scholarly journals The Regulator of the Yeast Proline Utilization Pathway Is Differentially Phosphorylated in Response to the Quality of the Nitrogen Source

2000 ◽  
Vol 20 (3) ◽  
pp. 892-899 ◽  
Author(s):  
Hoching L. Huang ◽  
Marjorie C. Brandriss
Keyword(s):  
Nitrogen Source ◽  
Target Genes ◽  
Nitrogen Sources ◽  
Sole Source ◽  
Wild Type ◽  
Mutant Proteins ◽  
Phosphatase Treatment ◽  
Proline Utilization ◽  
Utilization Pathway

ABSTRACT The proline utilization pathway in Saccharomyces cerevisiae is regulated by the Put3p transcriptional activator in response to the presence of the inducer proline and the quality of the nitrogen source in the growth medium. Put3p is constitutively bound to the promoters of its target genes, PUT1 andPUT2, under all conditions studied but activates transcription to the maximum extent only in the absence of rich nitrogen sources and in the presence of proline (i.e., when proline serves as the sole source of nitrogen). Changes in target gene expression therefore occur through changes in the activity of the DNA-bound regulator. In this report, we demonstrate by phosphatase treatment of immunoprecipitates of extracts metabolically labeled with32P or 35S that Put3p is a phosphoprotein. Examination of Put3p isolated from cells grown on a variety of nitrogen sources showed that it was differentially phosphorylated as a function of the quality of the nitrogen source: the poorer the nitrogen source, the slower the gel migration of the phosphoforms. The presence of the inducer does not detectably alter the phosphorylation profile. Activator-defective and activator-constitutive Put3p mutants have been analyzed. One activator-defective mutant appears to be phosphorylated in a pattern similar to that of the wild type, thus separating its ability to be phosphorylated from its ability to activate transcription. Three activator-constitutive mutant proteins from cells grown on an ammonia-containing medium have a phosphorylation profile similar to that of the wild-type protein in cells grown on proline. These results demonstrate a correlation between the phosphorylation status of Put3p and its ability to activate its target genes and suggest that there are two signals, proline induction and quality of nitrogen source, impinging on Put3p that act synergistically for maximum expression of the proline utilization pathway.

scholarly journals Evidence for Positive Regulation of the Proline Utilization Pathway in Saccharomyces cerevisiae

Genetics ◽  
1987 ◽  
Vol 117 (3) ◽  
pp. 429-435
Author(s):  
Marjorie C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Sole Source ◽  
Lacz Gene ◽  
Wild Type ◽  
Proline Utilization ◽  
Constitutive Mutation ◽  
Lacz Gene Fusions ◽  
Utilization Pathway ◽  
Gene Maps

ABSTRACT A mutation has been identified that prevents Saccharomyces cerevisiae cells from growing on proline as the sole source of nitrogen, causes noninducible expression of the PUT1 and PUT2 genes, and is completely recessive. In the put3-75 mutant, the basal level of expression (ammonia as nitrogen source) of PUT1-lacZ and PUT2-lacZ gene fusions as measured by β-galactosidase activity is reduced 4- and 7-fold, respectively, compared with the wild-type strain. Normal regulation is not restored when the cells are grown on arginine as the sole nitrogen source and put3-75 cells remain sensitive to the proline analog, l-azetidine-2-carboxylic acid, indicating that the block is not at the level of transport of the inducer, proline. In a cross between the put3-75 strain and the semidominant, constitutive mutation PUT3c-68, only parental ditype tetrads were found, indicating allelism of the two mutations. Further support for allelism derives from the comparison of enzyme levels in heteroallelic and heterozygous diploid strains. The constitutive allele appears to be fully dominant to the noninducible allele but only partially dominant to the wild type, suggesting an interaction between the wild-type and PUT3c-68 gene products. The PUT3 gene maps on chromosome XI, about 5.7 cM from the centromere. The phenotypes of alleles of the PUT3 gene, either recessive and noninducible (the put3-75 phenotype) or semidominant and constitutive (the PUT3c-68 phenotype), and their pleiotropy suggest that the PUT3 gene product is a positive activator of the proline utilization pathway.

The roles of PUT3, URE2, and GLN3 regulatory proteins in the proline utilization pathway of Saccharomyces cerevisiae

1995 ◽  
Vol 73 (S1) ◽  
pp. 153-159 ◽  
Author(s):  
Marjorie C. Brandriss ◽  
Darlene A. Falvey ◽  
Shelley Ann G. des Etages ◽  
Shiwei Xu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Transcriptional Activation ◽  
Regulatory Protein ◽  
Nitrogen Sources ◽  
Yeast Saccharomyces Cerevisiae ◽  
Molecular Tests ◽  
Proline Utilization ◽  
Nitrogen Repression ◽  
Utilization Pathway

The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as allantoin, urea, γ-aminobutyrate, or proline when preferred nitrogen sources such as asparagine, glutamine, or ammonium ions are unavailable in the environment. To use proline as the sole nitrogen source, cells must activate the expression of the proline transporters and the genes that encode the catabolic enzymes proline oxidase (PUT1) and Δ1-pyrroline-5-carboxylate dehydrogenase (PUT2). Transcriptional activation of the PUT genes requires the PUT3 regulatory protein, proline, and relief from nitrogen repression. PUT3 is a 979 amino acid protein that binds a short DNA sequence in the promoters of PUT1 and PUT2, independent of the presence of proline. The functional domains of PUT3 have been studied by biochemical and molecular tests and analysis of activator-constitutive and activator-defective mutant proteins. Mutations in the URE2 gene relieve nitrogen repression, permitting inducer-independent transcription of the PUT genes in the presence of repressing nitrogen sources. The GLN3 protein that activates the expression of many genes in alternative nitrogen source pathways is not required for the expression of the PUT genes under inducing, derepressing conditions (proline) or noninducing, repressing conditions (ammonia). Although it has been speculated that the URE2 protein antagonizes the action of GLN3 in the regulation of many nitrogen assimilatory pathways, URE2 appears to act independently of GLN3 in the proline-utilization pathway. Key words: Saccharomyces cerevisiae, proline utilization, nitrogen repression.

scholarly journals Functional Analysis of the PUT3 Transcriptional Activator of the Proline Utilization Pathway in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (4) ◽  
pp. 1069-1082
Author(s):  
Shelley Ann G des Etages ◽  
Darlene A Fahey ◽  
Richard J Reece ◽  
Marjorie C Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Target Genes ◽  
Yeast Saccharomyces Cerevisiae ◽  
Central Domain ◽  
Mutant Proteins ◽  
Molecular Tests ◽  
Defective Mutant ◽  
Proline Utilization ◽  
Genes Encoding ◽  
Utilization Pathway

Abstract Proline can serve as a nitrogen source for the yeast Saccharomyces cerevisiae when preferred sources of nitrogen are absent from the growth medium. PUT3, the activator of the proline utilization pathway, is required for the transcription of the genes encoding the enzymes that convert proline to glutamate. PUT3 is a 979 amino acid protein that constitutively binds a short DNA sequence in the promoters of its target genes, but does not activate their expression in the absence of induction by proline and in the presence of preferred sources of nitrogen. To understand how PUT3 is converted from an inactive to an active state, a dissection of its functional domains has been undertaken. Biochemical and molecular tests, domain swapping experiments, and an analysis of activator-constitutive and activator-defective mutant proteins indicate that PUT3 is dimeric and activates transcription with its negatively charged carboxyterminus, which does not appear to contain a proline-responsive domain. A mutation in the conserved central domain found in many fungal activators interferes with activation without affecting DNA binding or protein stability. Intragenic suppressors of the central domain mutation have been isolated and analyzed.

scholarly journals Mutants affecting histidine utilization inAspergillus nidulans

1975 ◽  
Vol 25 (2) ◽  
pp. 119-135 ◽  
Author(s):  
Meryl Polkinghorne ◽  
M. J. Hynes
Keyword(s):  
Carbon Source ◽  
Nitrogen Source ◽  
Sole Carbon Source ◽  
Nitrogen Sources ◽  
Limiting Factor ◽  
Sole Nitrogen Source ◽  
Wild Type ◽  
Exogenous Substrate ◽  
Growth Properties ◽  
Type Strains

SUMMARYWild-type strains ofAspergillus nidulansgrow poorly onL-histidine as a sole nitrogen source. The synthesis of the enzyme histidase (EC. 4.3.1.3) appears to be a limiting factor in the growth of the wild type, as strains carrying the mutantareA102 allele have elevated histidase levels and grow strongly on histidine as a sole nitrogen source.L-Histidine is an extremely weak sole carbon source for all strains.Ammonium repression has an important role in the regulation of histidase synthesis and the relief of ammonium repression is dependent on the availability of a good carbon source. The level of histidase synthesis does not respond to the addition of exogenous substrate.Mutants carrying lesions in thesarA orsarB loci (suppressor ofareA102) have been isolated. The growth properties of these mutants on histidine as a sole nitrogen source correlate with the levels of histidase synthesized. Mutation at thesarA andsarB loci also reduces the utilization of a number of other nitrogen sources. The data suggest that these two genes may code for regulatory products involved in nitrogen catabolism. No histidase structural gene mutants were identified and possible explanations of this are discussed.

scholarly journals Production characteristics of the diatom Cylindrotheca closterium (Ehrenb.) Reimann et Lewin grown in an intensive culture at various nitrogen sources in the medium

2019 ◽  
Vol 4 (1) ◽  
pp. 33-44 ◽  
Author(s):  
S. N. Zheleznova
Keyword(s):  
Stationary Phase ◽  
Nitrogen Source ◽  
Nutrient Medium ◽  
Maximum Yield ◽  
Nitrogen Sources ◽  
Dry Weight ◽  
Sole Source ◽  
Active Growth ◽  
Cylindrotheca Closterium ◽  
Production Characteristics

The diatom Cylindrotheca closterium (Ehrenberg) Reimann et Levin is characterized by high productivity (up to 1.5 g·l-1·day-1) and the ability to accumulate a valuable carotenoid fucoxanthin (up to 2 % of dry weight). In the development of biotechnology based on microalgae, the key issue is the creation of concentrated nutrient medium. Nitrogen is one of the most important components in the nutrient medium that significantly affects the production characteristics of all microalgae. The aim of this study is to compare the production characteristics of C. closterium in an intensive storage culture using different forms of nitrogen in the medium. In the first experiment, nitrate and sodium nitrite, urea, and nitrogen in the form of ammonium were used as a source of nitrogen. The amount of nitrates, nitrites, ammonium, and urea in the medium was calculated from the nitrogen content of the RS nutrient medium, with a nitrogen to phosphorus ratio of 15 : 1. In the second experiment, amino acids were used as a nitrogen source – arginine, asparagine, cysteine. The possibility of using the microalgae C. closterium for the growth of various organic sources of nitrogen (urea, cysteine, asparagine) was shown. Productive characteristics in the intensive storage culture of C. closterium using urea, cysteine, and asparagine as the sole source of nitrogen in the RS nutrient medium were determined. It is shown that when urea was used, the productivity reached its maximum values and amounted to 1.5 g·l-1·day-1. Thus, the expediency of using urea in the medium for obtaining the maximum yield of biomass was shown. The use of cysteine in the stationary phase of growth to achieve a long stationary phase with minimal concentrations of the nitrogen source in the nutrient medium is also advisable. It was found that C. closterium was able to grow and vegetate at sufficiently high concentrations of nitrite, and the addition of nitrogen in ammonium form to the nutrient medium during the active growth of C. closterium led to inhibition of all metabolic processes and to the death of the culture.

scholarly journals The Escherichia coli gabDTPC Operon: Specific γ-Aminobutyrate Catabolism and Nonspecific Induction

2002 ◽  
Vol 184 (24) ◽  
pp. 6976-6986 ◽  
Author(s):  
Barbara L. Schneider ◽  
Stephen Ruback ◽  
Alexandros K. Kiupakis ◽  
Hillary Kasbarian ◽  
Christine Pybus ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Nitrogen Limitation ◽  
Nitrogen Sources ◽  
Wild Type ◽  
Gaba A ◽  
Specific Induction ◽  
Related Compounds ◽  
Type Strains

ABSTRACT Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen. Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and γ-aminobutyrate (GABA). We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism. We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC. A ΔgabDT mutant grew normally on all of the nitrogen sources tested except GABA. The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes. Nac was required for gab transcription in vivo and in vitro. Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally. A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor. GabC is proposed to reduce nitrogen source-specific modulation of expression. Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required σS. Previous studies showing σS-dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains. The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction. We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis.

scholarly journals Nitrogen source-dependent inhibition of yeast growth by glycine and its N-methylated derivatives

2019 ◽  
Vol 113 (3) ◽  
pp. 437-445
Author(s):  
Tomas Linder
Keyword(s):  
Growth Inhibition ◽  
Nitrogen Source ◽  
Minimal Medium ◽  
Kluyveromyces Lactis ◽  
Nitrogen Sources ◽  
Growth Efficiency ◽  
Sole Source ◽  
Yeast Growth ◽  
Glycine Species ◽  
Dependent Inhibition

Abstract The effect of nitrogen source on the inhibitory properties of glycine and its N-methylated derivatives N-methylglycine (sarcosine), N,N-dimethylglycine, N,N,N-trimethylglycine (glycine betaine) on yeast growth was investigated. On solid minimal medium, all four glycine species completely or partially inhibited growth of Kluyveromyces lactis, Komagataella pastoris, Ogataea arabinofermentans, Spathaspora passalidarum and Yamadazyma tenuis at concentrations 5–10 mM when 10 mM NH4Cl was the sole source of nitrogen. If NH4Cl was substituted by sodium L-glutamate as the sole source of nitrogen, obvious growth inhibition by glycine and its N-methylated derivatives was generally not observed in any of these species. No obvious growth inhibition by any of the glycine species at a concentration of 10 mM was observed in Cyberlindnera jadinii, Lipomyces starkeyi, Lodderomyces elongisporus, Scheffersomyces stipitis or Yarrowia lipolytica on solid minimal medium irrespective of whether the nitrogen source was NH4Cl or sodium L-glutamate. Growth inhibition assays of K. pastoris in liquid minimal medium supplemented with increasing concentrations of N,N-dimethylglycine demonstrated inhibitory effects for nine tested nitrogen sources. In most cases, N,N-dimethylglycine supplementation caused a decrease in growth efficiency that appeared to be proportional to the concentration of N,N-dimethylglycine. The biological relevance of these results is discussed.

scholarly journals Rapamycin Treatment Results in GATA Factor-Independent Hyperphosphorylation of the Proline Utilization Pathway Activator in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (3) ◽  
pp. 552-559 ◽  
Author(s):  
Deepti Saxena ◽  
K. B. Kannan ◽  
Marjorie C. Brandriss
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nitrogen Source ◽  
Regulatory Elements ◽  
Immunosuppressive Drug ◽  
Nitrogen Deprivation ◽  
Gata Factor ◽  
Treatment Results ◽  
Rapamycin Treatment ◽  
Proline Utilization

ABSTRACT Treatment of Saccharomyces cerevisiae cells with the immunosuppressive drug rapamycin results in a variety of cellular changes in response to perceived nutrient deprivation. Among other effects, rapamycin treatment results in the nuclear localization of the global nitrogen activators Gln3p and Nil1p/Gat1p, which leads to expression of nitrogen assimilation genes. The proline utilization (Put) pathway genes were shown to be among the genes induced by rapamycin. Having previously shown that the Put pathway activator Put3p is differentially phosphorylated in response to the quality of the nitrogen source, we examined the phosphorylation status of Put3p after rapamycin treatment. Treatment with rapamycin resulted in the hyperphosphorylation of Put3p, which was independent of Gln3p, Nil1p, and Ure2p. The relative contributions of global nitrogen (Gln3p and Nil1p) and pathway-specific (Put3p) activators to rapamycin-induced expression of the target gene PUT1 were also examined. We found that Nil1p and Put3p, but not Gln3p, play major roles in rapamycin-induced PUT1 expression. Our findings show that perceived nitrogen deprivation triggered by rapamycin treatment and steady-state growth in nitrogen-derepressing conditions are associated with hyperphosphorylation of Put3p and increased PUT1 expression. Rapamycin treatment and nitrogen derepression may share some, but not all, regulatory elements, since Gln3p and Nil1p do not participate identically in both processes and are not required for hyperphosphorylation. A complex relationship exists among the global and pathway-specific regulators, depending on the nature and quality of the nitrogen source.

scholarly journals Nuclear Accumulation of the GATA Factor AreA in Response to Complete Nitrogen Starvation by Regulation of Nuclear Export

2005 ◽  
Vol 4 (10) ◽  
pp. 1646-1653 ◽  
Author(s):  
Richard B. Todd ◽  
James A. Fraser ◽  
Koon Ho Wong ◽  
Meryl A. Davis ◽  
Michael J. Hynes
Keyword(s):  
Gene Expression ◽  
Nitrogen Source ◽  
Nuclear Export ◽  
Nitrogen Starvation ◽  
Nitrogen Sources ◽  
Nuclear Accumulation ◽  
Gata Factor ◽  
Regulated Gene Expression ◽  
Activation Capacity

ABSTRACT Both the availability and the quality of nutrients affect cellular functions by controlling gene activity. AreA, a member of the GATA family of transcription factors, globally activates expression of genes involved in nitrogen source utilization in Aspergillus nidulans. The quality of the nitrogen source determines the level and activation capacity of AreA through controls at the level of areA mRNA stability and by interaction of AreA with the corepressor NmrA. The availability of potential nitrogen sources also affects the activation capacity of AreA. We show that the complete absence of a nitrogen source results in an enhanced level of AreA-dependent gene expression and that this response is independent of mechanisms regulating AreA activity in response to nitrogen source quality. During nitrogen starvation AreA accumulates in the nucleus, but the presence of a potential nitrogen source or carbon starvation prevents this accumulation. Furthermore, accumulated AreA is rapidly lost from the nuclei of nitrogen-starved cells when a nitrogen source is supplied or when a carbon source is absent, and this accompanies arrest of the AreA-dependent nitrogen starvation response on regulated gene expression. By the generation of a leptomycin B-sensitive mutant, we have been able to show that nuclear exit occurs via the CrmA exportin. We conclude that sensing mechanisms discriminate between starvation and the presence of potential nutrients that can signal to the AreA transcription factor. Nitrogen source availability, but not quality, affects nuclear accumulation by regulating nuclear exit of AreA, providing a rapid response to changes in the supply of nutrients.

scholarly journals Improvement of Nitrogen Assimilation and Fermentation Kinetics under Enological Conditions by Derepression of Alternative Nitrogen-Assimilatory Pathways in an Industrial Saccharomyces cerevisiae Strain

1998 ◽  
Vol 64 (10) ◽  
pp. 3831-3837 ◽  
Author(s):  
Jean-Michel Salmon ◽  
Pierre Barre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Grape Juice ◽  
Nitrogen Sources ◽  
Fermentation Kinetics ◽  
Nitrogen Levels ◽  
Amino Acid Utilization ◽  
Proline Utilization ◽  
Grape Juices ◽  
Utilization Pathway ◽  
Grape Musts

ABSTRACT Metabolism of nitrogen compounds by yeasts affects the efficiency of wine fermentation. Ammonium ions, normally present in grape musts, reduce catabolic enzyme levels and transport activities for nonpreferred nitrogen sources. This nitrogen catabolite repression severely impairs the utilization of proline and arginine, both common nitrogen sources in grape juice that require the proline utilization pathway for their assimilation. We attempted to improve fermentation performance by genetic alteration of the regulation of nitrogen-assimilatory pathways in Saccharomyces cerevisiae. One mutant carrying a recessive allele ofure2 was isolated from an industrial S. cerevisiae strain. This mutation strongly deregulated the proline utilization pathway. Fermentation kinetics of this mutant were studied under enological conditions on simulated standard grape juices with various nitrogen levels. Mutant strains produced more biomass and exhibited a higher maximum CO2 production rate than the wild type. These differences were primarily due to the derepression of amino acid utilization pathways. When low amounts of dissolved oxygen were added, the mutants could assimilate proline. Biomass yield and fermentation rate were consequently increased, and the duration of the fermentation was substantially shortened. S. cerevisiae strains lacking URE2 function could improve alcoholic fermentation of natural media where proline and other poorly assimilated amino acids are the major potential nitrogen source, as is the case for most fruit juices and grape musts.

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