A functional link between NAD+ homeostasis and N-terminal protein acetylation in Saccharomyces cerevisiae

Abstract

(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...

N-terminal protein acetylation by NatB modulates the levels of Nmnats, the NAD+biosynthetic enzymes inSaccharomyces cerevisiae

“…Interestingly, yeast NatB substrates are predominantly, near 100%, found in the acetylated state [28,29]. In line with these observations, we showed that Nma1 and Nma2 peptides identified from WT cells were 100% acetylated whereas Nma1 or Nma2 peptides identified from NatB mutants were 95%-100% non-acetylated [21]. Therefore, Nt-acetylation appears to be critical for maintaining proper Nmnat protein levels and thus NAD + biosynthesis.…”

“…and Nma2 may be critical for the co-regulation of de novo, NA/NAM salvage and NR salvage pathways. In fact, we have previously shown that overexpression of NMA1 caused a significant increase in the total NAD + content of the cell [21]. It was also shown in a recent study that Nma1 is the only NAD + biosynthesis enzyme that upon overexpression can increase the NAD + content in the cell, and modulates NAD + to correlate with the concentrations of ATP [12].…”

“…We previously reported altered NAD + homeostasis in NatB deletion mutants, nat3∆ and mdm20∆ [21]. These NatB deletion mutants have low levels of NAD + with an increased flux of NAD + precursors through the vacuolar NR salvage branch, which promotes the formation and release of NAM and NA.…”

“…Moreover, yeast cells also release and re-uptake small NAD + precursors such as NA, NAM, QA and NR (Fig. 1A) [11,19,21]. Specific transporters Tna1 (for NA and QA) [22,23] and Nrt1 (for NR) [24] are responsible for the uptake of NAD + precursors whereas the mechanisms of precursor release remain unclear.…”

“…(regulatory subunit), as a potential regulator of NAD + biosynthesis, possibly through Nterminal (Nt-) acetylation of Nma1 and Nma2 [21]. The levels of Nma1 and Nma2 proteins were reduced in NatB deletion mutants compared to wild type (WT).…”