NAD
+ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD
+ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD
+ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD
+-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD
+ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD
+ precursors. Together, our studies provide a molecular basis for how NAD
+ homeostasis factors confer metabolic flexibility.The pyridine nucleotide NAD
+ and its reduced form NADH are primary redox carriers involved in metabolism. In addition to serving as a coenzyme in redox reactions, NAD
+ also acts as a cosubstrate in protein modification reactions including deacetylation and ADP-ribosylation (
1,
2). NAD
+ also plays an important role in calorie restriction (CR)
2-mediated life span extension via regulating NAD
+-dependent longevity factors (
3,
4). CR is the most effective regimen known to extend life span in various species (
5,
6). CR also ameliorates many age-related diseases such as cancer and diabetes (
5). The Sir2 family proteins are NAD
+-dependent protein deacetylases, which have been shown to play important roles in several CR models in yeast (
3,
7) and higher eukaryotes (
8,
9). By coupling the cleavage of NAD
+ and deacetylation of target proteins, the Sir2 family proteins serve as a molecular link relaying the cellular energy state to the machinery of life span regulation. Mammalian Sir2 family proteins (SIRT1–7) have also been implicated in stress response, cell survival, and insulin and fat metabolism (
8–
10), supporting a role for SIRT proteins in age-related metabolic diseases and perhaps human aging.In eukaryotes, NAD
+ is generated by
de novo synthesis and by salvaging various intermediary precursors (see
A). In yeast, the
de novo pathway is mediated by Bna1–5 and Qpt1 (Bna6), which produces nicotinic acid mononucleotide (NaMN) from tryptophan (
11). Because the
de novo pathway requires molecular oxygen as a substrate, cells grown under anaerobic growth conditions would rely on exogenous NAD
+ precursors for the nicotinamide (Nam) moiety (
11). Yeast cells also salvage Nam from NAD
+ consuming reactions or nicotinic acid (NA) from environment via Tna1, Pnc1, and Npt1, leading to NaMN production. NaMN is then converted to NAD
+ via Nma1/2 and Qns1 (see
A). Nma1/2 are adenylyltransferases with dual specificity toward NMN and NaMN (
12,
13), and Qns1 is a glutamine-dependent NAD
+ synthetase. Recent studies also showed that supplementing nicotinamide riboside (NmR) and nicotinic acid riboside (NaR) to growth medium rescued the lethality of NAD
+ auxotrophic mutants (
14–
16). Assimilations of exogenous NmR and NaR are mainly mediated by a conserved NmR kinase (Nrk1) and three nucleosidases (Urh1, Pnp1, and Meu1). Nrk1 phosphorylates NmR and NaR to produce nicotinamide mononucleotide (NMN) and NaMN, respectively (
14,
16). Urh1, Pnp1, and Meu1 catabolize NmR and NaR to generate Nam and NA (
15,
16).
Open in a separate windowNicotinamide riboside (NmR) is an endogenous metabolite in yeast.
A, the current model of the NAD
+ biosynthesis pathways. Extracellular NmR enters the salvage cycle through Nrk1, Urh1, Pnp1, and Meu1.
B, NAD
+ prototrophic cells release metabolites into growth medium to cross-feed NAD
+ auxotrophic cells (the
npt1Δ
qpt1Δ and
qns1Δ mutants). Micro-colonies of the NAD
+ auxotrophic mutants become visible after 2-day incubation at 30 °C, which show “gradient” growth patterns descending from the side adjacent to WT.
C, Nrk1 is required for NAD
+ auxotrophic cells to utilize NmR. Anaerobic growth conditions (−O
2) are utilized to block
de novo NAD
+ biosynthesis in the
npt1Δ and
npt1Δ
nrk1Δ mutants.
D, Nrk1 is required to utilize cross-feeding metabolites.
E, cross-feeding activity is modulated by factors in NmR metabolism. Cells defective in NmR utilization (
left panel) or transport (
middle panel) show increased cross-feeding in spot assays. Overexpressing Nrk1 decreases cross-feeding activity (
right panel). The results show growth of the
npt1Δ
qpt1Δ recipient (plated on YPD at a density of ∼9000 cells/cm
2) supported by feeder cells (∼2 × 10
4 cells spotted directly onto the recipient lawn).
oe, overexpression.NmR supplementation has recently been shown to be a promising strategy for prevention and treatment of certain diseases (
17). For example, NmR protected neurons from axonal degeneration via functioning as a NAD
+ precursor (
18,
19). Given that several NmR assimilating enzymes and NmR transporters have been characterized and many are conserved from fungi to mammals (
14,
15,
20–
22), NmR has been speculated to be an endogenous NAD
+ precursor (
17,
23). Here, we provided direct evidence for endogenous NmR as an integral part of NAD
+ metabolism in yeast. We also determined the biological significance of salvaging endogenous NmR and studied its role in CR-induced life span extension. Moreover, we demonstrated that the NmR salvage machinery was also required for utilizing exogenous NMN, which has recently been shown to increase NAD
+ levels in mammalian cells (
24). Finally, we discussed the role of Sir2 in modulating the flux of pyridine nucleotides between alternate routes.
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