Regulation of NAD⁺ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae

Abstract

Pyridine nucleotides are essential coenzymes in many cellular redox reactions in all living systems. In addition to functioning as a redox carrier, NAD⁺ is also a required co-substrate for the conserved sirtuin deacetylases. Sirtuins regulate transcription, genome maintenance and metabolism and function as molecular links between cells and their environment. Maintaining NAD⁺ homeostasis is essential for proper cellular function and aberrant NAD⁺ metabolism has been implicated in a number of metabolic- and age-associated diseases. Recently, NAD⁺ metabolism has been linked to the phosphate-responsive signaling pathway (PHO pathway) in the budding yeast Saccharomyces cerevisiae. Activation of the PHO pathway is associated with the production and mobilization of the NAD⁺ metabolite nicotinamide riboside (NR), which is mediated in part by PHO-regulated nucleotidases. Cross-regulation between NAD⁺ metabolism and the PHO pathway has also been reported; however, detailed mechanisms remain to be elucidated. The PHO pathway also appears to modulate the activities of common downstream effectors of multiple nutrient-sensing pathways (Ras-PKA, TOR, Sch9/AKT). These signaling pathways were suggested to play a role in calorie restriction-mediated beneficial effects, which have also been linked to Sir2 function and NAD⁺ metabolism. Here, we discuss the interactions of these pathways and their potential roles in regulating NAD⁺ metabolism. In eukaryotic cells, intracellular compartmentalization facilitates the regulation of enzymatic functions and also concentrates or sequesters specific metabolites. Various NAD⁺-mediated cellular functions such as mitochondrial oxidative phosphorylation are compartmentalized. Therefore, we also discuss several key players functioning in mitochondrial, cytosolic and vacuolar compartmentalization of NAD⁺ intermediates, and their potential roles in NAD⁺ homeostasis. To date, it remains unclear how NAD⁺ and NAD⁺ intermediates shuttle between different cellular compartments. Together, these studies provide a molecular basis for how NAD⁺ homeostasis factors and the interacting signaling pathways confer metabolic flexibility and contribute to maintaining cell fitness and genome stability.

1. Introduction

Pyridine nucleotides NAD⁺(H) and NADP⁺(H) are essential coenzymes participating in many cellular redox reactions in all living systems. In addition, NAD⁺ and its derivatives also function as substrates and signaling molecules in key cellular processes such as regulation of Ca²⁺ signaling, chromatin structure, DNA repair and life span (1–5). Aberrant NAD⁺ metabolism has also been linked to cancer, metabolic disorders and neurodegenerative diseases such as Parkinson’s and Alzheimer’s disease (6–10).

Although redox reactions do not alter the total cellular NAD⁺ levels, some cellular processes such as Sir2-mediated protein deacetylation, consume NAD⁺; therefore constant replenishment of NAD⁺ is essential for maintaining cellular fitness. Cells have developed complex interconnecting biosynthetic and signaling pathways to regulate intracellular NAD⁺ levels. However, factors regulating NAD⁺ metabolism and homeostasis remain unclear due to the dynamic and complex nature of the NAD⁺ synthesis pathways. In this review, we summarize recent studies on the link between NAD⁺ metabolism and other signaling pathways in the budding yeast Saccharomyces cerevisiae, and discuss our perspectives on these findings in the mechanisms underlying the regulation of NAD⁺ homeostasis.

2. Overview of NAD⁺ biosynthesis

Cellular NAD⁺ is synthesized from a number of precursor molecules. Eukaryotes utilize the amino acid tryptophan for de novo biosynthesis of NAD⁺. NAD⁺ metabolites such as nicotinamide (Nam), nicotinic acid (NA) and nicotinamide riboside (NR) can also be salvaged and re-assimilated into NAD⁺. In S. cerevisiae, NAD⁺ is synthesized from two key intermediates nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN) via the de novo and NA/Nam/NR salvaging pathways (Fig. 1). NaMN is produced by transferring the phosphoribose moiety of phosphoribosyl pyrophosphate (PRPP) to nicotinic acid (NA) or to tryptophan-derived quinolinic acid (QA), which is catalyzed by phosphoribosyltransferases Npt1 (NA phosphoribosyltransferase) and Bna6 (biosynthesis of nicotinic acid 6), respectively (11–13). QA is mainly derived from the amino acid tryptophan via five enzymatic reactions and a spontaneous cyclization (14,15). The five enzymatic reactions are mediated by Bna2 (tryptophan 2, 3-dioxygenase), Bna7 (arylformamidase), Bna1 (3-hydroxyanthranilic acid dioxygenase), Bna4 (kynurenine 3-mono oxygenase) and Bna5 (kynureninase). NA can be generated from Nam mediated by the Nam deamidase Pnc1 (pyrazineamidase and nicotinamidase) as part of the salvaging reactions. Both NA and QA can also be acquired from the environment by the NA transporter Tna1 (transporter of nicotinic acid) (16,17). De novo synthesis and NA/Nam salvaging pathways converge at NaMN, which is converted to deamido-NAD⁺ (NaAD) by transferring the AMP moiety of ATP by Nma1 and Nma2 (NMNATs). Amidation of NaAD by Qns1 (glutamine (Q)-dependent NAD⁺ synthetase) completes NAD⁺ synthesis (18). NMN is produced from NR by Nrk1 (nicotinamide riboside kinase) -catalyzed phosphorylation (19,20) and is subsequently adenylylated to NAD⁺ by Nma1 and Nma2 (nicotinamide mononucleotide adenylyltransferase) (21–25). Nrk1 can also convert nicotinic acid riboside (NaR), a deamidated form of NR, to NaMN (20). Unlike higher eukaryotes including mammals, yeasts do not possess the gene coding for Nampt (Nam phosphorybosyltransferase), the enzyme that converts Nam to NMN. Instead, Nam is deamidated to NA by Pnc1 (26).

Figure 1.

A simplified model of the Saccharomyces cerevisiae NAD⁺ synthesis pathway. In yeast, NAD⁺ is synthesized de novo from tryptophan (Trp) and by salvaging nicotinic acid (NA), nicotinamide (Nam), quinolinic acid (QA) and nicotinamide riboside (NR). Cells can also salvage nicotinic acid riboside (NaR) by converting it to NA or NaMN (nicotinic acid mononucleotide). For simplicity, NaR salvaging is not shown in this figure. NaAD, deamido NAD⁺. NMN, nicotinamide mononucleotide. NAD⁺ and NAD⁺ intermediates are italicized. Abbreviations of protein names catalyzing each step are shown in bold.

Yeast cells appear to produce NAD⁺ predominantly via the NA/Nam salvage pathway during exponential growth (27) because standard growth media contain high concentrations (400 μg/l or ~ 3 μM) of niacin (NA and Nam) and budding yeast has a very efficient NA transporter Tna1 (Km for NA = ~ 1.7 μM) (17). Interestingly, although NR is an endogenous metabolite (28), standard yeast growth media do not contain NR because they do not support the growth of NAD⁺ synthesis mutants defective in both de novo and NA/Nam salvaging (for example, the qns1Δ mutant). It is possible that NR is degraded during the production of components of yeast media (for example, yeast extract) (29). Unlike NA and Nam, NR is more susceptible to heat and perhaps other stresses (Kato et al., unpublished results). Nevertheless, NR has been shown to be an efficient NAD⁺ precursor that contributes to the NAD⁺ pool and supports NAD⁺-dependent reactions (19,20). NR can be acquired from the environment by the high-affinity NR transporter Nrt1 (nicotinamide riboside transporter) (Km for NR = ~ 22 μM) (30) or by salvaging intracellular intermediates. Intriguingly, yeast cells constantly release NR to the growth medium and retrieve it back to cells (28) allowing NR to traffic between intracellular and extracellular compartments. It is possible that maintaining a flexible NR/NAD⁺ pool may facilitate prompt adjustments of cells in response to metabolic stresses (31). Intracellular level of NR in average wild type yeast cell is ~ 2–10 μM (28,32). Level of released NR in the growth medium varies depending on the growth conditions and genetic background. For example, in a standard late log phase cell culture in YPD (10 mL, total cell number ~3×10⁹), NR in the culture is estimated to be ~0.5 μM. Salvaging endogenously produced NR has been shown to be essential for maintaining NAD⁺ homeostasis and cellular fitness (28,33). An NR assimilation mutant (nrk1Δpnp1Δurh1Δ) is estimated to lose up to 0.3 mM of NR (28) and its NAD⁺ level is decreased by ~50% (20).

Recent studies have identified additional NR salvaging factors connecting NR metabolism to the NA/Nam salvaging pathway (Fig. 1). The nucleosidases Pnp1 (purine nucleoside phosphorylase), Urh1 (uridine hydrolase), and Meu1 (methylthioadenosine phosphorylase, originally identified as multicopy enhancer of UAS2) have been shown to convert NR to Nam, or NaR to NA (20). The nucleotidases Isn1 (IMP-specific 5′-nucleotidase) and Sdt1 (suppressor of disruption of TFIIS) (33) and phosphatases Pho5 and Pho8 (28,34) have also been shown to contribute to NR metabolism by converting NMN to NR. Additional routes may exist to assist the interconversion of NAD⁺ intermediates. For example, although purified recombinant Pnc1 appeared to be specific for the conversion of Nam to NA (33), a cell lysate-based study indicated that Pnc1 might also deamidate NMN and NR (28). In addition, we recently observed unexpected opposite expression patterns of functionally linked PNC1 (Nam deamidase, converts Nam to NA) and NPT1 (converts NA to NaMN) (Fig. 1) in late log phase. A significant up-regulation of PNC1 gene expression was accompanied by down-regulation of NPT1 (Kato et al., unpublished results), which contradicts the expectation that the expression levels of NPT1 and PNC1 are positively correlated based on the known function of these two genes. These observations suggest that Pnc1 might have additional substrates or functions, and further studies are required to explore these possibilities.

3. Crosstalk between NAD⁺ metabolism and nutrient/stress sensing pathways

3.1 Sir2, calorie restriction, and NAD⁺ metabolism

The Sir2 (silent information regulator 2) family proteins are highly conserved from bacteria to human (35,36). These proteins, collectively called sirtuins, harbor a conserved core domain required for its enzymatic activity. Many sirtuins have been biochemically characterized as NAD⁺-dependent protein deacetylases or ADP-ribosylases (37). Sir2 was first identified as a component of the Sir1/2/3/4 silencing complexes in yeast (38,39). The Sir silencing complexes are required for the maintenance and repression of three repetitive genomic regions: telomeres, the cryptic mating type loci (HML and HMR), and the ribosomal DNA loci (40–43). Each locus employs a unique set of DNA binding factors to recruit specific Sir proteins to mediate transcriptional silencing by generating a compact chromatin configuration. Among these Sir proteins, only Sir2 is required for maintaining the silencing of all three repetitive regions. The NAD⁺-dependent histone deacetylase activity of Sir2 provides a molecular mechanistic basis for Sir complex-mediated transcriptional silencing (44–46).

Sir2 homologs in yeast include Hst1, Hst2, Hst3 and Hst4 (homologous to Sir two) (35). Among those, Hst1 has the closest homology to Sir2 and has been shown to suppress silencing defects at the mating type loci of a sir2Δ mutant when overexpressed (35). However, it is suggested that Hst1 mediates transcriptional regulation independent of the SIR silencing complex by interacting with another transcription regulator Sum1 (47,48). Hst2 is the most abundantly expressed Sir2 homolog in yeast and accounts for most of the intracellular NAD⁺-dependent deacetylase activity (45). Hst2 appears to shuttle between the nucleus and the cytoplasm to regulate rDNA and telomeric silencing (45,49–52). Hst3 and Hst4 have also been linked to the regulation of genome stability (35,53).

Overexpression (one-extra copy) of Sir2 has been shown to extend yeast replicative life span (54). In metazoa, tissue-specific overexpression of Sir2 homologs has been shown to extend life span and ameliorate functional defects associated with aging (55–60). It appears that the expression of metazoan Sir2 must be temporally regulated and controlled at a moderate level in specific tissues to exert beneficial effects (55–57,59,61). The role of Sir2 in yeast life span regulation is also complex. Yeast life span has been defined and studied in two distinct ways: replicative lifespan (RLS) measures the division potential of individual cells (62), whereas chronological lifespan (CLS) measures the rate of post-mitotic survival of a non-dividing cell population (63,64). Various longevity factors (which affect either RLS, CLS or both) have been identified in S. cerevisiae, and some of these factors are associated with Sir2 function. These factors include mitochondrial function (65–70), stress response/hormesis/mitohormesis (64,66,68,71–74), the NAD⁺-dependent deacetylase Sir2 family (75,76), partitioning of damaged proteins (77,78), genome stability (79–81), homeostasis of NAD⁺ and other metabolic factors (1,6,31,82,83), vacuolar function (84,85), ribosome biogenesis (86), cell hypertrophy (87–89), proteostasis (90–92) and metabolites-induced toxicity (93–97). Although whether and how these factors contribute to Sir2 mediated life span extension remain debatable, it is clear that Sir2 promotes RLS under normal conditions. The role of Sir2 in CLS is less clear and in certain genetic backgrounds, deleting SIR2 appeared to extend CLS (98), highlighting the complex role Sir2 in life span regulation.

Sir2 has also been described as a key factor that mediates the beneficial effects of calorie restriction (CR); however, its role in CR is also highly debatable. In yeast, CR is often defined as the moderate reduction of glucose concentration in the growth medium from 2% to 0.5% (1,51,99–103). This moderate CR regimen extends both RLS and CLS. Interestingly, although moderate CR largely requires the Sir2 family proteins for RLS extension (51), Sir2 appears to be dispensable for RLS extension mediated by more severe CR condition (0.05% glucose, 10X further reduction of glucose compared with moderate CR, 0.5%)(104). In fact, severe CR extends RLS even more significantly without the Sir2 family (100). This phenomenon is reminiscent of the Sir2 effect on CLS: under nutrient deprivation, deleting SIR2 further extends CLS in certain genetic backgrounds (98). It is likely that cells respond to these two CR regimens differently. For example, severe CR may additionally trigger gluconeogenesis, since gluconeogenesis is activated upon glucose starvation. It has been suggested that gluconeogenesis is induced in the sir2Δ mutant and therefore contributes to CLS extension (98). Supporting this, a key gluconeogenesis enzyme Pck1 was found to be a substrate of Sir2. In sir2Δ mutant, Pck1 remains active and gluconeogenesis is promoted (105). On the other hand, age-enhanced gluconeogenesis has been shown to shorten CLS and Hst3 and Hst4 down-regulate age-enhanced gluconeogenesis (106). In addition, human Sirt1 has been reported to deacetylate PGC1-α, which leads to induction of gluconeogenesis (107). Together, these studies demonstrate the complex roles of gluconeogenesis and Sir2 in CR, which are not surprising for metabolic longevity factors. In addition to the Sir2 family, CR is suggested to function through reducing the activities of conserved nutrient-sensing pathways to extend life span. Such pathways include the Ras-cAMP/PKA (cyclic-AMP activated protein kinase A) pathway, the nutrient-responsive Sch9/AKT (homolog of mammalian S6K kinases) and TOR (target of rapamycin) kinase pathways (Fig. 2) (108,109). CR also induces the shunting of carbon metabolism from fermentation towards the mitochondrial TCA cycle, and a concomitant increase of respiration (110). The Sir2 family proteins have been shown to interact with or modulate the activity of these CR-associated longevity factors. We await future studies to further detail the mechanisms underlying the complexity of Sir2 functions in CR.

Figure 2.

Interactions of NAD⁺ metabolism and multiple cellular signaling pathways contribute to the regulation of cell function. NAD⁺ and NAD⁺ intermediates are italicized. Abbreviations of protein names catalyzing specific steps are shown in bold. Dashed lines indicate that additional evidence is required to reveal the molecular mechanisms.

NAD⁺ and its derivatives are important Sir2 regulators. NAD⁺ levels affect Sir2 activity and consequently the downstream events. Mutations that cause deficiency in NAD⁺ production affect Sir2-mediated processes. Deleting the NPT1 gene significantly reduces NAD⁺ levels and abolishes Sir2-mediated silencing (45) and CR-mediated life span extension (1). Nam is a by-product generated during Sir2-mediated deacetylation, which is also a potent non-competitive inhibitor of Sir2 (111–113). The nicotinamidase Pnc1 is responsible for the clearance of Nam by converting it to nicotinic acid (NA) (26). Increasing Nam concentration in the growth medium or blocking Nam clearance by deleting the PNC1 reduced Sir2-mediated silencing and shortened life span (Fig. 2) (111,114), suggesting that preventing Nam accumulation is important for Sir2 function. NADH was reported to be a competitive inhibitor of Sir2, and lowering the NADH level by genetic modifications or CR increased Sir2-mediated silencing and life span (115). However, NADH is a weak inhibitor of Sir2, and reported in vivo NADH levels are too low to inhibit Sir2 activity (113). Therefore, it is possible that intracellular compartmentalization of NAD⁺ and NADH and/or specific protein-protein interactions create locally high NAD⁺/NADH ratios, thereby activating Sir2 in vivo. It has been suggested that the affinity/sensitivity of Sir2 towards its substrates and inhibitors varied when Sir2 was in complex with different interacting partners (116). Interestingly, high concentrations of NMN could inhibit Sir2 activity in vitro (IC₅₀ = ~ 3 mM) (113). However, this inhibition is unlikely to be physiologically relevant considering low intracellular concentration of NMN (~ 60 μM) (32).

In addition to being regulated by NAD⁺ and its derivatives, both Sir2 and Hst1 have also been shown to regulate NAD⁺ metabolism. It has been reported that de novo NAD⁺ synthesis is regulated by Hst1-mediated repression and that deletion of HST1 raises the steady-state level of NAD⁺ (117). Hst1 has also been shown to repress the expression of NR transporter Nrt1 (118). Our recent studies suggested that Sir2 modulates the flux of NR salvage (28). The Sir2 family has been proposed to support the NA/Nam salvage pathway via producing Nam in deacetylation reactions (Fig. 1 and Fig. 2) (82). However, the precise role of Sir2 family proteins in NAD⁺ metabolism remained unclear. Increased NR release observed in the sir2Δ mutant suggested a specific role of Sir2 in NR salvage (28). It is possible that Sir2 helps to preserve the NAD⁺ pool by decreasing the pyridine nucleotide flux into the NR branch. Indeed, deleting SIR2 in cells lacking NR assimilation activity further decreased the size of the NAD⁺ pool (28). We await future studies to determine the molecular and biochemical roles of the Sir2 family in NR and NAD⁺ metabolism.

3.2 The PHO pathway and NAD⁺ metabolism

Our recent studies have uncovered a link between the regulation of NAD⁺ metabolism and phosphate signaling (34). In S. cerevisiae, phosphate homeostasis is regulated by the phosphate-responsive signaling pathway (PHO pathway), which monitors and responds to changes in the phosphate availability (119,120). The PHO pathway consists of regulatory components and downstream target genes whose expression is regulated by transcription factors Pho4 and Pho2. Activation of PHO pathway is influenced by intracellular phosphate (Pi) levels and phosphorylation state of Pho4 determines the gene expression of the downstream targets. In high Pi conditions, the cyclin-cyclin-dependent kinase (cyclin-CDK) complex Pho80-Pho85 phosphorylates Pho4, which is then exported out of the nucleus. Upon Pi starvation, the CDK inhibitor Pho81 binds the Pho80-Pho85 complex and inhibits Pho4 phosphorylation, allowing Pho4 to translocate to the nucleus and activate the expression of its target genes (119,120). Pho4-induced genes include those encoding repressible acid and alkaline phosphatases Pho5 and Pho8, high affinity phosphate transporters Pho84 and Pho89, and vacuolar proteins that mediate the utilization of stored polyphosphates (121).

We previously reported that production of NR correlates with activation of PHO signaling (34). Intracellular NR level is increased under conditions that activate the PHO pathway (such as in low Pi growth media and deletion of PHO84), and is decreased in cells defective in PHO signaling (such as the pho4Δ mutant). Increased NR production under high PHO activity conditions is largely due to the activation of the vacuolar phosphatase Pho8, since deletion of PHO8 gene significantly decreases intracellular NR level (34). Our studies also demonstrated that utilization of extracellular NMN requires prior conversion to NR mediated by the PHO-regulated periplasmic phosphatase Pho5 (31). The cytosolic nucleotidase Sdt1, which plays an important role in NR production (by hydrolyzing NMN) (33), has also been suggested to be under PHO regulation (122). Together, these studies show evidence for PHO regulation of several components of NAD⁺ metabolism in yeast. Some of these studies may be extrapolated to understanding NR/NAD⁺ metabolism in mammalian cells. For example, “conversion to NR” (and other smaller NAD⁺ intermediates) may represent a strategy for the transport and assimilation of large impermeable NAD⁺ precursors. Supplementing NAD⁺ precursors and intermediates has been shown to replenish NAD⁺ level and ameliorate functional defects associated with abnormal NAD⁺ metabolism in mammalian models (59,123–125). Because the structures of many NAD⁺ precursors are unfavorable for direct diffusion across the cell membrane, either specific transporters or extracellular catabolizing enzymes would be required to utilize exogenous NAD⁺ precursors. In a study using a system where Nam utilization is blocked (by Nampt inhibitors), it was suggested that extracellular NAD⁺ and NMN need to be converted to NR for cells to utilize them. Interfering with the enzymatic activities that mediate the degradation of extracellular NAD⁺ to NMN, or NMN to NR reduced the ability of cells to synthesize NAD⁺. Although the transport mechanism of NR has not been characterized in mammalian cells, pharmacological inhibition of plasma membrane nucleoside transporters reduced intracellular NAD⁺ production (126). Supporting this model, a recent study showed that CD73, an extracellular (periplasmic) nucleotidase orthologous to H. influenza NadN, could convert NAD⁺ and NMN to NR (127). In addition, it has been suggested that NR is circulated in the peripheral bloodstream in mammals (128). To date, the molecular aspects of phosphate signaling in mammalian cells remain unclear. Interesting future studies would be to determine whether NAD⁺ metabolism is also linked to phosphate signaling in higher eukaryotes.

In addition to regulating NAD⁺ metabolism, the PHO pathway was suggested to respond to intracellular NaMN levels (34). Npt1 is the major enzyme that mediates the production of NaMN from NA. Increased activity of a PHO-regulated acid phosphatase Pho5 was observed specifically in npt1Δ mutant, suggesting that depletion of NaMN leads to PHO activation (34). Mechanisms underlying the cross-regulation between PHO signaling and NAD⁺ metabolism remain unclear. The coupling of these two pathways may render a more efficient metabolic support under specific conditions. For example, the phosphate moiety of NMN is a putative target for phosphate scavenging during Pi limitation. Moreover, since NaMN is an important intermediate for NAD⁺ biosynthesis, low level of NaMN might reflect impaired NAD⁺ biosynthesis and lead to activation of an alternative NAD⁺ salvage route. Since NR-mediated NAD⁺ synthesis requires phosphate (in the form of ATP), a coordinated activation of the PHO pathway is expected in supporting NAD⁺ synthesis and homeostasis.

3.3 Other PHO-interacting nutrient/stress sensing pathways and their potential roles in NAD⁺ metabolism

Research in recent years has also connected PHO signaling to other nutrient sensing pathways (Fig. 2). For example, it has been shown that, after prolonged Pi starvation, the repletion of Pi to cells leads to degradation of the high-affinity phosphate transporter Pho84 via the activation of protein kinase A (PKA) (129–133), demonstrating a connection between the glucose and phosphate signaling pathways. These studies also support a role for Pho84 as a Pi transceptor, which can sense as well as transport Pi. Other components in the PHO pathway have also been associated with glucose metabolism (134,135). In addition, PHO signaling and Sch9/Akt and TOR pathways appear to converge on Rim15 to regulate stress response transcription factors such as Msn2 and Msn4 (103,136,137). However, these pathways also affect different targets and often synergistically or oppositely regulate specific targets (138). Complex crosstalk between these pathways may allow cells to quickly adapt to environmental changes.

How may these nutrient sensing pathways regulate NAD⁺ metabolism? The stress-responsive transcriptional factors Msn2 and Msn4 have been shown to regulate PNC1 expression, and are required for CR-mediated life span extension (139). Increased expression of PNC1 was also observed under various mild stresses such as glucose or amino acid restriction, and heat or salt stresses (140). In addition, correlation between mRNA mistranslation and PNC1 up-regulation has been shown (141). Given that mild stresses can mediate life span extension (99,140,142) and that CR induces mild stress signals (73), it is possible that Pnc1 is a key factor that connects stress signals (including CR) to the regulation of Sir2 (143) as well as NAD⁺ metabolism (Fig. 2). Interestingly, Nampt (nicotinamide phosphoribosyltransferase), the functional homolog of Pnc1 in higher eukaryotes, is also responsive to stresses and suggested to be the mediator of CR-induced benefits (144,145)

Considering Nam is a potent Sir2 inhibitor, mechanisms to control Nam concentration would be key to maintain Sir2 activity. In yeast, Nam clearance is also achieved by methylation catalyzed by the Nam methyltransferase Nnt1, a homolog of human NNMT (N-nicotinamide methyltransferase) (115,140). NNT1 overexpression was shown to rescue a PNC1 null mutation and restore CR-mediated life span extension in this genetic background (115). A recent study in C. elegans suggests that methylated Nam can induce a transient hormetic response, which leads to life span extension (146). The worm amine N-methyltransferase ANMT-1 contributes to the production of N-methylnicotinamide. Methylated Nam is a substrate of aldehyde oxidase, an H₂O₂-generating enzyme thereby inducing transient increase of ROS (reactive oxygen species) signal. Moreover, specific NAD⁺ synthesis components have also been shown to play a role in other cellular processes. For example, NMNAT family proteins were reported to affect cellular functions independent of their enzymatic function. Salmonella NadR is a multi-functional NMNAT with a role in transcriptional regulation (147). Drosophila NMNAT was shown to possess chaperone activity that contributes to axonal protection (7). In S. cerevisiae, Nma1 and Nma2 have been shown to alleviate proteotoxicity in yeast models of proteinopathies (148). Overall, these studies have demonstrated that specific NAD⁺ intermediates as well as NAD⁺ metabolism components also play a role in other cellular processes, further highlighting the importance of NAD⁺ homeostasis factors.

4. Intracellular compartmentalization of the NAD⁺ intermediate pools

4.1 Mitochondrial compartmentalization of NAD⁺ (H)

In yeast, NAD⁺ synthesis seems to occur mainly in the cytosol. It remains unclear how NAD⁺ and NAD⁺ derivatives are transported in and out of various intracellular compartments. Transport of NAD⁺ into the mitochondria can be mediated by the NAD⁺ carrier proteins encoded by NDT1 and NDT2 (149). Our understanding of mitochondrial NAD⁺(H) homeostasis mostly derives from studying the NAD⁺/NADH shuttle systems. Since the mitochondrial inner membrane is impermeable to NAD⁺ and NADH, the NADH shuttle systems function to move small permeable redox equivalents of NAD⁺ and NADH across the mitochondrial membrane to balance the NAD⁺/NADH ratio between the mitochondrial and the cytosolic/nuclear pools (150,151). For example, respiration-induced increase in NAD⁺/NADH ratio in the mitochondria can be transmitted to the cytosol by the malate-aspartate shuttle (Fig. 3). In yeast, CR induces a metabolic shift from fermentation to mitochondrial respiration (110) and a concomitant increase in the NAD⁺/NADH ratio (115). Because respiration produces NAD⁺ from NADH and NADH can function as a competitive inhibitor of Sir2 activity (115), it has been suggested that increased NAD⁺/NADH ratio may activate Sir2 during CR. Supporting this model, genetic manipulations that decrease NADH levels are shown to increase Sir2 activity and extend life span (110,115). In addition, deleting NADH shuttle components abolishes CR-mediated life span extension whereas overexpressing NADH shuttle components extends life span (101). This model has remained controversial because reported in vivo NADH levels are probably too low to inhibit Sir2 activity (113). It is possible that intracellular compartmentalization of NAD⁺ and NADH creates locally high NAD⁺/NADH ratios thereby activating Sir2 in vivo. It is also possible that when Sir2 forms specific protein-protein interactions with different partners, the affinity/sensitivity of Sir2 toward its substrates and inhibitors may vary (116). Another model (not mutually exclusive) is that the level of Nam, a much more potent Sir2 inhibitor, is decreased by CR (111) leading to Sir2 activation. CR has been shown to increase the expression of PNC1 (a Nam deamidase that converts Nam to NA) (140), which is expected to decrease Nam level, leading to Sir2 activation. However, since Nam is also a key NAD⁺ precursor in the NA/Nam salvage pathway, it is unclear whether CR-induced Sir2 activation is due to increased NAD⁺ availability, reduced Nam or both.

Figure 3.

Compartmentalization of intracellular NAD⁺ and its derivates regulates NAD⁺ homeostasis. Intracellular compartments contribute to the storage and supply of NAD⁺ intermediates such as NR and NMN. Vacuole is a plausible site for NR storage supported by the discovery of a putative NR transporter Fun26 and a NR producing nucleotidase Pho8. Periplasmic nucelotidase Pho5 and plasma membrane localized NR transporter Nrt1 facilitate the utilization of extracellular NMN and NR pools, respectively. The NADH shuttle systems do not directly affect NAD⁺ metabolism, instead, they function to balance redox equivalents between the mitochondrial and the cytosolic/nuclear pools to regulate the NAD⁺/NADH ratio. The malate-aspartate shuttle is shown here as an example. Abbreviations of protein names catalyzing specific steps are shown in bold. Dashed lines indicate that additional evidence is required to reveal the molecular mechanisms.

In mammals, CR has also been reported to affect NAD⁺/NADH ratio in a tissue-specific manner, and these changes correlate with the levels of SIRT1 expression/activity (152,153). Molecular mechanisms underling CR-induced changes in the NAD⁺/NADH ratios and the subsequent events remain to be elucidated. In yeast, CR increases NAD⁺/NADH ratio by decreasing the NADH level (115), whereas in mouse, CR triggers different responses in different tissues (152,153). In muscle and white adipose tissues, the increase in NAD⁺/NADH ratios appeared to be due to increased NAD⁺ levels (152). In liver, CR actually decreases NAD⁺/NADH ratio by increasing the NADH level (152). In pancreas, fasting decreases NAD⁺/NADH ratio by decreasing NAD⁺ level (153). Despite the complexity of the regulation of NAD⁺/NADH ratios and Sir2 activities as well as the controversies whether Sir2 is activated by CR, there is no doubt that sufficient NAD⁺ level (i.e. functional NAD⁺ biosynthesis) is required for Sir2 activity and certain CR-induced beneficial effects. Functional NADH shuttles may confer metabolic flexibility to support multiple CR-induced molecular changes. In mammals, impairments of mitochondrial metabolism and NADH shuttles have also been implicated in age-associated diseases such as diabetes (154,155). Overall, these studies suggest that the NADH shuttles play important roles in mitochondrial metabolism and metabolic fitness by regulating NAD⁺ homeostasis. Identification of additional mitochondrial NAD⁺ homeostasis factors including the pyridine nucleotide transporters will provide insights into the understanding of the roles of mitochondria in NAD⁺ homeostasis.

4.2 Vacuolar and cytosolic compartmentalization of NMN and NR

Our recent studies suggest that NAD⁺ metabolism is connected to the PHO pathway. In particular, PHO-activated Pho8 and Pho5 contribute directly to NR production by releasing phosphate from NMN (28). Since Pho8 is a vacuolar membrane protein whose activation requires a vacuolar peptidase (156), we speculate that the vacuole is a cellular compartment for NR production and storage (Fig. 3). Supporting this possibility, our previous genetic screens searching for mutants that show altered NR release have identified factors involved in vacuolar ATP synthase (v-ATPase) assembly (34). These v-ATPase-related factors are known to function in establishing and maintaining the acidity of the vacuolar matrix. We found that 12 mutants harboring deficiencies in the regulation or the structure of v-ATPase complexes released more NR (2- to 3-fold higher than the wild type). Furthermore, 19 mutants with deficiencies in vesicle-mediated transport also manifested increased NR release (2- to 3-fold higher than the wild type). Collectively, these results reinforced the idea that the vacuole and vesicle-mediated transport might play a role in NR metabolism and homeostasis.

If the vacuole is a cellular compartment for NR production and storage, how do cells salvage vacuolar NR to synthesize NAD⁺ in the cytosol? Fun26 may function to balance NR between the cytosolic and the vacuolar pools, enabling assimilation of NR originating from the vacuole into cytosolic NAD⁺ synthesis (Fig. 3) (34,157,158). Fun26 is the only yeast homolog of the human equilibrative nucleoside transporter (hENT) protein family, which mediates bi-directional transport of specific nucleosides across the plasma membrane and intracellular membranes (159–161). In yeast, the Fun26 protein was reported to have a minor role in NR transport relative to Nrt1 (30). However, the fun26Δ mutant displayed a level of NR release increase (3- to 4-fold higher than the wild type) comparable to that seen for the nrt1Δ mutant (34). In addition, a significant increase in intracellular NR level was also observed in the fun26Δ mutant (4- to 5-fold higher than the wild type) but not in the nrt1Δ mutant (similar to the wild type) (34). Therefore Fun26 may play a significant role in balancing the NR pools produced and stored in the vacuole and the NR pools used for NAD⁺ synthesis in the cytosol.

These studies also suggest that for each individual cell, the level of released NR reflects the size of its dynamic cytosolic NR pool, and that the steady state NR level (determined in total cell extract) reflects the size of the stored pool (Fig. 3) (34). Supporting this idea, deletions of genes encoding cytosolic nucleotidases Isn1 and Sdt1 (33) significantly decreased NR release (~40 % decrease) (33,162) but only slightly affected intracellular NR level (~15 % decrease) (34,162). Conversely, deleting PHO8 largely decreased intracellular NR level (50–70 % decrease) but only slightly affected the level of NR release (20–30 % decrease) (34,162). To date, the mechanisms of NR uptake and its transport between cellular compartments in higher eukaryotes are still unknown. Four Fun26-related human ENT (equilibrative nucleoside transporter) proteins have been characterized (159). Given the role of Fun26 in NR metabolism in yeast, it is possible that the plasma membrane-localized hENT1 and hENT2 and lysosome membrane-resided hENT3 also participate in NR homeostasis and NAD⁺ metabolism in human. One additional potential NAD⁺ homeostasis compartment is the peroxisome. Peroxisomal Npy1 has been shown to function as a NAD⁺(H) pyrophosphatase (or Nudix hydrolase) which could produce NMN(H) from NAD⁺(H) (163) and contribute to NAD⁺ homeostasis. Future studies on the identification of novel NAD⁺ homeostasis factors will further our understanding of how these compartments and associated factors regulate NAD⁺ metabolism and cell functions.

5. Conclusion and Perspectives

Intracellular concentrations of many of the NAD⁺ intermediates are maintained at low levels (32), which is characteristic of signaling molecules. NR and NMN, similar to other NAD⁺ intermediates, may function as signaling molecules to regulate NAD⁺ homeostasis or other cellular processes. We have previously discovered that low NaMN level is associated with the activation of the PHO pathway (34). Moreover, nicotinic acid adenine dinucleotide phosphate (NAADP) has been shown to function as a signaling molecule to regulate calcium homeostasis in variety of organisms (164,165). In addition to functioning as signaling molecules, high concentrations of intracellular NAD⁺ intermediates may be unfavorable for certain cellular processes. For example, NAD⁺-dependent DNA ligase in bacteria is inhibited by NMN and it is suggested that NMN deamidase contributes to maintaining small intracellular NMN pool (166). As discussed earlier, Nam is known as an inhibitor of Sir2 and clearance of Nam is critical for maintaining Sir2 activity and life span (114). Nam clearance is facilitated by a Nam deamidase Pnc1 in yeast or Nampt, a Nam phosphoribosyltransferase in mammals (111,167). Also, methylation of Nam offers beneficial effects by clearing Nam and providing stress resistance (146). Studies in yeast suggest that cells may constantly convert NMN to NR, which is more mobile and can be readily excreted, stored or re-assimilated (28,34). Ohashi et al. showed that quinolinic acid (QA) is also produced and excreted like NR (16). Thus, it is possible that pyridine nucleotides and their metabolites are involved in a variety of cellular processes and balancing their concentrations would be critical for the regulation of these processes. Future studies on the identification and characterization of additional signaling factors that regulate and/or crosstalk with NAD⁺ homeostasis are highly anticipated. Overall, these studies have contributed to the understanding of the complex NAD⁺ homeostasis pathways, and may also provide insights into the underlying mechanisms of diseases related to defects in NAD⁺ metabolism.

Highlights.

• Pyridine nucleotides and their metabolites play important roles in variety of cellular processes

• Crosstalk between sirtuins, NAD⁺- and nutrient-sensing pathways renders metabolic flexibility

• Compartmentalization helps regulate the homeostasis of pyridine nucleotides