Abstract
Nicotinamide adenine dinucleotide (NAD(+)) is an essential cofactor involved in various cellular biochemical reactions. To date the signaling pathways that regulate NAD(+) metabolism remain unclear due to the dynamic nature and complexity of the NAD(+) metabolic pathways and the difficulty of determining the levels of the interconvertible pyridine nucleotides. Nicotinamide riboside (NmR) is a key pyridine metabolite that is excreted and re-assimilated by yeast and plays important roles in the maintenance of NAD(+) pool. In this study we establish a NmR-specific reporter system and use it to identify yeast mutants with altered NmR/NAD(+) metabolism. We show that the phosphate-responsive signaling (PHO) pathway contributes to control NAD(+) metabolism. Yeast strains with activated PHO pathway show increases in both the release rate and internal concentration of NmR. We further identify Pho8, a PHO-regulated vacuolar phosphatase, as a potential NmR production factor. We also demonstrate that Fun26, a homolog of human ENT (equilibrative nucleoside transporter), localizes to the vacuolar membrane and establishes the size of the vacuolar and cytosolic NmR pools. In addition, the PHO pathway responds to depletion of cellular nicotinic acid mononucleotide (NaMN) and mediates nicotinamide mononucleotide (NMN) catabolism, thereby contributing to both NmR salvage and phosphate acquisition. Therefore, NaMN is a putative molecular link connecting the PHO signaling and NAD(+) metabolic pathways. Our findings may contribute to the understanding of the molecular basis and regulation of NAD(+) metabolism in higher eukaryotes.
Highlights
Inorganic phosphate (Pi) is essential for biomolecule synthesis, energy metabolism, and protein modification
We found that the level of Nicotinamide riboside (NmR) was significantly reduced in the pho8⌬ mutant (Fig. 3A), suggesting Pho8 might play an important role in NmR production
Because our studies showed that phosphate-responsive signaling (PHO) signaling and NADϩ metabolism are closely connected, it was possible that the changes of NADϩ and/or specific NADϩ intermediates in the qns1⌬ mutant triggered PHO activation
Summary
Inorganic phosphate (Pi) is essential for biomolecule synthesis, energy metabolism, and protein modification. To understand whether the PHO pathway activity correlates with NmR production, we determined intracellular NmR levels of the pho84⌬ (increased PHO activity) and pho4⌬ (decreased PHO activity) mutants by a liquid-based cross-feeding bioassay (“Experimental Procedures”). Active) mutant was not further increased by Pi limitation, suggesting the alterations in NmR levels induced by low Pi were signaled through the PHO pathway (Fig. 2C).
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