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• W2519379390 endingPage "6845" @default.
• W2519379390 startingPage "6831" @default.
• W2519379390 abstract "ABSTRACT Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the in vivo intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we were able to measure the intracellular ammonium concentration in N-limited aerobic chemostat cultivations using three different N sources (ammonium, urea, and glutamate) at the same growth rate (0.05 h −1 ). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH 4 + /liter IC , is required to supply the reactions in the central N metabolism, independent of the N source. Based on the experimental results and different assumptions, the vacuolar and cytosolic ammonium concentrations were estimated. Furthermore, we identified a futile cycle caused by NH 3 leakage into the extracellular space, which can cost up to 30% of the ATP production of the cell under N-limited conditions, and a futile redox cycle between Gdh1 and Gdh2 reactions. Finally, using shotgun proteomics with protein expression determined relative to a labeled reference, differences between the various environmental conditions were identified and correlated with previously identified N compound-sensing mechanisms. IMPORTANCE In our work, we studied central N metabolism using quantitative approaches. First, intracellular ammonium was measured under different N sources. The results suggest that Saccharomyces cerevisiae cells maintain a constant NH 4 + concentration (around 3 mmol NH 4 + /liter IC ), independent of the applied nitrogen source. We hypothesize that this amount of intracellular ammonium is required to obtain sufficient thermodynamic driving force. Furthermore, our calculations based on thermodynamic analysis of the transport mechanisms of ammonium suggest that ammonium is not equally distributed, indicating a high degree of compartmentalization in the vacuole. Additionally, metabolomic analysis results were used to calculate the thermodynamic driving forces in the central N metabolism reactions, revealing that the main reactions in the central N metabolism are far from equilibrium. Using proteomics approaches, we were able to identify major changes, not only in N metabolism, but also in C metabolism and regulation." @default.
• W2519379390 created "2016-09-23" @default.
• W2519379390 creator A5013283177 @default.
• W2519379390 creator A5017592884 @default.
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• W2519379390 creator A5053105874 @default.
• W2519379390 creator A5081992425 @default.
• W2519379390 creator A5090214575 @default.
• W2519379390 date "2016-12-01" @default.
• W2519379390 modified "2023-09-24" @default.
• W2519379390 title "<i>In Vivo</i> Analysis of NH ₄ ⁺ Transport and Central Nitrogen Metabolism in Saccharomyces cerevisiae during Aerobic Nitrogen-Limited Growth" @default.
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