Phosphate-responsive signaling pathway is a novel component of NAD+ metabolism in Saccharomyces cerevisiae

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.     

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not β-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn plays roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis.     

NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD⁺ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm^−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.     

The histone deacetylases Rpd3 and Hst1 antagonistically regulate de novo NAD+ metabolism in the budding yeast Saccharomyces cerevisiae

NAD⁺ is a cellular redox cofactor involved in many essential processes. The regulation of NAD⁺ metabolism and the signaling networks reciprocally interacting with NAD⁺-producing metabolic pathways are not yet fully understood. The NAD⁺-dependent histone deacetylase (HDAC) Hst1 has been shown to inhibit de novo NAD⁺ synthesis by repressing biosynthesis of nicotinic acid (BNA) gene expression. Here, we alternatively identify HDAC Rpd3 as a positive regulator of de novo NAD⁺ metabolism in the budding yeast Saccharomyces cerevisiae. We reveal that deletion of RPD3 causes marked decreases in the production of de novo pathway metabolites, in direct contrast to deletion of HST1. We determined the BNA expression profiles of rpd3Δ and hst1Δ cells to be similarly opposed, suggesting the two HDACs may regulate the BNA genes in an antagonistic fashion. Our chromatin immunoprecipitation analysis revealed that Rpd3 and Hst1 mutually influence each other’s binding distribution at the BNA2 promoter. We demonstrate Hst1 to be the main deacetylase active at the BNA2 promoter, with hst1Δ cells displaying increased acetylation of the N-terminal tail lysine residues of histone H4, H4K5, and H4K12. Conversely, we show that deletion of RPD3 reduces the acetylation of these residues in an Hst1-dependent manner. This suggests that Rpd3 may function to oppose spreading of Hst1-dependent heterochromatin and represents a unique form of antagonism between HDACs in regulating gene expression. Moreover, we found that Rpd3 and Hst1 also coregulate additional targets involved in other branches of NAD⁺ metabolism. These findings help elucidate the complex interconnections involved in effecting the regulation of NAD⁺ metabolism.     

Mitochondrial iron metabolism in the yeast Saccharomyces cerevisiae

Iron is fundamental to many biological processes, but is also detrimental as it fosters the synthesis of destructive oxygen radicals. Recent experiments have increased our knowledge of the critical process of regulation of mitochondrial iron metabolism. A number of genes directly involved in iron homeostasis in this organelle have been identified. Intriguingly, a minor Hsp70 molecular chaperone of the mitochondrial matrix has been implicated as a player in this process as well.     

Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides, and cofactors across the inner mitochondrial membrane. In Saccharomyces cerevisiae, NAD+ is synthesized outside the mitochondria and must be imported across the permeability barrier of the inner mitochondrial membrane. However, no protein responsible for this transport activity has ever been isolated or identified. In this report, the identification and functional characterization of the mitochondrial NAD+ carrier protein (Ndt1p) is described. The NDT1 gene was overexpressed in bacteria. The purified protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized. It transported NAD+ and, to a lesser extent, (d)AMP and (d)GMP but virtually not alpha-NAD+, NADH, NADP+, or NADPH. Transport was saturable with an apparent Km of 0.38 mM for NAD+. The Ndt1p-GFP was found to be targeted to mitochondria. Consistently with Ndt1p localization and its function as a NAD+ transporter, cells lacking NDT1 had reduced levels of NAD+ and NADH in their mitochondria and reduced activity of mitochondrial NAD+-requiring enzymes. Similar results were also found in the mitochondria of cells lacking NDT2 that encodes a protein (Ndt2p) displaying 70% homology with Ndt1p. The delta ndt1 delta ndt2 double mutant exhibited lower mitochondrial NAD+ and NADH levels than the single deletants and a more pronounced delay in growth on nonfermentable carbon sources. The main role of Ndt1p and Ndt2p is to import NAD+ into mitochondria by unidirectional transport or by exchange with intramitochondrially generated (d)AMP and (d)GMP.     

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction.     

Regulation of proliferation by the budding yeast Saccharomyces cerevisiae

Mutations in the budding yeast Saccharomyces cerevisiae define regulatory activities both for the mitotic cell cycle and for resumption of proliferation from the quiescent stationary-phase state. In each case, the regulation of proliferation occurs in the prereplicative interval that precedes the initiation of DNA replication. This regulation is particularly responsive to the nutrient environment and the biosynthetic capacity of the cell. Mutations in components of the cAMP-mediated effector pathway and in components of the biosynthetic machinery itself affect regulation of proliferation within the mitotic cell cycle. In the extreme case of nutrient starvation, cells cease proliferation and enter stationary phase. Mutations in newly defined genes prevent stationary-phase cells from reentering the mitotic cell cycle, but have no effect on proliferating cells. Thus stationary phase represents a unique developmental state, with requirements to resume proliferation that differ from those for the maintenance of proliferation in the mitotic cell cycle.     

Regulation of cell size in the yeast Saccharomyces cerevisiae.

For cells of the yeast Saccharomyces cerevisiae, the size at initiation of budding is proportional to growth rate for rates from 0.33 to 0.23 h-1. At growth rates lower than 0.23 h-1, cells displayed a minimum cell size at bud initiation independent of growth rate. Regardless of growth rate, cells displayed an increase in volume each time budding was initiated. When abnormally small cells, produced by starvation for nitrogen, were placed in fresh medium containing nitrogen but with different carbon sources, they did not initiate budding until they had grown to the critical size characteristic of that medium. Moreover, when cells were shifted from a medium supporting a low growth rate and small size at bud initiation to a medium supporting a higher growth rate and larger size at bud initiation, there was a transient accumulation of cells within G1. These results suggest that yeast cells are able to initiate cell division at different cell sizes and that regulation of cell size occurs within G1.     

Regulation of sugar and ethanol metabolism in Saccharomyces cerevisiae

This review briefly surveys the literature on the nature, regulation, genetics, and molecular biology of the major energy-yielding pathways in yeasts, with emphasis on Saccharomyces cerevisiae. While sugar metabolism has received the lion's share of attention from workers in this field because of its bearing on the production of ethanol and other metabolites, more attention is now being paid to ethanol metabolism and the regulation of aerobic metabolism by fermentable and nonfermentable substrates. The utility of yeast as a highly manipulable organism and the discovery that yeast metabolic pathways are subject to the same types of control as those of higher cells open up many opportunities in such diverse areas as molecular evolution and cancer research.