sequences required for complementation of dur1,2 mutations. The ROAM phenotype of the DUR1,2-Oh mutation is sharply different from that of cis-dominant, DUR80 mutations, which enhance DUR1,2 expression but do not affect the normal control pattern of the gene. There is evidence that DUR80 mutations may also be Ty insertions, which generate phenotypes that are different from those in DUR1,2-Oh mutations.

Requirement of upstream activation sequences for nitrogen catabolite repression of the allantoin system genes in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (12) ◽  
pp. 5440-5444
Author(s):  
T G Cooper ◽  
R Rai ◽  
H S Yoo
Keyword(s):  
Catabolite Repression ◽  
Control Process ◽  
Nitrogen Sources ◽  
Transport Systems ◽  
Nitrogen Catabolite Repression ◽  
Widespread Occurrence ◽  
Nitrogenous Compounds ◽  
Cis Acting ◽  
Steady State Levels ◽  
Flanking Regions

Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.

scholarly journals Requirement of upstream activation sequences for nitrogen catabolite repression of the allantoin system genes in Saccharomyces cerevisiae.

1989 ◽  
Vol 9 (12) ◽  
pp. 5440-5444 ◽  
Author(s):  
T G Cooper ◽  
R Rai ◽  
H S Yoo
Keyword(s):  
Catabolite Repression ◽  
Control Process ◽  
Nitrogen Sources ◽  
Transport Systems ◽  
Nitrogen Catabolite Repression ◽  
Widespread Occurrence ◽  
Nitrogenous Compounds ◽  
Cis Acting ◽  
Steady State Levels ◽  
Flanking Regions

Synthesis of the transport systems and enzymes mediating uptake and catabolism of nitrogenous compounds is sensitive to nitrogen catabolite repression. In spite of the widespread occurrence of the control process, little is known about its mechanism. We have previously demonstrated that growth of cells on repressive nitrogen sources results in a dramatic decrease in the steady-state levels of mRNA encoded by the allantoin and arginine catabolic pathway genes and of the transport systems associated with allantoin metabolism. The present study identified the upstream activation sequences in the 5'-flanking regions of the allantoin system genes as the cis-acting sites through which nitrogen catabolite repression is exerted.

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