On‐line measurements of oscillating mitochondrial membrane potential in glucose‐fermenting <i>Saccharomyces cerevisiae</i>

Andersen, Ann Zahle; Poulsen, Allan K.; Brasen, Jens Christian; Olsen, Lars Folke

doi:10.1002/yea.1508

Cited by 30 publications

(38 citation statements)

References 33 publications

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“…As for the F 0 F 1 ATPase the activity needs to be tightly controlled for oscillations to occur. Both inhibition of the ATPase by azide or stimulation of the ATPase by FCCP will result in the disappearance of the oscillations (16,21). Presumably the same applies to PMA1, although it has not yet been documented that an increase in the activity of this enzyme will make the oscillations disappear.…”

Section: Discussionmentioning

confidence: 95%

“…Previous experiments suggested that the two main ATP consuming processes are ATP hydrolysis by the mitochondrial F 0 F 1 ATPase and the plasma membrane ATPase (PMA1) (16). ATP hydrolysis by the F 0 F 1 ATPase is associated with pumping of protons out of the mitochondria which again results in the generation of a mitochondrial membrane potential (16,21) and because the intracellular ATP concentration is oscillating the mitochondrial membrane potential is oscillating too as shown in Fig. 3D.…”

Section: A New Atp Nanobiosensor To Measure Intracellular Atp-mentioning

confidence: 90%

“…Measurements of Mitochondrial Membrane PotentialMeasurements of the mitochondrial membrane potential in the yeast cells were done in the presence of 5 M DiOC 2 (3) as described previously (21). Excitation wavelength was 488/3 nm and emission at 600/3 nm.…”

Section: Methodsmentioning

confidence: 99%

“…Oscillations only occur when the cell density is beyond a certain critical value (15,(17)(18)(19). The oscillations in glycolysis are usually monitored as oscillations in the autofluorescence of NADH, but also time-resolved oscillations in CO 2 evolution (20), mitochondrial membrane potential (21,22), and intracellular pH (pH i ) 2 (16) have been recorded. The oscillations in mitochondrial membrane potential and in intracellular pH were suggested to be caused by oscillating activities of the F 0 F 1 ATPase and the plasma membrane ATPase (PMA1) caused by oscillations in intracellular ATP (16).…”

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confidence: 99%

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Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Özalp

Pedersen

Nielsen

et al. 2010

Journal of Biological Chemistry

107

109

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Adenosine 5-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F 0 F 1 ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.Adenosine 5Ј-triphosphate (ATP) is a highly important biomolecule in living cells: It plays a central role in cell energy metabolism and also serves directly or indirectly in a number of cell signaling processes (1-3). The intracellular concentration of ATP is believed to oscillate in some eukaryotic cells, e.g. in ␤-cells (4) and in cells of the yeast Saccharomyces cerevisiae (5). However, changes in cytoplasmic ATP concentration with high time resolution have so far only been measured in a few circumstances (6, 7), mainly because methods for such continuous measurements are not generally available, or, in the case of NMR, require very high densities of cells or tissue (8).The lack of time-resolved measurements has prohibited the understanding of how the level of ATP and other intracellular metabolites are regulated in the cell and how ATP in turn regulates a number of cellular processes. Most current measurements of ATP in cells use extraction of the cell content and measure the concentration of ATP in the extract by various off-line methods such as HPLC (9), luciferase (10), or other enzyme-based methods (11). A few protein-based sensors exist (6,7,(12)(13)(14), which in principle allow for time-resolved measurements of intracellular ATP or ADP, but some of these methods entail expression of the sensor molecule in situ, which is not always possible. Hence, although it is expected that the intracellular concentration of ATP is anything but stationary, this has not been verified by real-time measurements, except in a few cases where ...

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Section: Discussionmentioning

confidence: 95%

Section: A New Atp Nanobiosensor To Measure Intracellular Atp-mentioning

confidence: 90%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Özalp

Pedersen

Nielsen

et al. 2010

Journal of Biological Chemistry

107

109

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“…These include levels of nicotinamide adenine dinucleotide (NAD) which can be seen to oscillate between the oxidized (NAD + ) and reduced forms (NADH), as well as other glycolytic intermediates, including glucose-6-phosphate, fructose-6-phosphate and fructose 1,6-bisphosphate [1]. Glycolytic oscillations are accompanied by oscillations in mitochondrial membrane potential (Δψ m ) [2]. This phenomenon has been observed for over 40 years [3] and is known as glycolytic oscillations [4].…”

Section: Introductionmentioning

confidence: 99%

Exploring the genetic control of glycolytic oscillations in Saccharomyces Cerevisiae

et al. 2012

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BackgroundA well known example of oscillatory phenomena is the transient oscillations of glycolytic intermediates in Saccharomyces cerevisiae, their regulation being predominantly investigated by mathematical modeling. To our knowledge there has not been a genetic approach to elucidate the regulatory role of the different enzymes of the glycolytic pathway.ResultsWe report that the laboratory strain BY4743 could also be used to investigate this oscillatory phenomenon, which traditionally has been studied using S. cerevisiae X2180. This has enabled us to employ existing isogenic deletion mutants and dissect the roles of isoforms, or subunits of key glycolytic enzymes in glycolytic oscillations. We demonstrate that deletion of TDH3 but not TDH2 and TDH1 (encoding glyceraldehyde-3-phosphate dehydrogenase: GAPDH) abolishes NADH oscillations. While deletion of each of the hexokinase (HK) encoding genes (HXK1 and HXK2) leads to oscillations that are longer lasting with lower amplitude, the effect of HXK2 deletion on the duration of the oscillations is stronger than that of HXK1. Most importantly our results show that the presence of beta (Pfk2) but not that of alpha subunits (Pfk1) of the hetero-octameric enzyme phosphofructokinase (PFK) is necessary to achieve these oscillations. Furthermore, we report that the cAMP-mediated PKA pathway (via some of its components responsible for feedback down-regulation) modulates the activity of glycoytic enzymes thus affecting oscillations. Deletion of both PDE2 (encoding a high affinity cAMP-phosphodiesterase) and IRA2 (encoding a GTPase activating protein- Ras-GAP, responsible for inactivating Ras-GTP) abolished glycolytic oscillations.ConclusionsThe genetic approach to characterising the glycolytic oscillations in yeast has demonstrated differential roles of the two types of subunits of PFK, and the isoforms of GAPDH and HK. Furthermore, it has shown that PDE2 and IRA2, encoding components of the cAMP pathway responsible for negative feedback regulation of PKA, are required for glycolytic oscillations, suggesting an enticing link between these cAMP pathway components and the glycolysis pathway enzymes shown to have the greatest role in glycolytic oscillation. This study suggests that a systematic genetic approach combined with mathematical modelling can advance the study of oscillatory phenomena.

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Current awareness on yeast

2008

Yeast

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In order to keep subscribers up‐to‐date with the latest developments in their field, this current awareness service is provided by John Wiley & Sons and contains newly‐published material on yeasts. Each bibliography is divided into 10 sections. 1 Reviews; 2 General; 3 Biochemistry; 4 Biotechnology; 5 Cell Biology; 6 Gene Expression; 7 Genetics; 8 Physiology; 9 Medical Mycology; 10 Recombinant DNA Technology. Within each section, articles are listed in alphabetical order with respect to author. If, in the preceding period, no publications are located relevant to any one of these headings, that section will be omitted. (4 weeks journals ‐ search completed 4th. Oct. 2007)

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On‐line measurements of oscillating mitochondrial membrane potential in glucose‐fermenting Saccharomyces cerevisiae

Cited by 30 publications

References 33 publications

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

Exploring the genetic control of glycolytic oscillations in Saccharomyces Cerevisiae

Current awareness on yeast

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