Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

2005 ◽  
Vol 51 (10) ◽  
pp. 833-839 ◽  
Author(s):  
Adam Tylicki ◽  
Jan Czerniecki ◽  
Pawel Dobrzyn ◽  
Agnieszka Matanowska ◽  
Anna Olechno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Yeast Cells ◽  
Thiamine Pyrophosphate ◽  
Decarboxylase Activity ◽  
Mitochondrial Enzymes ◽  
Oxoglutarate Dehydrogenase ◽  
Cytosolic Enzymes

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phos phate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS–PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger in hibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.Key words: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, transketolase, pyruvate decarboxylase, activity, oxythiamine, inhibition.

scholarly journals Mitochondrial Superoxide Dismutase and Yap1p Act as a Signaling Module Contributing to Ethanol Tolerance of the Yeast Saccharomyces cerevisiae

2016 ◽  
Vol 83 (3) ◽  
Author(s):  
Anna N. Zyrina ◽  
Ekaterina A. Smirnova ◽  
Olga V. Markova ◽  
Fedor F. Severin ◽  
Dmitry A. Knorre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Hydrogen Peroxide ◽  
Superoxide Dismutase ◽  
Ethanol Tolerance ◽  
Yeast Cells ◽  
Ethanol Stress ◽  
Mitochondrial Enzymes ◽  
Mitochondrial Superoxide ◽  
Mitochondrial Superoxide Dismutase

ABSTRACT There are two superoxide dismutases in the yeast Saccharomyces cerevisiae—cytoplasmic and mitochondrial enzymes. Inactivation of the cytoplasmic enzyme, Sod1p, renders the cells sensitive to a variety of stresses, while inactivation of the mitochondrial isoform, Sod2p, typically has a weaker effect. One exception is ethanol-induced stress. Here we studied the role of Sod2p in ethanol tolerance of yeast. First, we found that repression of SOD2 prevents ethanol-induced relocalization of yeast hydrogen peroxide-sensing transcription factor Yap1p, one of the key stress resistance proteins. In agreement with this, the levels of Trx2p and Gsh1p, proteins encoded by Yap1 target genes, were decreased in the absence of Sod2p. Analysis of the ethanol sensitivities of the cells lacking Sod2p, Yap1p, or both indicated that the two proteins act in the same pathway. Moreover, preconditioning with hydrogen peroxide restored the ethanol resistance of yeast cells with repressed SOD2. Interestingly, we found that mitochondrion-to-nucleus signaling by Rtg proteins antagonizes Yap1p activation. Together, our data suggest that hydrogen peroxide produced by Sod2p activates Yap1p and thus plays a signaling role in ethanol tolerance. IMPORTANCE Baker's yeast harbors multiple systems that ensure tolerance to high concentrations of ethanol. Still, the role of mitochondria under severe ethanol stress in yeast is not completely clear. Our study revealed a signaling function of mitochondria which contributes significantly to the ethanol tolerance of yeast cells. We found that mitochondrial superoxide dismutase Sod2p and cytoplasmic hydrogen peroxide sensor Yap1p act together as a module of the mitochondrion-to-nucleus signaling pathway. We also report cross talk between this pathway and the conventional retrograde signaling cascade activated by dysfunctional mitochondria.

scholarly journals The Saccharomyces cerevisiae Leu3 protein activates expression of GDH1, a key gene in nitrogen assimilation.

1995 ◽  
Vol 15 (1) ◽  
pp. 52-57 ◽  
Author(s):  
Y Hu ◽  
T G Cooper ◽  
G B Kohlhaw
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Glutamate Dehydrogenase ◽  
Specific Activity ◽  
Yeast Cells ◽  
Homologous Sequence ◽  
Biosynthetic Pathways ◽  
Lacz Expression ◽  
Branched Chain Amino Acid ◽  
Branched Chain

The Leu3 protein of Saccharomyces cerevisiae has been shown to be a transcriptional regulator of genes encoding enzymes of the branched-chain amino acid biosynthetic pathways. Leu3 binds to upstream activating sequences (UASLEU) found in the promoters of LEU1, LEU2, LEU4, ILV2, and ILV5. In vivo and in vitro studies have shown that activation by Leu3 requires the presence of alpha-isopropylmalate. In at least one case (LEU2), Leu3 actually represses basal-level transcription when alpha-isopropylmalate is absent. Following identification of a UASLEU-homologous sequence in the promoter of GDH1, the gene encoding NADP(+)-dependent glutamate dehydrogenase, we demonstrate that Leu3 specifically interacts with this UASLEU element. We then show that Leu3 is required for full activation of the GDH1 gene. First, the expression of a GDH1-lacZ fusion gene is three- to sixfold lower in a strain lacking the LEU3 gene than in an isogenic LEU3+ strain. Expression is restored to near-normal levels when the leu3 deletion cells are transformed with a LEU3-bearing plasmid. Second, a significant decrease in GDH1-lacZ expression is also seen when the UASLEU of the GDH1-lacZ construct is made nonfunctional by mutation. Third, the steady-state level of GDH1 mRNA decreases about threefold in leu3 null cells. The decrease in GDH1 expression in leu3 null cells is reflected in a diminished specific activity of NADP(+)-dependent glutamate dehydrogenase. We also demonstrate that the level of GDH1-lacZ expression correlates with the cells' ability to generate alpha-isopropylmalate and is lowest in cells unable to produce alpha-isopropylmalate. We conclude that GDH1, which plays an important role in the assimilation of ammonia in yeast cells, is, in part, activated by a Leu3-alpha-isopropylmalate complex. This conclusion suggests that Leu3 participates in transcriptional regulation beyond the branched-chain amino acid biosynthetic pathways.

scholarly journals Expression of the rat GLUT1 glucose transporter in the yeast Saccharomyces cerevisiae

1996 ◽  
Vol 315 (1) ◽  
pp. 177-182 ◽  
Author(s):  
Toshiko KASAHARA ◽  
Michihiro KASAHARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Specific Activity ◽  
Expression System ◽  
Yeast Cells ◽  
Transport Activity ◽  
Yeast Saccharomyces Cerevisiae ◽  
Crude Membrane

We expressed the rat GLUT1 facilitative glucose transporter in the yeast Saccharomyces cerevisiae with the use of a galactose-inducible expression system. Confocal immunofluorescence microscopy indicated that a majority of this protein is retained in an intracellular structure that probably corresponds to endoplasmic reticulum. Yeast cells expressing GLUT1 exhibited little increase in glucose-transport activity. We prepared a crude membrane fraction from these cells and made liposomes with this fraction using the freeze–thaw/sonication method. In this reconstituted system, D-glucose-transport activity was observed with a Km for D-glucose of 3.4±0.2 mM (mean±S.E.M.) and was inhibited by cytochalasin B (IC50 = 0.44±0.03 μM), HgCl2 (IC50 = 3.5±0.5 μM), phloretin (IC50 = 49±12 μM) and phloridzin (IC50 = 355±67 μM). To compare these properties with native GLUT1, we made reconstituted liposomes with a membrane fraction prepared from human erythrocytes, in which the Km of D-glucose transport and ICs of these inhibitors were approximately equal to those obtained with GLUT1 made by yeast. When the relative amounts of GLUT1 in the crude membrane fractions were measured by quantitative immunoblotting, the specific activity of the yeast-made GLUT1 was 110% of erythrocyte GLUT1, indicating that GLUT1 expressed in yeast is fully active in glucose transport.

Expression of Rous sarcoma virus transforming protein pp60v-src in Saccharomyces cerevisiae cells

1987 ◽  
Vol 7 (6) ◽  
pp. 2180-2187
Author(s):  
J S Brugge ◽  
G Jarosik ◽  
J Andersen ◽  
A Queral-Lustig ◽  
M Fedor-Chaiken ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rous Sarcoma Virus ◽  
Mammalian Cells ◽  
Specific Activity ◽  
Constitutive Expression ◽  
Single Copy ◽  
Yeast Cells ◽  
Rous Sarcoma ◽  
Src Gene ◽  
Sarcoma Virus

The Rous sarcoma virus (RSV) pp60v-src protein was expressed in Saccharomyces cerevisiae cells either from a plasmid vector carrying the v-src gene or in yeast cells containing a single-copy v-src gene chromosomally integrated. In both yeast strains, v-src gene transcription is regulated by the galactose-inducible GAL10 promoter. Growth in galactose-containing medium resulted in constitutive expression of pp60v-src in the integrated strain and transient expression of higher levels of pp60v-src in the plasmid-bearing strain. The concentration of pp60v-src in the plasmid-bearing strain at its peak of expression was approximately threefold lower than that found in RSV-transformed mammalian cells. pp60v-src synthesized in yeast cells was phosphorylated in vivo on sites within the amino and carboxyl halves of the molecule. In immune complex kinase assays, the yeast pp60v-src was autophosphorylated on tyrosine and was able to phosphorylate exogenous substrates such as casein and enolase. The specific activity of pp60v-src synthesized in yeast cells was approximately 5- to 10-fold higher than that made in mammalian cells. Induction of pp60v-src caused the death of the plasmid-bearing yeast strain and transient inhibition of growth of the single-copy strain. Concomitantly, this induction resulted in high levels of tyrosine phosphorylation of yeast cell proteins. This indicates that pp60v-src functions as a tyrosine-specific phosphotransferase in yeast cells and suggests that hyperphosphorylation of yeast proteins is inimical to cell growth.

scholarly journals Physiological Characterization of the ARO10-Dependent, Broad-Substrate-Specificity 2-Oxo Acid Decarboxylase Activity of Saccharomyces cerevisiae

2005 ◽  
Vol 71 (6) ◽  
pp. 3276-3284 ◽  
Author(s):  
Zeynep Vuralhan ◽  
Marijke A. H. Luttik ◽  
Siew Leng Tai ◽  
Viktor M. Boer ◽  
Marcos A. Morais ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Substrate Specificity ◽  
Nitrogen Source ◽  
Constitutive Expression ◽  
Thiamine Pyrophosphate ◽  
Acid Decarboxylase ◽  
Decarboxylase Activity ◽  
Broad Substrate Specificity ◽  
Fusel Alcohols ◽  
Chemostat Cultures

ABSTRACT Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae CEN.PK113-7D were grown with different nitrogen sources. Cultures grown with phenylalanine, leucine, or methionine as a nitrogen source contained high levels of the corresponding fusel alcohols and organic acids, indicating activity of the Ehrlich pathway. Also, fusel alcohols derived from the other two amino acids were detected in the supernatant, suggesting the involvement of a common enzyme activity. Transcript level analysis revealed that among the five thiamine-pyrophospate-dependent decarboxylases (PDC1, PDC5, PDC6, ARO10, and THI3), only ARO10 was transcriptionally up-regulated when phenylalanine, leucine, or methionine was used as a nitrogen source compared to growth on ammonia, proline, and asparagine. Moreover, 2-oxo acid decarboxylase activity measured in cell extract from CEN.PK113-7D grown with phenylalanine, methionine, or leucine displayed similar broad-substrate 2-oxo acid decarboxylase activity. Constitutive expression of ARO10 in ethanol-limited chemostat cultures in a strain lacking the five thiamine-pyrophosphate-dependent decarboxylases, grown with ammonia as a nitrogen source, led to a measurable decarboxylase activity with phenylalanine-, leucine-, and methionine-derived 2-oxo acids. Moreover, even with ammonia as the nitrogen source, these cultures produced significant amounts of the corresponding fusel alcohols. Nonetheless, the constitutive expression of ARO10 in an isogenic wild-type strain grown in a glucose-limited chemostat with ammonia did not lead to any 2-oxo acid decarboxylase activity. Furthermore, even when ARO10 was constitutively expressed, growth with phenylalanine as the nitrogen source led to increased decarboxylase activities in cell extracts. The results reported here indicate the involvement of posttranscriptional regulation and/or a second protein in the ARO10-dependent, broad-substrate-specificity decarboxylase activity.

scholarly journals Induction of the branched-chain 2-oxo acid dehydrogenase complex in 3T3-L1 adipocytes during differentiation

1983 ◽  
Vol 214 (1) ◽  
pp. 177-181 ◽  
Author(s):  
D T Chuang ◽  
C W C Hu ◽  
M S Patel
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Common Mechanism ◽  
Acid Dehydrogenase ◽  
Oxoglutarate Dehydrogenase ◽  
Branched Chain ◽  
Kinetics Of ◽  
Acid Dehydrogenase Complex ◽  
Dehydrogenase Complex

The activities of 2-oxo acid dehydrogenase complexes were measured during hormone-mediated differentiation of 3T3-L1 preadipocytes into adipocytes. Specific activity of leucine-activated branched-chain 2-oxo acid dehydrogenase complex increased approx. 10-fold in 3T3-L1 adipocytes compared with 3T3-L1 preadipocytes. In contrast, specific activity of the 2-oxoglutarate dehydrogenase complex increased by only 3-fold in 3T3-L1 adipocytes. The three catalytic component enzymes of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex showed concomitant increases in their specific activities. A close similarity in kinetics of induction of the branched-chain 2-oxo acid dehydrogenase complex and the pyruvate dehydrogenase complex in 3T3-L1 adipocytes suggests that a common mechanism may be involved in hormone-dependent increases in the activities of the catalytic components of these two complexes in 3T3-L1 adipocytes during differentiation.

Isolierung und Charakterisierung eines membrangebundenen Pyruvatdehydrogenase-Komplexes aus dem phototrophen Bakterium Rhodospirillum rubrum /Isolation and Characterization of a Membrane-Bound Pyruvate Dehydrogenase Complex from the Phototrophic Bacterium Rhodospirillum rubrum

1977 ◽  
Vol 32 (5-6) ◽  
pp. 351-361 ◽  
Author(s):  
Rosemarie Lüderitz ◽  
Jobst-Heinrich Klemme
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Metal Ion ◽  
Rhodospirillum Rubrum ◽  
Coenzyme A ◽  
Specific Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Thiamine Pyrophosphate ◽  
Enzyme Complex ◽  
Isolation And Characterization ◽  
Dehydrogenase Complex

Abstract The pyruvate dehydrogenase complex from the photosynthetic bacterium Rhododospirillum rubrum was associated with the membrane fraction both in heterotrophically and photosynthetically grown cells. The complex was separated from the membranes and partially purified by precipitation with MgSO4 and gelfiltration through Sepharose 4B. The purified complex had a specific activity of 1.5 - 2 μmol/min-mg protein and contained the following partial activities: pyruvate dehydrogenase (EC 1.2.4.1), dihydrolipoamide transacetylase (EC 2.3.1.12) and dihydrolipoamide dehydrogenase (EC 1.6.4.3). Contrary to other bacterial pyruvate dehydrogenase complexes, the enzyme complex from R . rubrum revealed no cooperativity between pyruvate binding sites. The kinetic constants [Km) for the overall reaction were (in mм) : 0.14 (pyruvate), 0.07 (NAD) and 0.025 (coenzyme A). The Km for thiamine pyrophosphate was dependent on the nature and the concentration of the divalent metal ion (Mn or Mg) present in the reaction mixture, the values ranging from 0.5 to 3 μm . NADH was a potent inhibitor (Ki = 5 μm) of the enzyme complex and the dihydrolipo­ amide dehydrogenase. The inhibition was competitive with respect to NAD. In addition to its rapid inhibitory effect, NADH also inactivated the enzyme. Cysteine partially protected the enzyme complex against NADH-inactivation. Acetyl-coenzyme A also inhibited the overall reaction (Ki = 40 μм) . The inhibition was dependent on the concentration of coenzyme A, but independent of the concentration of pyruvate. Sugar phosphates, phosphoenolpyruvate, citric acid cycle intermediates and nucleosidephosphates (1 mм) had no pronounced effect on the overall reaction.

In vivo Evidence for a Functional Glycolytic Compartment in Synchronous Yeast Cells

1981 ◽  
Vol 36 (7-8) ◽  
pp. 615-618 ◽  
Author(s):  
Stefan Postius
Keyword(s):  
Yeast Cell ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Decarboxylase ◽  
Yeast Cells ◽  
Cell Populations ◽  
Aerobic Conditions ◽  
Metabolic Behaviour

Abstract The different metabolic behaviour of endogenously produced [1-14C] pyruvate derived from [3,4-14C]glucose and of exogenously added [1-14C]pyruvate were studied with synchronous yeast cell populations, under conditions which differentially influenced the activities of pyruvate decarboxylase in the cytoplasm and pyruvate dehydrogenase in the mitochondria. Endogenously produced [1-14C]pyruvate is decarboxylated almost exclusively by PDC under anaerobic condi­ tions, in contrast to added [1-14C]pyruvate which is decarboxylated under aerobic conditions by the action of PDH mainly. Whereas 14CO 2 evolution from exogenous [l-14C]pyruvate can be diluted proportionally by addition of pyruvate, this is not the case for 14CO 2 evolution from endogenous [1-14C]pyruvate. It is suggested that glycolysis or at least some constituents of it might be arranged in such a manner that the resulting complex behaves as a functional compartment, meaning that it is almost inaccessible to exogenously added pyruvate.

Identification, cloning and characterisation of a new gene required for full pyruvate decarboxylase activity in Saccharomyces cerevisiae

1989 ◽  
Vol 15 (3) ◽  
pp. 171-175 ◽  
Author(s):  
Anthony P. H. Wright ◽  
Hong-Lan Png ◽  
Brian S. Hartley
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Decarboxylase ◽  
Decarboxylase Activity ◽  
New Gene
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