Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics

2013 ◽  
Vol 454 (2) ◽  
pp. 227-237 ◽  
Author(s):  
Thomas Walther ◽  
Narjes Mtimet ◽  
Ceren Alkim ◽  
Amélie Vax ◽  
Marie-Odile Loret ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Metabolic Response ◽  
Adenine Nucleotides ◽  
Carbon Sources ◽  
Amp Deaminase ◽  
Cytosolic Ph ◽  
Fermentative Metabolism ◽  
Encoding Gene

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H+-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H+-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism.

Effects of bafilomycin A1 on cytosolic pH of sheep alveolar and peritoneal macrophages: evaluation of the pH-regulatory role of plasma membrane V-ATPases.

1995 ◽  
Vol 198 (8) ◽  
pp. 1711-1715 ◽  
Author(s):  
T A Heming ◽  
D L Traber ◽  
F Hinder ◽  
A Bidani
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Initial Rate ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Bafilomycin A1 ◽  
Atpase Inhibitor ◽  
Cytosolic Ph ◽  
Phi Regulation

The role of plasma membrane V-ATPase activity in the regulation of cytosolic pH (pHi) was determined for resident alveolar and peritoneal macrophages (m theta) from sheep. Cytosolic pH was measured using 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). The baseline pHi of both cell types was sensitive to the specific V-ATPase inhibitor bafilomycin A1. Bafilomycin A1 caused a significant (approximately 0.2 pH units) and rapid (within seconds) decline in baseline pHi. Further, bafilomycin A1 slowed the initial rate of pHi recovery (dpHi/dt) from intracellular acid loads. Amiloride had no effects on baseline pHi, but reduced dpHi/dt (acid-loaded pHi nadir < 6.8) by approximately 35%. Recovery of pHi was abolished by co-treatment of m theta with bafilomycin A1 and amiloride. These data indicate that plasma membrane V-ATPase activity is a major determinant of pHi regulation in resident alveolar and peritoneal m theta from sheep. Sheep m theta also appear to possess a Na+/H+ exchanger. However, Na+/H+ exchange either is inactive or can be effectively masked by V-ATPase-mediated H+ extrusion at physiological pHi values.

scholarly journals The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids

2008 ◽  
Vol 42 (1) ◽  
pp. 215-228 ◽  
Author(s):  
Hanna Forsberg ◽  
C. Fredrik Gilstring ◽  
Arezou Zargari ◽  
Paula Martínez ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Metabolic Response ◽  
Yeast Plasma Membrane

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

1986 ◽  
Vol 32 (12) ◽  
pp. 969-972 ◽  
Author(s):  
Albert J. Wilson ◽  
J. K. Bhattacharjee
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Kinase ◽  
Growth Medium ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Carbon Sources ◽  
Absolute Requirement ◽  
Fructose 1,6 Bisphosphatase ◽  
Futile Cycling ◽  
Respiratory Deficient

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present sumultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

scholarly journals Interaction of the Srb10 Kinase with Sip4, a Transcriptional Activator of Gluconeogenic Genes in Saccharomyces cerevisiae

2001 ◽  
Vol 21 (17) ◽  
pp. 5790-5796 ◽  
Author(s):  
Olivier Vincent ◽  
Sergei Kuchin ◽  
Seung-Pyo Hong ◽  
Robert Townley ◽  
Valmik K. Vyas ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Repression ◽  
Transcriptional Control ◽  
Carbon Sources ◽  
Transcriptional Activator ◽  
Glucose Limitation ◽  
Cell Extracts ◽  
Rna Polymerase Ii Holoenzyme

ABSTRACT Sip4 is a Zn2Cys6 transcriptional activator that binds to the carbon source-responsive elements of gluconeogenic genes in Saccharomyces cerevisiae. The Snf1 protein kinase interacts with Sip4 and regulates its phosphorylation and activator function in response to glucose limitation; however, evidence suggested that another kinase also regulates Sip4. Here we examine the role of the Srb10 kinase, a component of the RNA polymerase II holoenzyme that has been primarily implicated in transcriptional repression but also positively regulates Gal4. We show that Srb10 is required for phosphorylation of Sip4 during growth in nonfermentable carbon sources and that the catalytic activity of Srb10 stimulates the ability of LexA-Sip4 to activate transcription of a reporter. Srb10 and Sip4 coimmunoprecipitate from cell extracts and interact in two-hybrid assays, suggesting that Srb10 regulates Sip4 directly. We also present evidence that the Srb10 and Snf1 kinases interact with different regions of Sip4. These findings support the view that the Srb10 kinase not only plays negative roles in transcriptional control but also has broad positive roles during growth in carbon sources other than glucose.

scholarly journals Mig1 localization exhibits biphasic behavior which is controlled by both metabolic and regulatory roles of the sugar kinases

2020 ◽  
Vol 295 (6) ◽  
pp. 1489-1500
Author(s):  
Gregor W. Schmidt ◽  
Niek Welkenhuysen ◽  
Tian Ye ◽  
Marija Cvijovic ◽  
Stefan Hohmann
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Localization ◽  
Essential Role ◽  
Carbon Sources ◽  
Transcriptional Repressor ◽  
Energy Sources ◽  
Nucleocytoplasmic Shuttling

Abstract Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.

scholarly journals Fission yeast Opy1 is an endogenous PI(4,5)P2 sensor that binds to the phosphatidylinositol 4-phosphate 5-kinase Its3

2020 ◽  
Vol 133 (23) ◽  
pp. jcs247973
Author(s):  
Chloe E. Snider ◽  
Alaina H. Willet ◽  
HannahSofia T. Brown ◽  
Jun-Song Chen ◽  
Joshua M. Evers ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Cell Biology ◽  
Kinase Activity ◽  
Striking Difference ◽  
Evolutionary Divergence ◽  
Ph Domain ◽  
Phosphatidylinositol 4 ◽  
Related Proteins

ABSTRACTPhosphoinositides (PIPs) are a dynamic family of lipids that execute diverse roles in cell biology. PIP levels are regulated by numerous enzymes, but our understanding of how these enzymes are controlled in space and time is incomplete. One role of the PIP phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is to anchor the cytokinetic ring (CR) to the plasma membrane (PM) in Schizosaccharomyces pombe. While examining potential PI(4,5)P2-binding proteins for roles in CR anchoring, we identified the dual pleckstrin homology (PH) domain-containing protein Opy1. Although related proteins are implicated in PIP regulation, we found no role for S. pombe Opy1 in CR anchoring, which would be expected if it modulated PM PI(4,5)P2 levels. Our data indicate that although Opy1 senses PM PI(4,5)P2 levels and binds to the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, Opy1 does not regulate Its3 kinase activity or PM PI(4,5)P2 levels, a striking difference from its Saccharomyces cerevisiae homolog. However, overexpression of Opy1 resulted in cytokinesis defects, as might be expected if it sequestered PI(4,5)P2. Our results highlight the evolutionary divergence of dual PH domain-containing proteins and the need for caution when interpreting results based on their overexpression.This article has an associated First Person interview with the first author of the paper.

scholarly journals Plasma Membrane Localization of Ras Requires Class C Vps Proteins and Functional Mitochondria in Saccharomyces cerevisiae

2006 ◽  
Vol 26 (8) ◽  
pp. 3243-3255 ◽  
Author(s):  
Geng Wang ◽  
Robert J. Deschenes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Ras Proteins ◽  
Subcellular Targeting ◽  
Transport Pathways ◽  
Mitochondrial Defects ◽  
Protease Treatment ◽  
Class C

ABSTRACT Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.

scholarly journals Key Role of Ser562/661 in Snf1-Dependent Regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis

2004 ◽  
Vol 24 (10) ◽  
pp. 4083-4091 ◽  
Author(s):  
Godefroid Charbon ◽  
Karin D. Breunig ◽  
Ruddy Wattiez ◽  
Jean Vandenhaute ◽  
Isabelle Noël-Georis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Target Genes ◽  
Kluyveromyces Lactis ◽  
Active Form ◽  
Phosphorylation Site ◽  
Carbon Sources ◽  
Mutant Form ◽  
Zinc Cluster

ABSTRACT Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis.

Sign in / Sign up

Export Citation Format

Share Document