Abstract
Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite participating in cellular redox chemistry and signaling, and the complex regulation of NAD+ metabolism is not yet fully understood. To investigate this, we established a NAD+-intermediate specific reporter system to identify factors required for salvage of metabolically linked nicotinamide (NAM) and nicotinic acid (NA). Mutants lacking components of the NatB complex, NAT3 and MDM20, appeared as hits in this screen. NatB is an Nα-terminal acetyltransferase responsible for acetylation of the N terminus of specific Met-retained peptides. In NatB mutants, increased NA/NAM levels were concomitant with decreased NAD+ We identified the vacuolar pool of nicotinamide riboside (NR) as the source of this increased NA/NAM. This NR pool is increased by nitrogen starvation, suggesting NAD+ and related metabolites may be trafficked to the vacuole for recycling. Supporting this, increased NA/NAM release in NatB mutants was abolished by deleting the autophagy protein ATG14 We next examined Tpm1 (tropomyosin), whose function is regulated by NatB-mediated acetylation, and Tpm1 overexpression (TPM1-oe) was shown to restore some NatB mutant defects. Interestingly, although TPM1-oe largely suppressed NA/NAM release in NatB mutants, it did not restore NAD+ levels. We showed that decreased nicotinamide mononucleotide adenylyltransferase (Nma1/Nma2) levels probably caused the NAD+ defects, and NMA1-oe was sufficient to restore NAD+ NatB-mediated N-terminal acetylation of Nma1 and Nma2 appears essential for maintaining NAD+ levels. In summary, our results support a connection between NatB-mediated protein acetylation and NAD+ homeostasis. Our findings may contribute to understanding the molecular basis and regulation of NAD+ metabolism.
Highlights
Nicotinamide adenine dinucleotide (NAD؉) is an essential metabolite participating in cellular redox chemistry and signaling, and the complex regulation of NAD؉ metabolism is not yet fully understood
Our studies showed that two pathways downstream of NatB contribute to NADϩ homeostasis (Fig. 5)
NatB mutants have low NADϩ levels (Fig. 2C), and all NADϩ precursors examined failed to restore the NADϩ levels (Fig. 4, A and B). This suggests that a NADϩ biosynthesis factor(s) required for utilization of all NADϩ precursors is defected in NatB mutants
Summary
We first determined whether cells release more NA and/or NAM when NA/NAM salvage is blocked. Recipient cells cannot grow on niacin-free SD (NA/NAM-free), but when feeder cells are placed in proximity, feeder cell–released NA or NAM supports recipient cell growth by “cross-feeding.” As a result, this assay determines relative levels of total NA and NAM released by feeder cells. Npt1⌬ cells still supported the growth of bna6⌬nrk1⌬nrt1⌬pnc1⌬ These results confirmed that mutants with altered NA/NAM homeostasis could be identified using this system. To verify that observed phenotypes are due to the featured mutations and not to secondary cryptic mutations in the deletion collection, we reconstructed all deletion mutants used in this study We confirmed both nat3⌬ and mdm20⌬ released more NA/NAM (Fig. 1D, top).
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