(Fig. 1D, upper panel).Similar to pnc1∆ mutant (Fig. 1B), both nat3∆and mdm20∆ released mostly NAM not NA (Fig.1D, lower panel). Fig. 1E (Fig. 2E, top panel) had a lesser effect (Fig. 2E, bottom (Fig. 3B). Next, we tested whether deleting ATG14 was sufficient to restore NAD + levels in the NatB mutants. As shown in Fig. 3C (left panel), deleting ATG14did not rescue the low NAD + levels in nat3∆ cells. Similarly, deleting other factors contributing to NR and NAM production ( Fig. 1A and Fig. 2E) in nat3∆ cells also failed to restore the NAD + level back to wild type ( shown to restore actin cable formation in NatB mutants (23,24,28,29). Interestingly, both TPM1-oe and TPM1-5 largely blocked NA/NAM release in the nat3∆ mutant (Fig. 3D) 1A). N-terminal acetyltransferases have specific targets that are largely determined by the sequence of the first two amino acids. NatB acetylates proteins with a MET-retained residue, followed by ASP, GLU, GLN, or ASN as the second residue (25,26,39,40). Nma1 and Nma2 have a MET-ASP N-terminus, but neither protein has been identified as a NatB target.Similar to nat3∆ mutant, nma1∆ cells showed decreased NAD + levels, which could not be rescued by supplementing NR (Fig. 4B). As for the controls, NR efficiently restored the NAD + level in the npt1∆ mutant, which has functional NR salvage (Fig. 4B). The similarities between nma1∆ and nat3∆ mutants suggested that Nma1and Nma2 activities are likely reduced in nat3∆ cells. Since N-terminal acetylation may affect the turnover of target proteins, we first determined whether reduced Nma1/Nma2 activities were due to decreased Nma1/Nma2 protein levels. As shown in Fig. 4C, Nma1 and Nma2 protein levels were indeed decreased in nat3∆ cells (Fig. 4C). In addition to Nma1 and Nma2, NAD + homeostasis factors Bna2, Bna5and Hst1 are also potential NatB targets. Bna2and Bna5 are biosynthesis enzymes in the de novo pathway (Fig. 1A) DISCUSSIONIn this study we characterized NatB complex as a novel NAD + homeostasis factor.Mutants lacking components of the NatB complex, NAT3 and MDM20, produce and release excess amount of NA and NAM. Our studies showed two pathways downstream of NatB contribute to NAD + homeostasis (Fig. 5).In one, NatB is important for proper NAD + biosynthesis by regulating Nma1/Nma2. NatB mutants have low NAD + levels (Fig. 2C), and all NAD + precursors examined failed to restore the NAD + levels ( Fig. 4A and 4B). This suggests that a NAD + biosynthesis factor(s) required for utilization of all NAD + precursors is defected in NatB mutants. Nma1 and Nma2 are such targets because they are the only factors required for all three NAD + biosynthesis pathways (de novo, NA/NAM salvage, and NR salvage) (Fig. 1A), and the N-terminal amino acid sequences are a match for NatB acetylation. In addition, nma1∆and nat3∆ mutants showed similar NAD + utilization defects (Fig. 4B). Although Nma2 is present in both mutants, Nma2 is known to play a minor role in NAD + metabolism. Supporting this model, decreased Nma1 protein level was observed in t...

Edited by John M. Denu NADH (NAD ؉) is an essential metabolite involved in various cellular biochemical processes. The regulation of NAD ؉ metabolism is incompletely understood. Here, using budding yeast (Saccharomyces cerevisiae), we established an NAD ؉ intermediate-specific genetic system to identify factors that regulate the de novo branch of NAD ؉ biosynthesis. We found that a mutant strain (mac1⌬) lacking Mac1, a copper-sensing transcription factor that activates copper transport genes during copper deprivation, exhibits increases in quinolinic acid (QA) production and NAD ؉ levels. Similar phenotypes were also observed in the hst1⌬ strain, deficient in the NAD ؉-dependent histone deacetylase Hst1, which inhibits de novo NAD ؉ synthesis by repressing BNA gene expression when NAD ؉ is abundant. Interestingly, the mac1⌬ and hst1⌬ mutants shared a similar NAD ؉ metabolism-related gene expression profile, and deleting either MAC1 or HST1 de-repressed the BNA genes. ChIP experiments with the BNA2 promoter indicated that Mac1 works with Hst1-containing repressor complexes to silence BNA expression. The connection of Mac1 and BNA expression suggested that copper stress affects de novo NAD ؉ synthesis, and we show that copper stress induces both BNA expression and QA production. Moreover, nicotinic acid inhibited de novo NAD ؉ synthesis through Hst1-mediated BNA repression, hindered the reuptake of extracellular QA, and thereby reduced de novo NAD ؉ synthesis. In summary, we have identified and characterized novel NAD ؉ homeostasis factors. These findings will expand our understanding of the molecular basis and regulation of NAD ؉ metabolism. NAD ϩ and its reduced form NADH are primary redox carriers in cellular metabolism. NAD ϩ is also a cosubstrate in protein modifications, such as protein deacetylation mediated by the sirtuins (Sir2 family proteins) and ADP-ribosylation mediated by the poly(ADP-ribose) polymerases. These protein modifications contribute to the maintenance and regulation of chromatin structure, DNA repair, circadian rhythm, metabolic responses, and life span (1-4). NAD ϩ is also an NADP ϩ precursor, which, like NAD ϩ , is carefully balanced with its reduced form NADPH to maintain a favorable redox state. Aberrant NAD ϩ metabolism is associated with a number of diseases, including diabetes, cancer, and neuron degeneration (2, 3, 5-11). Administration of NAD ϩ precursors, such as nicotinamide mononucleotide (NMN), 2 nicotinamide (NAM), nicotinic acid riboside, and nicotinamide riboside (NR), has been shown to ameliorate deficiencies related to aberrant NAD ϩ metabolism in yeast, mouse, and human cells (3, 5-10, 12-15). However, the molecular mechanisms underlying the beneficial effects of NAD ϩ precursor supplementation are not yet completely understood. The NAD ϩ pool is maintained by multiple NAD ϩ biosynthesis pathways, which are conserved from bacteria to humans. Depending on the cell types, growth conditions, and availability of specific NAD ϩ precursors, one pathway may dominate the others. ...

NAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are incompletely understood. Yeast (Saccharomyces cerevisiae) cells lacking the N-terminal (Nt) protein acetyltransferase complex NatB exhibit an approximate 50% reduction in NAD+ levels and aberrant metabolism of NAD+ precursors, changes that are associated with a decrease in nicotinamide mononucleotide adenylyltransferase (Nmnat) protein levels. Here, we show that this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins and not of other NatB substrates. Nt-acetylation critically regulates protein degradation by the N-end rule pathways, suggesting that the absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t½) of non-Nt-acetylated Nmnats did not significantly differ from those of Nt-acetylated Nmnats. Accordingly, deletion or depletion of the N-end rule pathway ubiquitin E3 ligases in NatB mutants did not restore NAD+ levels. Next, we examined whether the status of Nt-acetylation would affect the translation of Nmnats, finding that the absence of Nt-acetylation does not significantly alter the polysome formation rate on Nmnat mRNAs. However, we observed that NatB mutants have significantly reduced Nmnat protein maturation. Our findings indicate that the reduced Nmnat levels in NatB mutants are mainly due to inefficient protein maturation. Nmnat activities are essential for all NAD+ biosynthesis routes, and understanding the regulation of Nmnat protein homeostasis may improve our understanding of the molecular basis and regulation of NAD+ metabolism.

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ABSTRACTNAD+ is an essential metabolite participating in cellular biochemical processes and signaling. The regulation and interconnection among multiple NAD+ biosynthesis pathways are not completely understood. We previously identified the N-terminal (Nt) protein acetyltransferase complex NatB as a NAD+ homeostasis factor. Cells lacking NatB show an approximate 50% reduction in the NAD+ level and aberrant metabolism of NAD+ precursors, which are associated with a decrease of nicotinamide mononucleotide adenylyltransferases (Nmnat) protein levels. Here we show this decrease in NAD+ and Nmnat protein levels is specifically due to the absence of Nt-acetylation of Nmnat (Nma1 and Nma2) proteins, and not other NatB substrates. Nt-acetylation is a critical regulator of protein degradation by the N-end rule pathways, indicating absence of Nt-acetylation may alter Nmnat protein stability. Interestingly, the rate of protein turnover (t1/2) of non-Nt-acetylated Nmnats does not significantly differ from Nt-acetylated Nmnats, suggesting reduced Nmnat levels in NmatB mutants are not due to increased post-translational degradation of non-Nt-acetylated Nmnats. In line with these observations, deletion or depletion of N-rule pathway ubiquitin E3 ligases in NatB mutants is not sufficient to restore NAD+ levels. Moreover, the status of Nt-acetylation does not alter the rate of translation initiation of Nmnats. Collectively our studies suggest absence of Nt-acetylation may increase co-translational degradation of nascent Nmnat polypeptides, which results in reduced Nmnat levels in NatB mutants. Nmnat activities are essential for all routes of NAD+ biosynthesis. Understanding the regulation of Nmnat protein homeostasis will facilitate our understanding of the molecular basis and regulation of NAD+ metabolism.