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 (npt1Delta) and NAD+ de novo pathway knockout (qpt1Delta) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized 14C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and 14C detection to quantitate the total amounts of NAD+ and NADH and the amounts derived from the salvage pathway. We observed that wild-type and qpt1Delta 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 qpt1Delta strains, while no increase in lifespan was observed for the npt1Delta strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR.
Sporty
Jennifer
J
Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA.
Lin
Su-Ju
SJ
Kato
Michiko
M
Ognibene
Ted
T
Stewart
Benjamin
B
Turteltaub
Ken
K
Bench
Graham
G
D002250:Carbon Radioisotopes / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000737:chemistry; D017353:Gene Deletion; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D009243:NAD / Q000096:biosynthesis*; D009525:Niacin / Q000378:metabolism; D010430:Pentosyltransferases / Q000172:deficiency; D011874:Radiometry; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013057:Spectrum Analysis; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)
D002250:Carbon Radioisotopes; D003470:Culture Media; D009243:NAD; D009525:Niacin; D010430:Pentosyltransferases; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating); C021864:nicotinate phosphoribosyltransferase; D005947:Glucose