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

Jennifer Sporty, Su Ju Lin, Michiko Kato, Ted Ognibene, Benjamin Stewart, Ken W Turteltaub, Graham Bench

Research output: Contribution to journalArticlepeer-review

25 Scopus citations

Abstract

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 (npt1Δ) 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 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 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.

Original languageEnglish (US)
Pages (from-to)363-369
Number of pages7
JournalYeast
Volume26
Issue number7
DOIs
StatePublished - 2009

Keywords

  • AMS
  • Calorie restriction
  • De novo
  • NAD
  • NADH
  • NPT1
  • QPT1
  • Salvage
  • Yeast

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Genetics
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Medicine(all)

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