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 journalArticle

21 Citations (Scopus)

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

Fingerprint

Salvaging
Biosynthesis
NAD
Yeast
Saccharomyces cerevisiae
Glucose
Acids
Niacin
Yeasts
Decomposition

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)

Cite this

Sporty, J., Lin, S. J., Kato, M., Ognibene, T., Stewart, B., Turteltaub, K. W., & Bench, G. (2009). Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae. Yeast, 26(7), 363-369. https://doi.org/10.1002/yea.1671

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae. / Sporty, Jennifer; Lin, Su Ju; Kato, Michiko; Ognibene, Ted; Stewart, Benjamin; Turteltaub, Ken W; Bench, Graham.

In: Yeast, Vol. 26, No. 7, 2009, p. 363-369.

Research output: Contribution to journalArticle

Sporty, J, Lin, SJ, Kato, M, Ognibene, T, Stewart, B, Turteltaub, KW & Bench, G 2009, 'Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae', Yeast, vol. 26, no. 7, pp. 363-369. https://doi.org/10.1002/yea.1671
Sporty, Jennifer ; Lin, Su Ju ; Kato, Michiko ; Ognibene, Ted ; Stewart, Benjamin ; Turteltaub, Ken W ; Bench, Graham. / Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae. In: Yeast. 2009 ; Vol. 26, No. 7. pp. 363-369.
@article{5fd6615d907c43fd94dfde665f237f40,
title = "Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae",
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.",
keywords = "AMS, Calorie restriction, De novo, NAD, NADH, NPT1, QPT1, Salvage, Yeast",
author = "Jennifer Sporty and Lin, {Su Ju} and Michiko Kato and Ted Ognibene and Benjamin Stewart and Turteltaub, {Ken W} and Graham Bench",
year = "2009",
doi = "10.1002/yea.1671",
language = "English (US)",
volume = "26",
pages = "363--369",
journal = "Yeast",
issn = "0749-503X",
publisher = "John Wiley and Sons Ltd",
number = "7",

}

TY - JOUR

T1 - Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

AU - Sporty, Jennifer

AU - Lin, Su Ju

AU - Kato, Michiko

AU - Ognibene, Ted

AU - Stewart, Benjamin

AU - Turteltaub, Ken W

AU - Bench, Graham

PY - 2009

Y1 - 2009

N2 - 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.

AB - 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.

KW - AMS

KW - Calorie restriction

KW - De novo

KW - NAD

KW - NADH

KW - NPT1

KW - QPT1

KW - Salvage

KW - Yeast

UR - http://www.scopus.com/inward/record.url?scp=68349089282&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=68349089282&partnerID=8YFLogxK

U2 - 10.1002/yea.1671

DO - 10.1002/yea.1671

M3 - Article

C2 - 19399913

AN - SCOPUS:68349089282

VL - 26

SP - 363

EP - 369

JO - Yeast

JF - Yeast

SN - 0749-503X

IS - 7

ER -