TY - JOUR
T1 - In vivo analysis of NH4 + transport and central nitrogen metabolism in Saccharomyces cerevisiae during aerobic nitrogen-limited growth
AU - Cueto-Rojas, H. F.
AU - Maleki Seifar, R.
AU - ten Pierick, A.
AU - van Helmond, W.
AU - Pieterse, M. M.
AU - Heijnen, J. J.
AU - Wahl, S. A.
PY - 2016
Y1 - 2016
N2 - 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 NH4 +/literIC, 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 NH3 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.
AB - 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 NH4 +/literIC, 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 NH3 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.
UR - http://resolver.tudelft.nl/uuid:c7151f9a-e95b-4496-84a7-b2824f533dbd
UR - http://www.scopus.com/inward/record.url?scp=84996865984&partnerID=8YFLogxK
U2 - 10.1128/AEM.01547-16
DO - 10.1128/AEM.01547-16
M3 - Article
AN - SCOPUS:84996865984
SN - 0099-2240
VL - 82
SP - 6831
EP - 6845
JO - Applied and Environmental Microbiology
JF - Applied and Environmental Microbiology
IS - 23
ER -