The regulation of NAD + metabolism and its interaction with other cellular pathways are not yet completely understood.Here we study why cells lacking the Pof1 NMNAT have high levels of nicotinamide riboside (NR), produced from NAD + turnover and in response to nutritional stress.To understand the mechanism, we identify novel NR-producing factors using genetic screens and further characterize Phm8 as an NMN nucleotidase.In addition, loss of Pof1 moderately delays the turnover of Sdt1, another nucleotidase contributing to NR production.Interestingly, the ATPase domain of Pof1 appears to play a minor role in NR production compared with the NAD + synthesis catalytic domain.We further study another NMNAT, Nma1, that also contains the conserved NAD + synthesis domain.Nma1 overexpression only partially reduces NR production in pof1 cells, implying the NAD + synthesis domain of Pof1 may affect ATPase activity or NMN binding.Moreover, an inverse correlation between the expression of NMNATs and nucleotidases under nutritional stress also contributes to pof1-associated NR production.Supporting this, NR production is enhanced upon moderate glucose depletion, while Pof1 protein is reduced.Overall, our data suggest that in pof1 cells, NMN is likely more accessible to the nucleotidases, resulting in NR production.Additionally, the similarity between NR production and nucleotide degradation indicates that NR production is likely important under replication stress.We show that NR levels positively correlate with resistance to replication stress.Our findings help understand the critical role of Pof1 in modulating NR production and the putative role of NR in overall nucleotide metabolism.
Lee et al. (Wed,) studied this question.