Assimilation of Endogenous Nicotinamide Riboside Is Essential for Calorie Restriction-mediated Life Span Extension in Saccharomyces cerevisiae
2009; Elsevier BV; Volume: 284; Issue: 25 Linguagem: Inglês
10.1074/jbc.m109.004010
ISSN1083-351X
AutoresShu-Ping Lu, Michiko Kato, Su-Ju Lin,
Tópico(s)Tryptophan and brain disorders
ResumoNAD+ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD+ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD+ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD+-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD+ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD+ precursors. Together, our studies provide a molecular basis for how NAD+ homeostasis factors confer metabolic flexibility. NAD+ (nicotinamide adenine dinucleotide) is an essential cofactor involved in various biological processes including calorie restriction-mediated life span extension. Administration of nicotinamide riboside (NmR) has been shown to ameliorate deficiencies related to aberrant NAD+ metabolism in both yeast and mammalian cells. However, the biological role of endogenous NmR remains unclear. Here we demonstrate that salvaging endogenous NmR is an integral part of NAD+ metabolism. A balanced NmR salvage cycle is essential for calorie restriction-induced life span extension and stress resistance in yeast. Our results also suggest that partitioning of the pyridine nucleotide flux between the classical salvage cycle and the NmR salvage branch might be modulated by the NAD+-dependent Sir2 deacetylase. Furthermore, two novel deamidation steps leading to nicotinic acid mononucleotide and nicotinic acid riboside production are also uncovered that further underscore the complexity and flexibility of NAD+ metabolism. In addition, utilization of extracellular nicotinamide mononucleotide requires prior conversion to NmR mediated by a periplasmic phosphatase Pho5. Conversion to NmR may thus represent a strategy for the transport and assimilation of large nonpermeable NAD+ precursors. Together, our studies provide a molecular basis for how NAD+ homeostasis factors confer metabolic flexibility. The pyridine nucleotide NAD+ and its reduced form NADH are primary redox carriers involved in metabolism. In addition to serving as a coenzyme in redox reactions, NAD+ also acts as a cosubstrate in protein modification reactions including deacetylation and ADP-ribosylation (1Lin S.J. Guarente L. Curr Opin Cell Biol. 2003; 15: 241-246Crossref PubMed Scopus (407) Google Scholar, 2Sauve A.A. Wolberger C. Schramm V.L. Boeke J.D. Annu. Rev. Biochem. 2006; 75: 435-465Crossref PubMed Scopus (606) Google Scholar). NAD+ also plays an important role in calorie restriction (CR) 2The abbreviations used are: CRcalorie restrictionLC-MSliquid chromatography-mass spectrometryNmRnicotinamide ribosideNaMNnicotinic acid mononucleotideNaRnicotinic acid ribosideNMNnicotinamide mononucleotideNamnicotinamideNAnicotinic acidWTwild typeCLSchronological life spanRLSreplicative life span. -mediated life span extension via regulating NAD+-dependent longevity factors (3Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1507) Google Scholar, 4Easlon E. Tsang F. Skinner C. Wang C. Lin S.J. Genes Dev. 2008; 22: 931-944Crossref PubMed Scopus (116) Google Scholar). CR is the most effective regimen known to extend life span in various species (5Weindruch W. Walford R.L. The Retardation of Aging and Diseases by Dietary Restriction. Charles C. Thomas, Springfield, IL1998Google Scholar, 6Roth G.S. Ingram D.K. Lane M.A. Ann. N.Y. Acad. Sci. 2001; 928: 305-315Crossref PubMed Scopus (197) Google Scholar). CR also ameliorates many age-related diseases such as cancer and diabetes (5Weindruch W. Walford R.L. The Retardation of Aging and Diseases by Dietary Restriction. Charles C. Thomas, Springfield, IL1998Google Scholar). The Sir2 family proteins are NAD+-dependent protein deacetylases, which have been shown to play important roles in several CR models in yeast (3Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1507) Google Scholar, 7Easlon E. Tsang F. Dilova I. Wang C. Lu S.P. Skinner C. Lin S.J. J. Biol. Chem. 2007; 282: 6161-6171Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and higher eukaryotes (8Dilova I. Easlon E. Lin S.J. Cell Mol. Life Sci. 2007; 64: 752-767Crossref PubMed Scopus (64) Google Scholar, 9Guarente L. Cell. 2008; 132: 171-176Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). By coupling the cleavage of NAD+ and deacetylation of target proteins, the Sir2 family proteins serve as a molecular link relaying the cellular energy state to the machinery of life span regulation. Mammalian Sir2 family proteins (SIRT1–7) have also been implicated in stress response, cell survival, and insulin and fat metabolism (8Dilova I. Easlon E. Lin S.J. Cell Mol. Life Sci. 2007; 64: 752-767Crossref PubMed Scopus (64) Google Scholar, 9Guarente L. Cell. 2008; 132: 171-176Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 10Guarente L. Nature. 2006; 444: 868-874Crossref PubMed Scopus (388) Google Scholar), supporting a role for SIRT proteins in age-related metabolic diseases and perhaps human aging. calorie restriction liquid chromatography-mass spectrometry nicotinamide riboside nicotinic acid mononucleotide nicotinic acid riboside nicotinamide mononucleotide nicotinamide nicotinic acid wild type chronological life span replicative life span. In eukaryotes, NAD+ is generated by de novo synthesis and by salvaging various intermediary precursors (see Fig. 1A). In yeast, the de novo pathway is mediated by Bna1–5 and Qpt1 (Bna6), which produces nicotinic acid mononucleotide (NaMN) from tryptophan (11Panozzo C. Nawara M. Suski C. Kucharczyka R. Skoneczny M. Bécam A.M. Rytka J. Herbert C.J. FEBS Lett. 2002; 517: 97-102Crossref PubMed Scopus (122) Google Scholar). Because the de novo pathway requires molecular oxygen as a substrate, cells grown under anaerobic growth conditions would rely on exogenous NAD+ precursors for the nicotinamide (Nam) moiety (11Panozzo C. Nawara M. Suski C. Kucharczyka R. Skoneczny M. Bécam A.M. Rytka J. Herbert C.J. FEBS Lett. 2002; 517: 97-102Crossref PubMed Scopus (122) Google Scholar). Yeast cells also salvage Nam from NAD+ consuming reactions or nicotinic acid (NA) from environment via Tna1, Pnc1, and Npt1, leading to NaMN production. NaMN is then converted to NAD+ via Nma1/2 and Qns1 (see Fig. 1A). Nma1/2 are adenylyltransferases with dual specificity toward NMN and NaMN (12Emanuelli M. Carnevali F. Lorenzi M. Raffaelli N. Amici A. Ruggieri S. Magni G. FEBS Lett. 1999; 455: 13-17Crossref PubMed Scopus (52) Google Scholar, 13Emanuelli M. Amici A. Carnevali F. Pierella F. Raffaelli N. Magni G. Protein Expr. Purif. 2003; 27: 357-364Crossref PubMed Scopus (32) Google Scholar), and Qns1 is a glutamine-dependent NAD+ synthetase. Recent studies also showed that supplementing nicotinamide riboside (NmR) and nicotinic acid riboside (NaR) to growth medium rescued the lethality of NAD+ auxotrophic mutants (14Bieganowski P. Brenner C. Cell. 2004; 117: 495-502Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 15Belenky P. Racette F.G. Bogan K.L. McClure J.M. Smith J.S. Brenner C. Cell. 2007; 129: 473-484Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 16Tempel W. Rabeh W.M. Bogan K.L. Belenky P. Wojcik M. Seidle H.F. Nedyalkova L. Yang T. Sauve A.A. Park H.W. Brenner C. PLoS Biol. 2007; 5: e263Crossref PubMed Scopus (117) Google Scholar). Assimilations of exogenous NmR and NaR are mainly mediated by a conserved NmR kinase (Nrk1) and three nucleosidases (Urh1, Pnp1, and Meu1). Nrk1 phosphorylates NmR and NaR to produce nicotinamide mononucleotide (NMN) and NaMN, respectively (14Bieganowski P. Brenner C. Cell. 2004; 117: 495-502Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 16Tempel W. Rabeh W.M. Bogan K.L. Belenky P. Wojcik M. Seidle H.F. Nedyalkova L. Yang T. Sauve A.A. Park H.W. Brenner C. PLoS Biol. 2007; 5: e263Crossref PubMed Scopus (117) Google Scholar). Urh1, Pnp1, and Meu1 catabolize NmR and NaR to generate Nam and NA (15Belenky P. Racette F.G. Bogan K.L. McClure J.M. Smith J.S. Brenner C. Cell. 2007; 129: 473-484Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 16Tempel W. Rabeh W.M. Bogan K.L. Belenky P. Wojcik M. Seidle H.F. Nedyalkova L. Yang T. Sauve A.A. Park H.W. Brenner C. PLoS Biol. 2007; 5: e263Crossref PubMed Scopus (117) Google Scholar). NmR supplementation has recently been shown to be a promising strategy for prevention and treatment of certain diseases (17Bogan K.L. Brenner C. Annu. Rev. Nutr. 2008; 28: 115-130Crossref PubMed Scopus (506) Google Scholar). For example, NmR protected neurons from axonal degeneration via functioning as a NAD+ precursor (18Araki T. Sasaki Y. Milbrandt J. Science. 2004; 305: 1010-1013Crossref PubMed Scopus (930) Google Scholar, 19Sasaki Y. Araki T. Milbrandt J. J. Neurosci. 2006; 26: 8484-8491Crossref PubMed Scopus (229) Google Scholar). Given that several NmR assimilating enzymes and NmR transporters have been characterized and many are conserved from fungi to mammals (14Bieganowski P. Brenner C. Cell. 2004; 117: 495-502Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar, 15Belenky P. Racette F.G. Bogan K.L. McClure J.M. Smith J.S. Brenner C. Cell. 2007; 129: 473-484Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 20Ma B. Pan S.J. Zupancic M.L. Cormack B.P. Mol. Microbiol. 2007; 66: 14-25Crossref PubMed Scopus (47) Google Scholar, 21Belenky P. Christensen K.C. Gazzaniga F. Pletnev A.A. Brenner C. J. Biol. Chem. 2009; 284: 158-164Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 22Belenky P.A. Moga T.G. Brenner C. J. Biol. Chem. 2008; 283: 8075-8079Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), NmR has been speculated to be an endogenous NAD+ precursor (17Bogan K.L. Brenner C. Annu. Rev. Nutr. 2008; 28: 115-130Crossref PubMed Scopus (506) Google Scholar, 23McClure J.M. Gallo C.M. Smith Jr., D.L. Matecic M. Hontz R.D. Buck S.W. Racette F.G. Smith J.S. Genetics. 2008; 180: 797-810Crossref PubMed Scopus (15) Google Scholar). Here, we provided direct evidence for endogenous NmR as an integral part of NAD+ metabolism in yeast. We also determined the biological significance of salvaging endogenous NmR and studied its role in CR-induced life span extension. Moreover, we demonstrated that the NmR salvage machinery was also required for utilizing exogenous NMN, which has recently been shown to increase NAD+ levels in mammalian cells (24Revollo J.R. Körner A. Mills K.F. Satoh A. Wang T. Garten A. Dasgupta B. Sasaki Y. Wolberger C. Townsend R.R. Milbrandt J. Kiess W. Imai S. Cell Metab. 2007; 6: 363-375Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). Finally, we discussed the role of Sir2 in modulating the flux of pyridine nucleotides between alternate routes. Yeast strain BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 was acquired from Open Biosystems (25Brachmann C.B. Davies A. Cost G.J. Caputo E. Li J. Hieter P. Boeke J.D. Yeast. 1998; 14: 115-132Crossref PubMed Scopus (2705) Google Scholar). Rich media YPD and synthetic media SD were made as described (26Burke D. Dawson D. Sterns T. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY2000: 171-174Google Scholar). The growth medium used for replicative life span analysis was YPD (2% bacto peptone, 1% yeast extract, 1.5% agar) supplemented with filter-sterilized glucose at a final concentration of 2 or 0.5%. Growth medium used for chronological life span analysis was either YPD or SD (supplemented with 4× auxotrophic amino acids leucine, histidine, lysine, and uracil). All gene deletions were made by replacing wild type genes with a reusable loxP-Kanr-loxP marker as described (27Güldener U. Heck S. Fielder T. Beinhauer J. Hegemann J.H. Nucleic Acids Res. 1996; 24: 2519-2524Crossref PubMed Scopus (1390) Google Scholar) and verified by PCR using oligonucleotides flanking genes of interest. Multiple deletions were carried out by popping out the Kanr marker using a galactose inducible Cre-recombinase. The Nrk1 overexpression construct was made as follows: a pair of oligonucleotides adding a NotI site to the 5′ end and a NheI site to the 3′ end of the NRK1 gene was designed to amplify the coding region of NRK1 via PCR. After PCR amplification, DNA was digested with NotI and NheI and then cloned into ppp81 (7Easlon E. Tsang F. Dilova I. Wang C. Lu S.P. Skinner C. Lin S.J. J. Biol. Chem. 2007; 282: 6161-6171Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), resulting in pADH1-NRK1, which was verified by DNA sequencing. Anaerobic growth conditions were achieved by using the BBL GasPak anaerobic chamber system. Total intracellular levels of NAD+ were determined using enzymatic cycling reaction as described (7Easlon E. Tsang F. Dilova I. Wang C. Lu S.P. Skinner C. Lin S.J. J. Biol. Chem. 2007; 282: 6161-6171Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). NmR and NaR were determined by LC-MS as described (28Chen P. Wolf W.R. Anal. Bioanal. Chem. 2007; 387: 2441-2448Crossref PubMed Scopus (56) Google Scholar) at the metabolomics core laboratory at University of California, Davis. The cell extracts were prepared from 1010 cells grown to late log phase by beads beating in ice-cold 50 mm ammonium acetate solution (29Sporty J.L. Kabir M.M. Turteltaub K.W. Ognibene T. Lin S.J. Bench G. J. Sep. Sci. 2008; 31: 3202-3211Crossref PubMed Scopus (67) Google Scholar). Culture supernatants (50 ml) were collected and lyophilized along with cell extracts at −80 °C. Lyophilized products were resuspended in 100 μl (cell lysate) or 2 ml (culture supernatant) of 13 mm ammonium acetate/acetonitrile (1:1, v/v). 10 μl was used for LC-MS analysis. Chemically synthesized NmR and NaR, which were kindly provided by Dr. A. Sauve (30Yang T. Chan N.Y. Sauve A.A. J. Med. Chem. 2007; 50: 6458-6461Crossref PubMed Scopus (95) Google Scholar), were used to establish standard curves. Detection and quantification of NmR and NaR were performed using the MS-multiple reaction mode methods (NmR, retention time 6 min; NaR, retention time 8 min). Four single colonies from each strain were analyzed in each experiment as described (31Fabrizio P. Longo V.D. Aging Cell. 2003; 2: 73-81Crossref PubMed Scopus (418) Google Scholar) with a few modifications. The cells were grown in YPD or SD supplemented with 2 or 0.5% glucose at starting A600 of 0.1. The cells were grown in 50-ml tubes on a roller drum set at maximum speed to ensure proper aeration. After 2 days, the cells were collected by centrifugation and washed three times with sterile water. The cells were then resuspended in 10 ml of sterile water to A600 of 1 and then were incubated at 30 °C. Cell viability was monitored every 1–2 days by plating a fraction of culture onto fresh growth medium to determine the colony-forming units. The cell survival rate was calculated by normalizing the colony-forming units to the cell number obtained at day 2 (maximum A600). The cells were grown in SD at a starting A600 of 0.1. After 2 days, the cells were spotted onto YPD plates in 5-fold serial dilutions (started at A600 of 1) and then were incubated at 55 or 25 °C for 45 or 60 min. After heat shock, the plates were transferred to 30 °C for another 2–3 days. All RLS analyses were carried out on YPD plates supplemented with glucose at a final concentration of 2 or 0.5% with 50 cells/strain/experiment (7Easlon E. Tsang F. Dilova I. Wang C. Lu S.P. Skinner C. Lin S.J. J. Biol. Chem. 2007; 282: 6161-6171Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) using a micromanipulator. Statistical analysis was carried out using the JMP statistics software (SAS), and the Wilcoxon rank sum test p values were calculated for each pair of life spans. 300 A600 unit cells grown overnight in YPD were harvested, and cell lysate was obtained by beads beating in breaking buffer (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, Roche protease inhibitors). Cell lysate containing 80 μg of total cellular proteins was added to 300 μl of deamidase reaction mix (10 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm MgCl2) (32Ghislain M. Talla E. François J.M. Yeast. 2002; 19: 215-224Crossref PubMed Scopus (111) Google Scholar, 33Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Sinclair D.A. Nature. 2003; 423: 181-185Crossref PubMed Scopus (619) Google Scholar) using 8 mm of NAD+, NMN, NmR, or Nam as substrates followed by incubation at 30 °C for 45 min. 100 μl of the deamidase reaction mix was then added to 1 ml of ammonia assay mix (3.4 mm α-ketoglutarate, 0.23 mm NADPH, 50 mm phosphate buffer, pH 7.4, 10 units of glutamate dehydrogenase) followed by a reaction at room temperature for 15 min (32Ghislain M. Talla E. François J.M. Yeast. 2002; 19: 215-224Crossref PubMed Scopus (111) Google Scholar, 33Anderson R.M. Bitterman K.J. Wood J.G. Medvedik O. Sinclair D.A. Nature. 2003; 423: 181-185Crossref PubMed Scopus (619) Google Scholar). The amount of ammonia was calculated by the decrease in absorbance at 340 nm using standard curve derived from the ammonia standard solutions (Sigma). Yeast cells lacking both the NPT1 and QPT1 genes or the QNS1 gene are inviable in regular growth media because a functional salvage or de novo NAD+ biosynthesis pathway is essential for growth (Fig. 1A) (3Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1507) Google Scholar, 14Bieganowski P. Brenner C. Cell. 2004; 117: 495-502Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar). Our previous studies (3Lin S.J. Defossez P.A. Guarente L. Science. 2000; 289: 2126-2128Crossref PubMed Scopus (1507) Google Scholar) led to a fortuitous discovery that the lethality of the npt1Δqpt1Δ and qns1Δ mutants could be rescued by growing these cells adjacent to wild type (WT) cells (Fig. 1B, left panel). These data suggested that NAD+ prototrophic yeast cells (feeders) constantly released certain NAD+ precursors, which rendered the growth of NAD+ auxotrophic cells (recipients) via cross-feeding (Fig. 1B, right panel). Because NmR supplementation could rescue the lethality of the qns1Δ mutant (14Bieganowski P. Brenner C. Cell. 2004; 117: 495-502Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar), it was possible that WT cells cross-fed the npt1Δqpt1Δ and the qns1Δ mutants with NmR. We first examined whether cross-feeding would be prevented by deleting NRK1 (NmR kinase) in recipient cells. Anaerobic growth conditions (−O2) were utilized to block de novo NAD+ biosynthesis (11Panozzo C. Nawara M. Suski C. Kucharczyka R. Skoneczny M. Bécam A.M. Rytka J. Herbert C.J. FEBS Lett. 2002; 517: 97-102Crossref PubMed Scopus (122) Google Scholar) in the npt1Δ mutants. As shown in Fig. 1C, npt1Δ cells grown anaerobically became auxotrophic for NAD+, and as expected, utilization of exogenous NmR required functional Nrk1 (Fig. 1C, left panel). Nrk1 was also required for the npt1Δ mutant to utilize cross-feeding molecules released by WT cells (Fig. 1D). Furthermore, deleting NmR assimilation enzymes Nrk1, Urh1, and Pnp1 in WT cells dramatically enhanced cross-feeding activity (Fig. 1E, left panel). Conversely, overexpressing NRK1 reduced the cross-feeding ability of WT cells (Fig. 1E, right panel). Interestingly, preventing NmR import by deleting NmR transporters NRT1 and FUN26 also conferred strong cross-feeding (Fig. 1E, middle panel), indicating that yeast cells constantly released NmR. Together, these data showed that cross-feeding activity of the feeder cells appeared to be inversely correlated with NmR salvage and import activities, supporting NmR as the major released NAD+ precursor that rescued the lethality of recipients. To understand the significance of the NmR pool, we first exploited our cross-feeding reporter system to determine the amount of NmR released by WT and NmR assimilation mutants. Fractions of cell-free culture supernatants of the feeders were collected to supplement the recipients. Fig. 2A showed that the magnitude of cross-feeding conferred by the supernatants of WT and nrk1Δurh1Δpnp1Δ and nrt1Δfun26Δ mutants in liquid culture-based assays was correlated with that determined by agar plate-based assays (Fig. 1E). Using a standard curve derived from defined concentrations of NmR and their corresponding abilities to support growth of the recipients (supplemental Fig. S1), the cumulative concentration of NmR released by the nrk1Δurh1Δpnp1Δ mutant was estimated to be ∼6.74 μm. The amount of NmR released by each nrk1Δurh1Δpnp1Δ mutant cell was about 250 × 10−10 nmol (∼0.3 mm). For comparison, the level of total NAD+ in a WT cell was ∼840 × 10−10 nmol (1.2 mm) (see Fig. 4B). These data highlighted the significance of the NmR pool in NAD+ metabolism.FIGURE 4Analyses of NmR release and NAD+ levels in the NA/Nam salvage mutants. A, the level of NmR released by the npt1Δ mutant is extremely low. The qpt1Δ mutant releases WT-level NmR. B, comparisons of the extent of NmR release (cross-feeding activities) and NAD+ levels in the NA/Nam salvage mutants. Upward arrows, increased NmR release; downward arrows, decreased NmR release; N, normal. The width of the arrows indicates the extent of NmR release compared with WT. One set of representative experiments conducted in triplicate is shown. The error bars denote standard deviations. The p values are calculated using Student's t test (***: p < 0.005). C, inactivation of NmR assimilating enzymes partially restores NmR release in the npt1Δ mutant (left panel). Deleting Sir2 slightly increases NmR release in WT (right panel) and further increases NmR release in the npt1Δnrk1Δurh1Δpnp1Δ mutant (left panel). The npt1Δqpt1Δ mutant is used as recipient in all cross-feeding assays.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We next directly quantitated the levels of intracellular and released NmR by LC-MS. Cell extracts (intracellular fractions) and culture supernatants (extracellular fractions) of WT and the nrk1Δurh1Δpnp1Δ mutant were prepared and analyzed as described (28Chen P. Wolf W.R. Anal. Bioanal. Chem. 2007; 387: 2441-2448Crossref PubMed Scopus (56) Google Scholar, 29Sporty J.L. Kabir M.M. Turteltaub K.W. Ognibene T. Lin S.J. Bench G. J. Sep. Sci. 2008; 31: 3202-3211Crossref PubMed Scopus (67) Google Scholar). As shown in Fig. 2C, NmR was detected in the intracellular fractions of WT and the nrk1Δurh1Δpnp1Δ mutant, which confirmed that NmR was an endogenous metabolite. As expected, the concentrations of both intracellular and released NmR were higher in the nrk1Δurh1Δpnp1Δ mutant compared with WT (Fig. 2C, left panel). In both strains, extracellular concentrations of NmR were maintained at much lower levels compared with the intracellular fractions, indicating that NmR transporters Nrt1 and Fun26 efficiently retrieved NmR back to the cell. Fig. 2C (right panel) showed that the amount of NmR released by each nrk1Δurh1Δpnp1Δ cell was ∼40-fold higher than that of WT cell. Overall, results obtained by LC-MS analysis correlated well with results acquired by cross-feeding assays (Figs. 1E and 2A). It has been reported that exogenous NaR could function as a NAD+ precursor, which also relied on Nrk1 and Urh1/Pnp1/Meu1 for assimilation (16Tempel W. Rabeh W.M. Bogan K.L. Belenky P. Wojcik M. Seidle H.F. Nedyalkova L. Yang T. Sauve A.A. Park H.W. Brenner C. PLoS Biol. 2007; 5: e263Crossref PubMed Scopus (117) Google Scholar). We therefore examined whether NaR was also present in the intracellular and the extracellular fractions. Interestingly, intracellular concentrations of NaR were higher (∼6-fold) than NmR in both WT and the nrk1Δurh1Δpnp1Δ mutant (Fig. 2D, left panel). However, extracellular NaR concentrations were extremely low in both strains. Consistent with a recent study (21Belenky P. Christensen K.C. Gazzaniga F. Pletnev A.A. Brenner C. J. Biol. Chem. 2009; 284: 158-164Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), we found that NaR was a much less efficient (∼100-fold less) NAD+ precursor supplement compared with NmR (supplemental Fig. S2). This was likely due to inefficient transport of NaR across the cell membrane (21Belenky P. Christensen K.C. Gazzaniga F. Pletnev A.A. Brenner C. J. Biol. Chem. 2009; 284: 158-164Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Collectively, our in vivo cross-feeding data and LC-MS quantitative results provide evidence that both NmR and NaR are endogenous metabolites. Because most NaR is retained intracellularly, NmR is likely the key NAD+ precursor that rescues the growth of recipients. The constant release and re-uptake cycle of NmR may represent a novel extended pool of NAD+. We next determined whether deficiencies in salvaging endogenous NmR would cause any growth defects. Because NmR was mainly produced during late log phase (Fig. 2B), NmR salvage might be central for cell survival in the stationary phase. Yeast chronological life span (CLS) is defined as the length of time cells remain viable in a nondividing state (stationary phase or post-diauxic shift) and is suggested to be a model for studying life span regulation of post-mitotic cells in metazoan (31Fabrizio P. Longo V.D. Aging Cell. 2003; 2: 73-81Crossref PubMed Scopus (418) Google Scholar). As shown in Fig. 3A, the nrk1Δurh1Δpnp1Δ mutant displayed short CLS. CR-induced CLS extension was largely abolished in the nrk1Δurh1Δpnp1Δ mutant. The nrt1Δfun26Δ mutant showed moderate decrease in CLS. However, CR-induced CLS extension was not affected in this mutant (Fig. 3A). NmR supplement has been shown to restore the NAD+ level and the life span of cells grown in media lacking NA (15Belenky P. Racette F.G. Bogan K.L. McClure J.M. Smith J.S. Brenner C. Cell. 2007; 129: 473-484Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar). However, NA supplement failed to rescue the short CLS of the nrk1Δurh1Δpnp1Δ mutant (Fig. 3B), indicating that NmR salvage played a more important role than the classical NA/Nam salvage in CLS. Yeast CLS extension has been attributed to enhanced stress resistance (31Fabrizio P. Longo V.D. Aging Cell. 2003; 2: 73-81Crossref PubMed Scopus (418) Google Scholar, 34Fabrizio P. Pozza F. Pletcher S.D. Gendron C.M. Longo V.D. Science. 2001; 292: 288-290Crossref PubMed Scopus (732) Google Scholar, 35Powers 3rd, R.W. Kaeberlein M. Caldwell S.D. Kennedy B.K. Fields S. Genes Dev. 2006; 20: 174-184Crossref PubMed Scopus (793) Google Scholar, 36Bonawitz N.D. Chatenay-Lapointe M. Pan Y. Shadel G.S. Cell Metab. 2007; 5: 265-277Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar). In line with these studies, the nrk1Δurh1Δpnp1Δ and the nrt1Δfun26Δ mutants were sensitive to heat shock stress (Fig. 3, C and D). Also consistent with CLS shown in Fig. 3A, CR-induced heat resistance was abolished in the nrk1Δurh1Δpnp1Δ mutant (Fig. 3C) but was unaffected in the nrt1Δfun26Δ mutant (Fig. 3D). We next examined whether NmR salvage was also required for the CR-induced RLS (division potential of individual cells) extension. As shown in Fig. 3E, CR-induced RLS extension was completely abolished in the nrk1Δurh1Δpnp1Δ mutant and was only partially prevented in the nrt1Δfun26Δ mutant. Collectively, our data demonstrated that the severity of growth defects observed in the nrt1Δfun26Δ and nrk1Δurh1Δpnp1Δ mutants correlated with the amount of NmR released (Fig. 2A) and that NmR assimilation was essential for CR-induced benefits. We next investigated the endogenous sources of NmR. Because the nicotinamide moiety of NmR is likely to originate from de novo synthesis or NA/Nam-mediated salvage (Fig. 1A), we compared the cross-feeding activities of the qpt1Δ and npt1Δ mutants. As shown in Fig. 4A, the npt1Δ mutant was unable to cross-feed the recipients, whereas the qpt1Δ mutant exhibited similar cross-feeding ability as the WT cells, suggesting that NA/Nam-mediated salvage was required for NmR production. However, it was also possible that in the npt1Δ mutant, NmR assimilation was activated, thereby resulting in decreased NmR levels. Deleting NmR assimilation enzymes in the npt1Δ mutant only slightly restored NmR release (Fig. 4C). Therefore, NmR production was indeed compromised in the npt1Δ mutant. Because the level of NAD+ in the npt1Δ mutant was significantly reduced compared with WT and the qpt1Δ mutant cells (Fig. 4B) (37Sandmeier J.J. Celic I. Boeke J.D. Smith J.S. Genetics. 2002; 160: 877-889Crossref PubMed Google Scholar), it appeared that the cross-feeding activities were determined by intracellular NAD+ levels. We therefore determined the contribution of each step of the NA/Nam salvage pathway to cross-feeding activities and NAD+ levels. Deleting TNA1 (the major NA transporter) (Fig. 1A) significantly decreased total cellular NAD+ levels (Fig. 4B); however,
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