scholarly journals The SWI/SNF KlSnf2 Subunit Controls the Glucose Signaling Pathway To Coordinate Glycolysis and Glucose Transport in Kluyveromyces lactis

2012 ◽  
Vol 11 (11) ◽  
pp. 1382-1390 ◽  
Author(s):  
Pascale Cotton ◽  
Alexandre Soulard ◽  
Micheline Wésolowski-Louvel ◽  
Marc Lemaire
Keyword(s):  
Signaling Pathway ◽  
Glucose Transport ◽  
Chromatin Remodeling ◽  
Kluyveromyces Lactis ◽  
Content Type ◽  
Glucose Signaling ◽  
Helix Loop Helix ◽  
Reverse Transcription Quantitative Pcr ◽  
Extracellular Glucose ◽  
Transcriptional Effect

ABSTRACTInKluyveromyces lactis, the expression of the major glucose permease geneRAG1is controlled by extracellular glucose through a signaling cascade similar to theSaccharomyces cerevisiaeSnf3/Rgt2/Rgt1 pathway. We have identified a key component of theK. lactisglucose signaling pathway by characterizing a new mutation,rag20-1, which impairs the regulation ofRAG1and hexokinaseRAG5genes by glucose. Functional complementation of therag20-1mutation identified theKlSNF2gene, which encodes a protein 59% identical toS. cerevisiaeSnf2, the major subunit of the SWI/SNF chromatin remodeling complex. Reverse transcription-quantitative PCR and chromatin immunoprecipitation analyses confirmed that the KlSnf2 protein binds toRAG1andRAG5promoters and promotes the recruitment of the basic helix-loop-helix Sck1 activator. Besides this transcriptional effect, KlSnf2 is also implicated in the glucose signaling pathway by controlling Sms1 and KlRgt1 posttranscriptional modifications. When KlSnf2 is absent, Sms1 is not degraded in the presence of glucose, leading to constitutiveRAG1gene repression by KlRgt1. Our work points out the crucial role played by KlSnf2 in the regulation of glucose transport and metabolism inK. lactis, notably, by suggesting a link between chromatin remodeling and the glucose signaling pathway.

scholarly journals Glycolysis Controls Plasma Membrane Glucose Sensors To Promote Glucose Signaling in Yeasts

2014 ◽  
Vol 35 (4) ◽  
pp. 747-757 ◽  
Author(s):  
Amélie Cairey-Remonnay ◽  
Julien Deffaud ◽  
Micheline Wésolowski-Louvel ◽  
Marc Lemaire ◽  
Alexandre Soulard
Keyword(s):  
Signaling Pathway ◽  
Glucose Sensor ◽  
Kluyveromyces Lactis ◽  
Glucose Sensors ◽  
Content Type ◽  
Glucose Signaling ◽  
Extracellular Glucose ◽  
Glucose Accumulation ◽  
The Stability ◽  
Intracellular Glucose

Sensing of extracellular glucose is necessary for cells to adapt to glucose variation in their environment. In the respiratory yeastKluyveromyces lactis, extracellular glucose controls the expression of major glucose permease geneRAG1through a cascade similar to theSaccharomyces cerevisiaeSnf3/Rgt2/Rgt1 glucose signaling pathway. This regulation depends also on intracellular glucose metabolism since we previously showed that glucose induction of theRAG1gene is abolished in glycolytic mutants. Here we show that glycolysis regulatesRAG1expression through theK. lactisRgt1 (KlRgt1) glucose signaling pathway by targeting the localization and probably the stability of Rag4, the single Snf3/Rgt2-type glucose sensor ofK. lactis. Additionally, the control exerted by glycolysis on glucose signaling seems to be conserved inS. cerevisiae. This retrocontrol might prevent yeasts from unnecessary glucose transport and intracellular glucose accumulation.

scholarly journals Sck1 Negatively Regulates Gpa2-Mediated Glucose Signaling in Schizosaccharomyces pombe

2013 ◽  
Vol 13 (2) ◽  
pp. 202-208 ◽  
Author(s):  
Dayna K. Mudge ◽  
Fan Yang ◽  
Brian M. Currie ◽  
James M. Kim ◽  
Kelly Yeda ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
G Protein ◽  
Free Form ◽  
Slight Reduction ◽  
Content Type ◽  
Glucose Signaling ◽  
Extracellular Glucose ◽  
The One ◽  
Two Hybrid

ABSTRACTSchizosaccharomyces pombedetects extracellular glucose via a G protein-mediated cyclic AMP (cAMP)-signaling pathway activating protein kinase A (PKA) and regulating transcription of genes involved in metabolism and sexual development. In this pathway, Gpa2 Gα binds to and activates adenylyl cyclase in response to glucose detection by the Git3 G protein-coupled receptor. Using a two-hybrid screen to identify extrinsic regulators of Gpa2, we isolated a clone that expresses codons 471 to 696 of the Sck1 kinase, which appears to display a higher affinity for Gpa2K270E-activated Gα relative to Gpa2+Gα. Deletion ofsck1+or mutational inactivation of the Sck1 kinase produces phenotypes reflecting increased PKA activity in strains expressing Gpa2+or Gpa2K270E, suggesting that Sck1 negatively regulates PKA activation through Gpa2. In contrast to the Gpa2K270EGDP-GTP exchange rate mutant, GTPase-defective Gpa2R176Hweakly binds Sck1 in the two-hybrid screen and a deletion ofsck1+in a Gpa2R176Hstrain confers phenotypes consistent with a slight reduction in PKA activity. Finally, deletingsck1+in agpa2Δ strain results in phenotypes consistent with a second role for Sck1 acting in parallel with PKA. In addition to this parallel role with PKA, our data suggest that Sck1 negatively regulates Gpa2, possibly targeting the nucleotide-free form of the protein that may expose the one and only AKT/PKB consensus site in Gpa2 for Sck1 to bind. This dual role for Sck1 may allowS. pombeto produce distinct biological responses to glucose and nitrogen starvation signals that both activate the Wis1-Spc1/StyI stress-activated protein kinase (SAPK) pathway.

scholarly journals Characterization of KlGRR1 and SMS1 Genes, Two New Elements of the Glucose Signaling Pathway of Kluyveromyces lactis

2008 ◽  
Vol 7 (8) ◽  
pp. 1299-1308 ◽  
Author(s):  
Martina Hnatova ◽  
Micheline Wésolowski-Louvel ◽  
Guenaëlle Dieppois ◽  
Julien Deffaud ◽  
Marc Lemaire
Keyword(s):  
Signaling Pathway ◽  
Glucose Transporter ◽  
Glucose Sensor ◽  
Kluyveromyces Lactis ◽  
Single Gene ◽  
Negative Regulator ◽  
Casein Kinase I ◽  
Glucose Signaling ◽  
F Box Protein

ABSTRACT The expression of the major glucose transporter gene, RAG1, is induced by glucose in Kluyveromyces lactis. This regulation involves several pathways, including one that is similar to Snf3/Rgt2-ScRgt1 in Saccharomyces cerevisiae. We have identified missing key components of the K. lactis glucose signaling pathway by comparison to the same pathway of S. cerevisiae. We characterized a new mutation, rag19, which impairs RAG1 regulation. The Rag19 protein is 43% identical to the F-box protein ScGrr1 of S. cerevisiae and is able to complement an Scgrr1 mutation. In the K. lactis genome, we identified a single gene, SMS1 (for similar to Mth1 and Std1), that encodes a protein showing an average of 50% identity with Mth1 and Std1, regulators of the ScRgt1 repressor. The suppression of the rag4 (glucose sensor), rag8 (casein kinase I), and rag19 mutations by the Δsms1 deletion, together with the restoration of RAG1 transcription in the double mutants, demonstrates that Sms1 is a negative regulator of RAG1 expression and is acting downstream of Rag4, Rag8, and Rag19 in the cascade. We report that Sms1 regulates KlRgt1 repressor activity by preventing its phosphorylation in the absence of glucose, and that SMS1 is regulated by glucose, both at the transcriptional and the posttranslational level. Two-hybrid interactions of Sms1 with the glucose sensor and KlRgt1 repressor suggest that Sms1 mediates the glucose signal from the plasma membrane to the nucleus. All of these data demonstrated that Sms1 was the K. lactis homolog of MTH1 and STD1 of S. cerevisiae. Interestingly, MTH1 and STD1 were unable to complement a Δsms1 mutation.

scholarly journals Derepression of INO1 Transcription Requires Cooperation between the Ino2p-Ino4p Heterodimer and Cbf1p and Recruitment of the ISW2 Chromatin-Remodeling Complex

2010 ◽  
Vol 9 (12) ◽  
pp. 1845-1855 ◽  
Author(s):  
Ameet Shetty ◽  
John M. Lopes
Keyword(s):  
Chromatin Remodeling ◽  
Binding Sites ◽  
De Novo ◽  
Bhlh Protein ◽  
Promoter Element ◽  
Content Type ◽  
Reading Frame ◽  
Helix Loop Helix ◽  
Chromatin Remodeling Complex ◽  
Binding Factor

ABSTRACT The Saccharomyces cerevisiae INO1 gene encodes the structural enzyme inositol-3-phosphate synthase for the synthesis de novo of inositol and inositol-containing phospholipids. The transcription of INO1 is completely derepressed in the absence of inositol and choline (I− C−). Derepression requires the binding of the Ino2p-Ino4p basic helix-loop-helix (bHLH) heterodimer to the UAS INO promoter element. We report here the requirement of a third bHLH protein, centromere-binding factor 1 (Cbf1p), for the complete derepression of INO1 transcription. We found that Cbf1p regulates INO1 transcription by binding to sites distal to the INO1 promoter and encompassing the upstream SNA3 open reading frame (ORF) and promoter. The binding of Cbf1p requires Ino2p-Ino4p binding to the UAS INO sites in the INO1 promoter and vice versa, suggesting a cooperative mechanism. Furthermore, Cbf1p binding to the upstream sites was required for the binding of the ISW2 chromatin-remodeling complex to the Ino2p-Ino4p-binding sites on the INO1 promoter. Consistent with this, ISW2 was also required for the complete derepression of INO1 transcription.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

scholarly journals Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes

2008 ◽  
Vol 198 (3) ◽  
pp. 561-569 ◽  
Author(s):  
Wenbin Shang ◽  
Ying Yang ◽  
Libin Zhou ◽  
Boren Jiang ◽  
Hua Jin ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Glucose Uptake ◽  
Signaling Pathway ◽  
Glucose Transport ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
C2c12 Myotubes ◽  
Maximal Effect ◽  
Dependent Manner ◽  
Active Constituent

A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb1 has been shown to regulate peroxisome proliferator-activated receptor γ activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb1 on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb1 significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 μM and a time of 3 h. In adipocytes, Rb1 promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb1 increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb1-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb1 stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides.

scholarly journals Streptomycin-Induced Expression in Bacillus subtilis of YtnP, a Lactonase-Homologous Protein That Inhibits Development and Streptomycin Production in Streptomyces griseus

2011 ◽  
Vol 78 (2) ◽  
pp. 599-603 ◽  
Author(s):  
Johannes Schneider ◽  
Ana Yepes ◽  
Juan C. Garcia-Betancur ◽  
Isa Westedt ◽  
Benjamin Mielich ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Signaling Pathway ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Induced Expression ◽  
Content Type ◽  
Homologous Protein

ABSTRACTBacillus subtilisinduces expression of the geneytnPin the presence of the antimicrobial streptomycin, produced by the Gram-positive bacteriumStreptomyces griseus.ytnPencodes a lactonase-homologous protein that is able to inhibit the signaling pathway required for the streptomycin production and development of aerial mycelium inS. griseus.

scholarly journals A Salmonella Virulence Factor Activates the NOD1/NOD2 Signaling Pathway

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
A. Marijke Keestra ◽  
Maria G. Winter ◽  
Daisy Klein-Douwel ◽  
Mariana N. Xavier ◽  
Sebastian E. Winter ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Type Iii Secretion ◽  
Salmonella Enterica ◽  
Intestinal Inflammation ◽  
Inflammatory Responses ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Content Type ◽  
Type Iii

ABSTRACTThe invasion-associated type III secretion system (T3SS-1) ofSalmonella entericaserotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasionin vitrobut required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responsesin vitroandin vivo.IMPORTANCESalmonella entericaserotype Typhimurium (S. Typhimurium) deploys a type III secretion system (T3SS-1) to induce intestinal inflammation and benefits from the ensuing host response, which enhances growth of the pathogen in the intestinal lumen. However, the mechanisms by which the T3SS-1 triggers inflammatory responses have not been resolved. Here we show that the T3SS-1 effector protein SipA induces NF-κB activation and intestinal inflammation by activating the NOD1/NOD2 signaling pathway. These data suggest that the T3SS-1 escalates innate responses through a SipA-mediated activation of pattern recognition receptors in the host cell cytosol.

scholarly journals Establishment and Application of Rapid Diagnosis for Reverse Transcription-Quantitative PCR of Newly Emerging Goose-Origin Nephrotic Astrovirus in China

mSphere ◽  
2018 ◽  
Vol 3 (6) ◽  
Author(s):  
Xiaoyuan Yuan ◽  
Kai Meng ◽  
Yuxia Zhang ◽  
Lihong Qi ◽  
Wu Ai ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Clinical Application ◽  
Reverse Transcription ◽  
Test Sample ◽  
Sensitivity Testing ◽  
Content Type ◽  
Reverse Transcription Quantitative Pcr ◽  
Emerging Virus ◽  
New Type ◽  
And Control

ABSTRACT In 2017, a new type of goose-origin astrovirus (GoAstV) that is completely different from previously identified avian astroviruses (which have only 30.0% to 50.5% homology with GoAstV) has been isolated from diseased geese in China. This disease can cause joint swelling in sick geese, and the anatomy shows a clear precipitation of urate in the kidney. The rate of death and culling can reach more than 30%, revealing the disease’s severe pathogenicity. To quickly and accurately diagnose the newly emerging disease, we established a highly specific reverse transcription-quantitative PCR (RT-qPCR) method of detecting GoAstV. Sensitivity testing showed that the minimum amount of test sample for this method is 52.5 copies/μl. Clinical application confirmed that this method can quickly and effectively detect GoAstV, providing a diagnostic platform for the prevention and control of goose disease. IMPORTANCE Goose-origin astrovirus (GoAstV), as a newly emerging virus in 2017, is different from previously known astroviruses in the genus Avastrovirus. So far, few studies have focused on the novel virus. Considering the infectious development of astrovirus (AstV), we established a reverse transcription-quantitative PCR (RT-qPCR) assay with a strong specificity to quickly and accurately diagnose GoAstV. Confirmed by clinical application, this method can quickly and accurately detect prevalent GoAstV. The assay is thus convenient for clinical operation and is applicable to the monitoring of GoAstV disease.

Assessment of extracellular glucose distribution and glucose transport activity in conscious rats

1995 ◽  
Vol 268 (4) ◽  
pp. E712-E721 ◽  
Author(s):  
J. H. Youn ◽  
J. K. Kim ◽  
G. M. Steil
Keyword(s):  
Glucose Transport ◽  
Insulin Infusion ◽  
Compartmental Analysis ◽  
Time Profile ◽  
Cellular Transport ◽  
Transport Activity ◽  
Conscious Rats ◽  
Distribution Kinetics ◽  
Extracellular Glucose

The effects of insulin on extracellular glucose distribution and cellular glucose transport activity were studied by simultaneously analyzing the plasma kinetics of L-[1-14C]glucose and 3-O-[3H]methylglucose after an intravenous injection during saline or insulin infusion (euglycemic glucose clamp) in conscious rats (n = 7 for each). The time profiles of plasma L-glucose were almost superimposable in the two protocols, and compartmental analysis showed that neither distribution volumes nor distribution rate constants were affected with insulin (P > 0.05 for all), suggesting that glucose distribution within the extracellular space was not influenced with insulin. In contrast, the time profile of plasma 3-O-methylglucose (3-MG) was markedly altered with insulin; the initial decrease was much faster during insulin infusion than during saline infusion, indicating stimulation of 3-MG transport into intracellular spaces with insulin. The 3-MG data were analyzed using a comprehensive model separately describing extracellular distribution and cellular transport of 3-MG by incorporating information on extracellular distribution kinetics obtained from L-glucose data. The combined L-glucose and 3-MG kinetic analysis precisely estimated insulin's effect in vivo to stimulate glucose transport into and out of intracellular spaces. We conclude that 1) insulin does not affect extracellular glucose distribution kinetics or volumes in conscious rats and 2) insulin's effects on cellular glucose transport in vivo can be assessed by simultaneous analysis of plasma L-glucose and 3-MG kinetics.

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