Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae
Nicotinamide adenine dinucleotide (NAD{sup +}) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or by the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD{sup +} decomposition products. NAD{sup +} biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD{sup +} biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD{sup +} and NADH (the reduced form of NAD{sup +}) analyses on BY4742 wild type, NAD+ salvage pathway knockout (npt1{Delta}), and NAD+ de novo pathway knockout (qpt1{Delta}) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized {sup 14}C labeled nicotinic acid in the culture media combined with HPLC speciation and both UV and {sup 14}C detection to quantitate the total amounts of NAD{sup +} and NADH and the amounts derived from the salvage pathway. We observe that wild type and qpt1{Delta} yeast exclusively utilize extracellular nicotinic acid for NAD{sup +} and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observe that NAD{sup +} concentrations decrease in all three strains under CR. However, unlike the wild type strain, NADH concentrations do not decrease and NAD{sup +}:NADH ratios do not increase under CR for either knockout strain. Lifespan analyses reveal that CR results in a lifespan increase of approximately 25% for the wild type and qpt1{Delta} strains, while no increase in lifespan is observed for the npt1{Delta} strain. In combination these data suggest that having a functional salvage pathway is more important than the absolute levels of NAD{sup +} or NADH for lifespan extension under CR.
- Research Organization:
- Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- W-7405-ENG-48
- OSTI ID:
- 966233
- Report Number(s):
- LLNL-JRNL-410719; TRN: US200921%%330
- Journal Information:
- Yeast, vol. 26, N/A, April 27, 2009, pp. 363-369, Vol. 26
- Country of Publication:
- United States
- Language:
- English
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