Quantitation of NAD + biosynthesis from the salvage pathway in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD + ) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD + decomposition products. NAD + 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 + biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD + and NADH (the reduced form of NAD + ) analyses on BY4742 wild‐type, NAD + salvage pathway knockout ( npt1 Δ) and NAD + de novo pathway knockout ( qpt1 Δ) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized 14 C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and 14 C detection to quantitate the total amounts of NAD + and NADH and the amounts derived from the salvage pathway. We observed that wild‐type and qpt1 Δ yeast exclusively utilized extracellular nicotinic acid for NAD + 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 observed that NAD + concentrations decreased in all three strains under CR. However, unlike the wild‐type strain, NADH concentrations did not decrease and NAD + : NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild‐type and qpt1 Δ strains, while no increase in lifespan was observed for the npt1 Δ strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR. Copyright © 2009 John Wiley & Sons, Ltd.