Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions

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The relationship between dNTP levels and DNA synthesis was investigated using ␣ factor-synchronized yeast treated with the ribonucleotide reductase inhibitor hydroxyurea (HU). Although HU blocked DNA synthesis and prevented the dNTP pool expansion that normally occurs at G 1 /S, it did not exhaust the levels of any of the four dNTPs, which dropped to about 80% of G 1 levels. When dbf4 yeast that are ts for replication initiation were allowed to preaccumulate dNTPs at 37°C before being released to 25°C in the presence of HU, they synthesized 0.3 genome equivalents of DNA and then arrested as dNTPs approached sub-G 1 levels. Accumulation of dNTPs at G 1 /S was not a prerequisite for replication initiation, since dbf4 cells incubated in HU at 25°C were able to replicate when subsequently switched to 37°C in the absence of HU. The replication arrest mechanism was not dependent on the Mec1/ Rad53 pathway, since checkpoint-deficient rad53 cells also failed to exhaust basal dNTPs when incubated in HU. The persistence of basal dNTP levels in HU-arrested cells and partial bypass of the arrest in cells that had preaccumulated dNTPs suggest that cells have a mechanism for arresting DNA chain elongation when dNTP levels are not maintained above a critical threshold. Hydroxyurea (HU)1 is a potent inhibitor of the enzyme ribonucleotide reductase (RNR) and inhibits DNA replication in a wide variety of cells, including Saccharomyces cerevisiae (1). The simplest explanation for HU inhibition of DNA synthesis is that it starves the DNA polymerase at the replication forks for dNTPs. HU treatment has been shown to reduce the purine dNTP pools in a variety of mammalian cells (2-7); however, conflicting data exist concerning its modulation of pyrimidine dNTP pool levels. Furthermore, even for purine dNTPs, HU has only rarely been shown to cause a complete depletion of the dGTP or dATP pools (2-4). More commonly, HU results in only partial depletion of the purine dNTP pools (5-7). The complicated, often reciprocal, changes in individual dNTP pools that occur in HU-treated mammalian cells may be due to the compensatory activities of deoxyribonucleotide salvage pathways in higher eukaryotes. Budding yeast offers a simpler system in which to study the mechanism by which HU affects replication. Yeast possess no deoxyribonucleoside kinase activities, and thus deoxyribonucleotide synthesis is entirely dependent on ribonucleotide reductase. Also, yeast can easily be synchronized in G 1 using mating pheromone, and the availability of several temperature-sensitive cdc mutations allows cell cycle progression to be reversibly halted at specific points throughout the cell cycle (8). Reciprocal switch experiments in yeast have ordered the execution point of several cdc genes with respect to the HU-sensitive step during the cell cycle (9). Furthermore, mutational screens in yeast have identified regulatory proteins, such as Mec1 and Rad53, that are necessary for proper execution of the HU-induced replication arrest checkpoint (10). Despite the gene...

Section: Discussionmentioning

confidence: 99%

Hydroxyurea Arrests DNA Replication by a Mechanism That Preserves Basal dNTP Pools

Koç

Wheeler²,

Mathews³

et al. 2004

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197

“…Thus, glucose is a metabolic substrate acting as an extracellular source of free energy powering life. In yeast, a strong negative correlation between glycolytic flux and intracellular ATP concentration has been widely documented (1)(2)(3). Because glucose catabolism is the main source of ATP-derived free energy, this observation (the higher the rate of glycolysis the lower the ATP content) is a counterintuitive phenomenon, which has been referred to as the ATP paradox.…”

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confidence: 99%

The ATP Paradox Is the Expression of an Economizing Fuel Mechanism

Aledo

Valle

2004

The strong negative correlation between glycolytic flux and intracellular ATP concentration observed in yeast has long been an intriguing and counterintuitive phenomenon, which has been referred to as the ATP paradox. Herein, using principles of irreversible thermodynamics it was shown that if the ATP-consuming pathways are more sensitive to extracellular glucose than glycolysis, then upon glucose addition glycolysis performance can switch from an efficient working regime to a dissipative regime, and vice versa, depending on glucose availability. The efficient regime represents a good compromise between high output power and low dissipation, whereas the dissipative working regime offers a higher output power although at a high glucose cost. The physiological and evolutionary implications of this switch strategy are discussed.Glycolysis is a metabolic pathway providing cells with energy in the form of ATP, which can be used to drive cellular processes. Thus, glucose is a metabolic substrate acting as an extracellular source of free energy powering life. In yeast, a strong negative correlation between glycolytic flux and intracellular ATP concentration has been widely documented (1-3). Because glucose catabolism is the main source of ATP-derived free energy, this observation (the higher the rate of glycolysis the lower the ATP content) is a counterintuitive phenomenon, which has been referred to as the ATP paradox. A sudden transition from glucose limitation to glucose excess leads to a new steady state at increased glycolytic flux with a sustained decrease in the ATP levels (3). Because ADP phosphorylation is tightly coupled to glucose breakdown, a higher glycolytic flux implies that more ATP is being formed. The subsequent increase in the flux throughout ATP-consuming reactions was thought to be brought about by an increased ATP/ADP ratio. Therefore, the decreased steady state level of ATP constitutes an intriguing observation. Somsen et al.(3), in an elegant work employing metabolic control analysis and a simple model, concluded that the experimental ATP changes can only be explained if the sensitivities of ATP-consuming routes to extracellular glucose are higher than that of the glycolytic pathway. Although this particular sensitivity of anabolic pathways solves the problem of how a drop in ATP concentration triggered by high glucose levels is possible, one question still remains open. What are the physiological implications of this behavior we have termed as the ATP paradox? In other words, what is the purpose of the stronger sensitivity to the level of extracellular fuel exhibited by ATP-utilizing pathways with respect to that of ATP-producing pathways?

“…2), and fructose-1,6-bisphosphatase (1) has been proposed, even though the overexpression of these key enzymes does not increase the glycolytic flux (3). Other mechanisms of control have been proposed such as futile cycle activity (4) and an inhibitory effect of ATP (5). Indeed, it seems likely that the regulation of glycolysis is a complex process involving different hierarchical events leading from gene expression to the metabolic fluxes via protein levels, enzyme activities, and metabolite effects (6,7).…”

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confidence: 99%

The Trehalose Pathway Regulates Mitochondrial Respiratory Chain Content through Hexokinase 2 and cAMP in Saccharomyces cerevisiae

Noubhani

Bunoust

Bonini

et al. 2009

In yeast, trehalose is synthesized by a multimeric enzymatic complex: TPS1 encodes trehalose 6-phosphate synthase, which belongs to a complex that is composed of at least three other subunits, including trehalose 6-phosphate phosphatase Tps2 and the redundant regulatory subunits Tps3 and Tsl1. The product of the TPS1 gene plays an essential role in the control of the glycolytic pathway by restricting the influx of glucose into glycolysis. In this paper, we investigated whether the trehalose synthesis pathway could be involved in the control of the other energy-generating pathway: oxidative phosphorylation. We show that the different mutants of the trehalose synthesis pathway (tps1⌬, tps2⌬, and tps1,2⌬) exhibit modulation in the amount of respiratory chains, in terms of cytochrome content and maximal respiratory activity. Furthermore, these variations in mitochondrial enzymatic content are positively linked to the intracellular concentration in cAMP that is modulated by Tps1p through hexokinase2. This is the first time that a pathway involved in sugar storage, i.e. trehalose, is shown to regulate the mitochondrial enzymatic content.The control of glycolysis in the yeast Saccharomyces cerevisiae has been extensively studied. First, allosteric regulation of the irreversible steps catalyzed by phosphofructokinase (1), pyruvate kinase (review in Ref.2), and fructose-1,6-bisphosphatase (1) has been proposed, even though the overexpression of these key enzymes does not increase the glycolytic flux (3). Other mechanisms of control have been proposed such as futile cycle activity (4) and an inhibitory effect of ATP (5). Indeed, it seems likely that the regulation of glycolysis is a complex process involving different hierarchical events leading from gene expression to the metabolic fluxes via protein levels, enzyme activities, and metabolite effects (6, 7). Among these actors, the product of the TPS1 gene has been shown to play an essential role in the control of the glycolytic pathway by restricting the influx of glucose into glycolysis (8). TPS1 encodes trehalose 6-phosphate (Tre6P) 3 synthase (9 -12). This enzyme is part of a multimeric protein complex composed of at least three other subunits, i.e. Tre6P phosphatase encoded by TPS2 (13) and the redundant regulatory subunits Tps3 and Tsl1 (14).A particularly intriguing finding is that tps1⌬ mutants are defective not only for Tre6P synthesis but also for growth on glucose or related rapidly fermented sugars (8,11,15). This may be explained by an uncontrolled influx of glucose into the glycolytic pathway. This phenomenon is characterized by hyperaccumulation of glucose 6-phosphate, fructose 6-phosphate, and fructose 1,6-bisphosphate (Fru1,6bP) (8, 16 -18) and depletion of ATP, P i , and all intermediates of glycolysis downstream of glyceraldehyde-3-phosphate dehydrogenase (19). Several mutations have been described that suppress the growth defect of tps1⌬ mutants apparently by reducing sugar influx into glycolysis (16,20) or by diverting the excess sugar phosphate into ...