scholarly journals Carbon Source-Dependent Phosphorylation of Hexokinase PII and Its Role in the Glucose-Signaling Response in Yeast

1998 ◽  
Vol 18 (5) ◽  
pp. 2940-2948 ◽  
Author(s):  
Francisca Randez-Gil ◽  
Pascual Sanz ◽  
Karl-Dieter Entian ◽  
Jose Antonio Prieto
Keyword(s):  
Carbon Source ◽  
Glucose Repression ◽  
Carbon Sources ◽  
Glucose Signaling ◽  
Regulatory Effects ◽  
Metabolic Signal ◽  
Signal Transductions ◽  
Mutant Cells

ABSTRACT The HXK2 gene is required for a variety of regulatory effects leading to an adaptation for fermentative metabolism inSaccharomyces cerevisiae. However, the molecular basis of the specific role of Hxk2p in these effects is still unclear. One important feature in order to understand the physiological function of hexokinase PII is that it is a phosphoprotein, since protein phosphorylation is essential in most metabolic signal transductions in eukaryotic cells. Here we show that Hxk2p exists in vivo in a dimeric-monomeric equilibrium which is affected by phosphorylation. Only the monomeric form appears phosphorylated, whereas the dimer does not. The reversible phosphorylation of Hxk2p is carbon source dependent, being more extensive on poor carbon sources such as galactose, raffinose, and ethanol. In vivo dephosphorylation of Hxk2p is promoted after addition of glucose. This effect is absent in glucose repression mutants cat80/grr1, hex2/reg1, andcid1/glc7. Treatment of a glucose crude extract fromcid1-226 (glc7-T152K) mutant cells with λ-phosphatase drastically reduces the presence of phosphoprotein, suggesting that CID1/GLC7 phosphatase together with its regulatory HEX2/REG1 subunit are involved in the dephosphorylation of the Hxk2p monomer. An HXK2 mutation encoding a serine-to-alanine change at position 15 [HXK2(S15A)] was to clarify the in vivo function of the phosphorylation of hexokinase PII. In this mutant, where the Hxk2 protein is unable to undergo phosphorylation, the cells could not provide glucose repression of invertase. Glucose induction ofHXT gene expression is also affected in cells expressing the mutated enzyme. Although we cannot rule out a defect in the metabolic state of the cell as the origin of these phenomena, our results suggest that the phosphorylation of hexokinase is essential in vivo for glucose signal transduction.

scholarly journals In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake in Streptomyces coelicolor

2003 ◽  
Vol 185 (3) ◽  
pp. 929-937 ◽  
Author(s):  
Harald Nothaft ◽  
Stephan Parche ◽  
Annette Kamionka ◽  
Fritz Titgemeyer
Keyword(s):  
Carbon Source ◽  
Glucose Repression ◽  
Streptomyces Coelicolor ◽  
Carbon Sources ◽  
Glycerol Kinase ◽  
Transcriptional Analysis ◽  
Phosphotransferase System ◽  
Gram Positive ◽  
High Affinity

ABSTRACT HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent Km of 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor.

An essential function of Grr1 for the degradation of Cln2 is to act as a binding core that links Cln2 to Skp1

1998 ◽  
Vol 111 (24) ◽  
pp. 3655-3661 ◽  
Author(s):  
T. Kishi ◽  
F. Yamao
Keyword(s):  
Glucose Repression ◽  
Dependent Manner ◽  
Binding Core ◽  
Essential Function ◽  
F Box Protein ◽  
Leucine Rich Repeats ◽  
Mutant Cells

In budding yeast, SCF complexes, composed of Skp1, Cdc53 and one of the F-box proteins, have been implicated in Cdc34-dependent ubiquitination. Grr1, which is required for degradation of G1 cyclins Cln1 and Cln2 as well as for regulation of glucose repression, is an F-box protein and interacts with Skp1 through the F-box motif. Grr1 also interacts in vitro with phosphorylated Cln1 and Cln2. However, ubiquitination of Cln1 has not been successful in an in vitro reconstituted system. In this study, domain analysis was performed to understand the role of Grr1 in the degradation of Cln2. Grr1 has another motif, leucine-rich repeats (LRR), in addition to the F-box. We found that the LRR is a domain for Cln2 binding. A deletion of half of the LRR abolished the interaction of Grr1 with phosphorylated Cln2 but not with Skp1 in vivo, and a deletion of the F-box abolished the interaction of Grr1 with Skp1 but not with phosphorylated Cln2 in vivo. Based on these results, we constructed grr1 mutants that are defective in association with either Skp1 or Cln2. Cln2 was highly stabilized and accumulated in the phosphorylated forms in the mutant cells. Furthermore, Skp1 associated in vivo with phosphorylated Cln2 in a Grr1-dependent manner. These data suggest that Grr1 is required for degradation of Cln2 through linking phosphorylated Cln2 to Skp1 in a SCFGrr1 complex.

Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Nitric Oxide Overproduction by cue1 Mutants Differs on Developmental Stages and Growth Conditions

Plants ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 1484 ◽  
Author(s):  
Tamara Lechón ◽  
Luis Sanz ◽  
Inmaculada Sánchez-Vicente ◽  
Oscar Lorenzo
Keyword(s):  
Nitric Oxide ◽  
Carbon Metabolism ◽  
Root Elongation ◽  
Developmental Stages ◽  
Primary Root ◽  
Carbon Sources ◽  
Growth Conditions ◽  
No Production

The cue1 nitric oxide (NO) overproducer mutants are impaired in a plastid phosphoenolpyruvate/phosphate translocator, mainly expressed in Arabidopsis thaliana roots. cue1 mutants present an increased content of arginine, a precursor of NO in oxidative synthesis processes. However, the pathways of plant NO biosynthesis and signaling have not yet been fully characterized, and the role of CUE1 in these processes is not clear. Here, in an attempt to advance our knowledge regarding NO homeostasis, we performed a deep characterization of the NO production of four different cue1 alleles (cue1-1, cue1-5, cue1-6 and nox1) during seed germination, primary root elongation, and salt stress resistance. Furthermore, we analyzed the production of NO in different carbon sources to improve our understanding of the interplay between carbon metabolism and NO homeostasis. After in vivo NO imaging and spectrofluorometric quantification of the endogenous NO levels of cue1 mutants, we demonstrate that CUE1 does not directly contribute to the rapid NO synthesis during seed imbibition. Although cue1 mutants do not overproduce NO during germination and early plant development, they are able to accumulate NO after the seedling is completely established. Thus, CUE1 regulates NO homeostasis during post-germinative growth to modulate root development in response to carbon metabolism, as different sugars modify root elongation and meristem organization in cue1 mutants. Therefore, cue1 mutants are a useful tool to study the physiological effects of NO in post-germinative growth.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

scholarly journals The Catabolite Repressor/Activator Cra Is a Bridge Connecting Carbon Metabolism and Host Colonization in the Plant Drought Resistance-Promoting Bacterium Pantoea alhagi LTYR-11Z

2018 ◽  
Vol 84 (13) ◽  
Author(s):  
Lei Zhang ◽  
Muhang Li ◽  
Qiqi Li ◽  
Chaoqiong Chen ◽  
Meng Qu ◽  
...  
Keyword(s):  
Carbon Source ◽  
Carbon Metabolism ◽  
Carbon Sources ◽  
Endophytic Bacterium ◽  
Bacterial Attachment ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Host Colonization ◽  
Content Type

ABSTRACT Efficient root colonization is a prerequisite for application of plant growth-promoting (PGP) bacteria in improving health and yield of agricultural crops. We have recently identified an endophytic bacterium, Pantoea alhagi LTYR-11Z, with multiple PGP properties that effectively colonizes the root system of wheat and improves its growth and drought tolerance. To identify novel regulatory genes required for wheat colonization, we screened an LTYR-11Z transposon (Tn) insertion library and found cra to be a colonization-related gene. By using transcriptome (RNA-seq) analysis, we found that transcriptional levels of an eps operon, the ydiV gene encoding an anti-FlhD 4 C 2 factor, and the yedQ gene encoding an enzyme for synthesis of cyclic dimeric GMP (c-di-GMP) were significantly downregulated in the Δ cra mutant. Further studies demonstrated that Cra directly binds to the promoters of the eps operon, ydiV , and yedQ and activates their expression, thus inhibiting motility and promoting exopolysaccharide (EPS) production and biofilm formation. Consistent with previous findings that Cra plays a role in transcriptional regulation in response to carbon source availability, the activating effects of Cra were much more pronounced when LTYR-11Z was grown within a gluconeogenic environment than when it was grown within a glycolytic environment. We further demonstrate that the ability of LTYR-11Z to colonize wheat roots is modulated by the availability of carbon sources. Altogether, these results uncover a novel strategy utilized by LTYR-11Z to achieve host colonization in response to carbon nutrition in the environment, in which Cra bridges a connection between carbon metabolism and colonization capacity of LTYR-11Z. IMPORTANCE Rapid and appropriate response to environmental signals is crucial for bacteria to adapt to competitive environments and to establish interactions with their hosts. Efficient colonization and persistence within the host are controlled by various regulatory factors that respond to specific environmental cues. The most common is nutrient availability. In this work, we unraveled the pivotal role of Cra in regulation of colonization ability of Pantoea alhagi LTYR-11Z in response to carbon source availability. Moreover, we identified three novel members of the Cra regulon involved in EPS synthesis, regulation of flagellar biosynthesis, and synthesis of c-di-GMP and propose a working model to explain the Cra-mediated regulatory mechanism that links carbon metabolism to host colonization. This study elucidates the regulatory role of Cra in bacterial attachment and colonization of plants, which raises the possibility of extending our studies to other bacteria associated with plant and human health.

scholarly journals Regulation of yeast COX6 by the general transcription factor ABF1 and separate HAP2- and heme-responsive elements.

1992 ◽  
Vol 12 (5) ◽  
pp. 2302-2314 ◽  
Author(s):  
J D Trawick ◽  
N Kraut ◽  
F R Simon ◽  
R O Poyton
Keyword(s):  
Transcription Factor ◽  
Carbon Source ◽  
Protein Binding ◽  
Glucose Repression ◽  
Protein Complexes ◽  
Carbon Sources ◽  
Major Protein ◽  
Mobility Shift ◽  
Gel Mobility Shift ◽  
In Cells

Transcription of the Saccharomyces cerevisiae COX6 gene is regulated by heme and carbon source. It is also affected by the HAP2/3/4 transcription factor complex and by SNF1 and SSN6. Previously, we have shown that most of this regulation is mediated through UAS6, an 84-bp upstream activation segment of the COX6 promoter. In this study, by using linker scanning mutagenesis and protein binding assays, we have identified three elements within UAS6 and one element downstream of it that are important. Two of these, HDS1 (heme-dependent site 1; between -269 and -251 bp) and HDS2 (between -228 and -220 bp), mediate regulation of COX6 by heme. Both act negatively. The other two elements, domain 2 (between -279 and -269 bp) and domain 1 (between -302 and -281 bp), act positively. Domain 2 is required for optimal transcription in cells grown in repressing but not derepressing carbon sources. Domain 1 is essential for transcription per se in cells grown on repressing carbon sources, is required for optimal transcription in cells grown on a derepressing carbon source, is sufficient for glucose repression-derepression, and is the element of UAS6 at which HAP2 affects COX6 transcription. This element contains the major protein binding sites within UAS6. It has consensus binding sequences for ABF1 and HAP2. Gel mobility shift experiments show that domain 1 binds ABF1 and forms different numbers of DNA-protein complexes in extracts from cells grown in repressing or derepressing carbon sources. In contrast, gel mobility shift experiments have failed to reveal that HAP2 or HAP3 binds to domain 1 or that hap3 mutations affect the complexes bound to it. Together, these findings permit the following conclusions: COX6 transcription is regulated both positively and negatively; heme and carbon source exert their effects through different sites; domain 1 is absolutely essential for transcription on repressing carbon sources; ABF1 is a major component in the regulation of COX6 transcription; and the HAP2/3/4 complex most likely affects COX6 transcription indirectly.

scholarly journals Nrg1 and Nrg2 Transcriptional Repressors Are Differently Regulated in Response to Carbon Source

2004 ◽  
Vol 3 (2) ◽  
pp. 311-317 ◽  
Author(s):  
Cristin D. Berkey ◽  
Valmik K. Vyas ◽  
Marian Carlson
Keyword(s):  
Carbon Source ◽  
Carbon Sources ◽  
Large Set ◽  
Transcriptional Repressors ◽  
Glucose Limitation ◽  
Dna Binding Domains ◽  
Protein Levels ◽  
Binding Domains ◽  
And Function ◽  
Mutant Cells

ABSTRACT The Nrg1 and Nrg2 repressors of Saccharomyces cerevisiae have highly similar zinc fingers and closely related functions in the regulation of glucose-repressed genes. We show that NRG1 and NRG2 are differently regulated in response to carbon source at both the RNA and protein levels. Expression of NRG1 RNA is glucose repressed, whereas NRG2 RNA levels are nearly constant. Nrg1 protein levels are elevated in response to glucose limitation or growth in nonfermentable carbon sources, whereas Nrg2 levels are diminished. Chromatin immunoprecipitation assays showed that Nrg1 and Nrg2 bind DNA both in the presence and absence of glucose. In mutant cells lacking the corepressor Ssn6(Cyc8)-Tup1, promoter-bound Nrg1, but not Nrg2, functions as an activator in a reporter assay, providing evidence that the two Nrg proteins have distinct properties. We suggest that the differences in expression and function of these two repressors, in combination with their similar DNA-binding domains, contribute to the complex regulation of the large set of glucose-repressed genes.

scholarly journals Carbon Source Dependence and Photostimulation of Conidiation in Hypocrea atroviridis

2007 ◽  
Vol 74 (1) ◽  
pp. 245-250 ◽  
Author(s):  
Martina A. Friedl ◽  
Christian P. Kubicek ◽  
Irina S. Druzhinina
Keyword(s):  
Carbon Source ◽  
Cyclic Amp ◽  
Parental Strain ◽  
Carbon Sources ◽  
Spore Formation ◽  
Blue Light Receptor ◽  
Small Set ◽  
Prime Determinant ◽  
Catalytic Role

ABSTRACT Hypocrea atroviridis is frequently used as a photomorphogenetic model due to its ability to conidiate upon exposure to light. Light is thereby believed to be the primary trigger for spore formation. In contrast, we show here that conidiation is primarily carbon source dependent and that illumination plays a catalytic role; of a total of 95 tested carbon sources, only a small set of carbohydrates, polyols, and sugar acids allowed conidiation in darkness, and on most of them, conidiation was significantly more strongly expressed in light. In addition, there are also a number of carbon sources on which H. atroviridis conidiates in darkness, but light does not further stimulate the process. Yet on another small set of carbon sources (l-sorbitol, d-fucose, d- and l-arabinose, and erythritol), H. atroviridis shows better sporulation in darkness than in light. No sporulation was observed on organic acids and amino acids. Mutants with deletions in the two blue-light receptor proteins BLR-1 and BLR-2 generally showed weaker conidiation on a smaller number of carbon sources than did the parental strain, yet they clearly sporulated on 15 and 27 of the 95 carbon sources tested, respectively. Of the carbon sources supporting sporulation, only 11 supported the conidiation of both mutants, suggesting that the BLR-1 and BLR-2 receptors are variously involved in the carbon source-dependent regulation of spore formation. The addition of cyclic AMP, which has been reported to lead to conidiation in darkness, both positively and negatively affected sporulation and resulted in different effects in the parental strain and the two Δblr mutants. Our data show that the carbon source is the prime determinant for conidiation and that it influences the organism's regulation of conidiation by means of BLR-1 and BLR-2 and their cross talk with cyclic AMP.

scholarly journals Restoration of a tumorigenic phenotype by beta 2-microglobulin transfection to EL-4 mutant cells.

1992 ◽  
Vol 175 (3) ◽  
pp. 843-846 ◽  
Author(s):  
R Glas ◽  
K Sturmhöfel ◽  
G J Hämmerling ◽  
K Kärre ◽  
H G Ljunggren
Keyword(s):  
Transfected Cells ◽  
Histocompatibility Complex ◽  
Tumorigenic Potential ◽  
Immunological Recognition ◽  
Beta 2 ◽  
Beta 2 Microglobulin ◽  
First Time ◽  
Mutant Cells

It has frequently been suggested that loss of beta 2-microglobulin (beta 2m) in tumor cells may lead to malignant progression due to escape from immunological recognition. Here, we directly tested the role of beta 2m expression in tumorigenicity. A beta 2 m loss mutant (C4.4-25-), selected from the murine lymphoma EL-4, showed a marked reduction in tumorigenicity as compared with EL-4 in normal C57B1/6 (B6) mice. The reduced tumorigenicity was directly related to beta 2 m expression. Transfection of an intact murine beta 2m gene markedly increased the tumorigenic potential. The reduced tumorigenicity of C4.4-25- compared with beta 2m transfected cells was observed also in athymic B6 nu/nu mice, but was abolished in B6 mice depleted of natural killer (NK) 1.1-positive cells. These results show that restoration of beta 2m expression can promote tumorigenicity and demonstrate for the first time that induction of major histocompatibility complex class I expression by transfection can lead to escape from NK cells in vivo.

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