Nrt1 and Tna1-Independent Export of NAD+ Precursor Vitamins Promotes NAD+ Homeostasis and Allows Engineering of Vitamin Production

NAD+ is both a co-enzyme for hydride transfer enzymes and a substrate of sirtuins and other NAD+ consuming enzymes. NAD+ biosynthesis is required for two different regimens that extend lifespan in yeast. NAD+ is synthesized from tryptophan and the three vitamin precursors of NAD+: nicotinic acid, nicotinamide and nicotinamide riboside. Supplementation of yeast cells with NAD+ precursors increases intracellular NAD+ levels and extends replicative lifespan. Here we show that both nicotinamide riboside and nicotinic acid are not only vitamins but are also exported metabolites. We found that the deletion of the nicotinamide riboside transporter, Nrt1, leads to increased export of nicotinamide riboside. This discovery was exploited to engineer a strain to produce high levels of extracellular nicotinamide riboside, which was recovered in purified form. We further demonstrate that extracellular nicotinamide is readily converted to extracellular nicotinic acid in a manner that requires intracellular nicotinamidase activity. Like nicotinamide riboside, export of nicotinic acid is elevated by the deletion of the nicotinic acid transporter, Tna1. The data indicate that NAD+ metabolism has a critical extracellular element in the yeast system and suggest that cells regulate intracellular NAD+ metabolism by balancing import and export of NAD+ precursor vitamins

Nicotinamide riboside promotes sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD(þ). Cell 129

Although NAD + biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD + precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD + precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD + synthesis through two pathways, the Nrk1 pathway and the Urh1/ Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD + metabolism in the absence of nicotinamideriboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD + and increases Sir2 function

Purification of NR from PAB076-Conditioned Media.

<p>A) HPLC trace of media collected from strain PAB076 grown to OD 60 in 2x YPD and supplemented with 5 mM NA. B) Preparative SP-Sephadex chromatography with fractions analyzed by HPLC. NR eluted at 20 to 50 mM NaCl in fractions 27 to 36. C) Intracellular NAD⁺ determination of strain BY4742 grown in NA-free SDC media and NA-free SDC media supplemented with 10 µM NR from fractions produced in panel B.</p

NR Export is Nrt1-Independent and Increased by NA and Nam Supplementation.

<p>A) NR exported by NR-accumulating strain PAB038 is not diminished but is rather increased by deletion of the NR transporter gene, <i>NRT1</i>, in strain PAB076. Conditioned media, collected from BY4742, PAB038 and PAB076 cells, grown in SDC media to a OD_{600 nm} of 3, were mixed 1∶1 with fresh media and evaluated for their support of <i>qns1</i> growth. The extent of <i>qns1</i> growth on these conditioned media samples was compared to <i>qns1</i> growth on fresh SDC supplemented with chemically synthesized NR (n = 3). B) NR export can be increased by supplementation of PAB076 with NA or Nam. Conditioned media, collected from BY4742 and PAB076 grown in SDC media supplemented with the indicated concentrations of NA or Nam, were evaluated for their support of <i>qns1</i> growth.</p