Abstract
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.
Highlights
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
Our results show that the intracellular ATP concentration is oscillating, but the amplitude of the oscillations is lower than suggested by previous experiments using quenching of cells and extraction of cell content [11]
A New ATP Nanobiosensor to Measure Intracellular ATP— In a previous report we described a new type of nanobiosensor design which can be used to measure intracellular metabolites, e.g. ATP [32]
Summary
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. Richard et al [11] demonstrated oscillations in a number of glycolytic intermediates, including ATP, ADP, and AMP, by quenching yeast cells and extracting their cell content. These measurements had a low time resolution (4 –5 s) and were inherently noisy because of the complex procedures in extraction and subsequent off-line measurements of metabolites. They were able to show that most glycolytic intermediates seem to oscillate with different phases compared with NADH. Our results suggest that 2-deoxyglucose, an inhibitor of glycolysis, inhibits the activity of PMA1
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