Less is more: Nutrient limitation induces cross-talk of nutrient sensing pathways with NAD⁺ homeostasis and contributes to longevity

Abstract

Nutrient sensing pathways and their regulation grant cells control over their metabolism and growth in response to changing nutrients. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as glucose-sensing PKA, nitrogen-sensing TOR and S6 kinase homolog Sch9 have been linked to increased life span in the yeast, Saccharomyces cerevisiae, and higher eukaryotes. Recently, reduced activity of amino acid sensing SPS pathway was also shown to increase yeast life span. Life span extension by reduced SPS activity requires enhanced NAD⁺ (nicotinamide adenine dinucleotide, oxidized form) and nicotinamide riboside (NR, a NAD⁺ precursor) homeostasis. Maintaining adequate NAD⁺ pools has been shown to play key roles in life span extension, but factors regulating NAD⁺ metabolism and homeostasis are not completely understood. Recently, NAD⁺ metabolism was also linked to the phosphate (Pi)-sensing PHO pathway in yeast. Canonical PHO activation requires Pi-starvation. Interestingly, NAD⁺ depletion without Pi-starvation was sufficient to induce PHO activation, increasing NR production and mobilization. Moreover, SPS signaling appears to function in parallel with PHO signaling components to regulate NR/NAD⁺ homeostasis. These studies suggest that NAD⁺ metabolism is likely controlled by and/or coordinated with multiple nutrient sensing pathways. Indeed, cross-regulation of PHO, PKA, TOR and Sch9 pathways was reported to potentially affect NAD⁺ metabolism; though detailed mechanisms remain unclear. This review discusses yeast longevity-related nutrient sensing pathways and possible mechanisms of life span extension, regulation of NAD⁺ homeostasis, and cross-talk among nutrient sensing pathways and NAD⁺ homeostasis.

Keywords

nutrient sensing / NAD⁺ homeostasis / yeast longevity

Introduction

Nutrient sensing pathways are integral to growth and survival of all living organisms. A multitude of nutrient sensing mechanisms allow organisms to regulate cell activity with fine control. To this end, many sensor pathways cross-regulate each other or cross-talk (Fig. 1). Much research has been done in the budding yeast, Saccharomyces cerevisiae, on sensing of nutrients such as amino acids, nitrogen, glucose and phosphate.

Reduced activity of nutrient sensing pathways such as glucose-sensing cyclic AMP-protein kinase A (PKA), nitrogen-sensing target of rapamycin (TOR) and the S6 kinase homolog Sch9 have been linked to increased life span in yeast and higher eukaryotes ( Lin et al., 2000; Fabrizio et al., 2001; Kaeberlein et al., 2005b; Wei et al., 2008; Wang et al., 2009; Longo and Fabrizio, 2012). Recent studies have proposed several molecular mechanisms for yeast life span extension (see the section “Factors contributing to life span extension”). Among these, metabolic factors such as NAD⁺ play an important role. In this review, we start by discussing longevity related nutrient sensing pathways and proposed mechanisms of life span extension, followed by NAD⁺ metabolism and signaling pathways connected to NAD⁺ metabolism.

PKA and carbon-source sensing pathways

Yeast cells can sense and uptake preferred carbon sources through many pathways. Although well studied, the exact mechanism of carbon-source activation in some pathways is still unclear. In this section, we introduce five interacting glucose-sensing pathways, which have been connected to life span: The cyclic AMP-protein kinase A (PKA) (cell growth and stress response), SNF1 (alternative carbon source utilization), HAP (mitochondrial function), Snf3-Rgt2 (glucose sensing and transport) and Sch9 (cell growth).

PKA

In the presence of external glucose, PKA is activated, increasing anabolic and decreasing catabolic activity of the cell. Two glucose-sensing pathways, the G-protein coupled receptor (GPCR) system and the monomeric small GTPase, RAS, protein system act upon PKA ( Broach 2012; Conrad et al., 2014). The major activator of PKA, RAS, is activated in the presence of glucose by the cytoplasmic GTP exchange factor (GEF), Cdc25. Cdc25 exchanges a GDP for a GTP on the GTPases, Ras1 and Ras2 (Ras1/2), which are tethered to the plasma membrane via post-translational palmitoylation at their C-termini. Ras1/2 are active in their GTP form (GTP-Ras1/2), and inactive in their GDP form (GDP-Ras1/2). Inactivation requires cytoplasmic GTPase activating proteins (GAPs), Ira1 and Ira2, which terminate RAS signaling by stimulating GTPase activity of GTP-Ras1/2, resulting in inactive GDP-Ras1/2. When activated, GTP-Ras1/2 directly activates plasma membrane associated adenylate cyclase (AC), stimulating AC catalytic activity. Activated AC produces cyclic AMP (cAMP) from ATP and the phosphodiesterases, Pde1 and Pde2, terminate cAMP signaling by converting cAMP into AMP. Once produced, cAMP can bind to Bcy1, the cytoplasmic regulatory subunit of PKA, which disassociates from and thus activates the catalytic subunits of PKA, Tpk1, Tpk2 and Tpk3 (Tpk1–3). How glucose activates the RAS protein system remains unclear. Activation appears to require glucose uptake/catabolism, hexokinase-mediated phosphorylation ( Rolland et al., 2000; Thevelein et al., 2000; Conrad et al., 2014) and possibly cellular pH changes ( Broach 2012). Once AC is stimulated by the RAS pathway, the GPCR system can also activate AC ( Conrad et al., 2014).

The GPCR system consists of the plasma membrane G-protein coupled receptor, Gpr1, which in the presence of glucose shifts conformation and activates Gpa2, a G protein alpha subunit. Gpa2 associates with the plasma membrane via post-translational palmitoylation and myristoylation ( Dohlman and Thorner, 2001; Overton et al., 2005). Gpa2 activates both cAMP-dependent and cAMP-independent pathways of PKA activation. In the cAMP-dependent pathway, Gpa2 activates AC, but only after AC has been activated by GTP-Ras1/2 via the RAS protein system. Activated AC then activates PKA as described above. It is peculiar that Gpa2 only activates AC after AC has been stimulated by RAS proteins, since this indicates that extracellular glucose sensing by the GPCR system requires intracellular glucose catabolism to activate its pathway. However, the GPCR system does have a cAMP-independent pathway via Krh1 and Krh2 (G β mimics), which directly activate PKA by lowering affinity between regulatory subunit, Bcy1, and catalytic subunits, Tpk1-3 ( Peeters et al., 2006). Perhaps PKA activation through both RAS protein and GPCR systems is an elegant method by which the cell integrates glucose sensing and transport with the levels of glucose catabolism, requiring availability of glycolytic enzymes and phosphate sources to amplify glucose transport. Reduced PKA pathway activity has been shown to extend life span and may mimic calorie restriction (CR) ( Lin et al., 2000; Wang et al., 2009) (see the section “Factors contributing to life span extension”).

Activated PKA, as shown in Fig. 1, increases various anabolic pathways including ribosome biogenesis, mass accumulation and cell growth and inhibits certain catabolic responses ( Broach, 2012). Also, PKA inhibits Rim15-mediated stress response pathway by phosphorylation of Rim15 (serine/threonine kinase). Phosphorylated Rim15 is anchored to the cytoplasm through its binding to 14-3-3 proteins (Bmh1/2) and kept inactive ( Wanke et al., 2005). Active Rim15 upregulates various genes whose expression depends on zinc finger transcription factors, Msn2, Msn4, and Gis1. Msn2/4 activate gene expression by binding to stress response element (STRE, AGGGG) in response to diverse stress conditions including nutrient starvation. Gis1 activates transcription through post diauxic shift (PDS, TT/AAGGGAT) element upon glucose depletion. Rim15 has been shown to directly phosphorylate and activate Msn2 ( Lee et al., 2013), but it remains unclear how Rim 15 activates Gis1 and Msn4. PKA also directly regulates Msn2/4 by controlling their nuclear localization: nuclear localization of Msn2/4 is correlated inversely to cAMP levels and PKA activity. Nuclear localization signal sequences (NLS) of Msn2/4 are direct targets of PKA and phosphorylated NLS prevents nuclear import of Msn2/4 ( Görner et al., 1998; Görner et al., 2002). PKA may also play a role in NAD⁺ metabolism via Msn2/4 regulated Pnc1 expression. Pnc1 is a nicotinamide deamidase, which converts nicotinamide (Nam) to nicotinic acid (NA), contributing to the Nam/NA salvage branch of NAD⁺ metabolism (see the subsection Overview of NAD⁺ biosynthesis). Nam is also a product of conserved Sir2 (silent information regulator 2) family proteins, which are NAD⁺-dependent deacetylases, whose activity is strongly inhibited by Nam. Pnc1 may thus enhance Sir2 activity both by promoting Nam clearance (removing Sir2 inhibitor) as well as by increasing NAD⁺ synthesis (providing substrate for Sir2) (see the subsection “Additional roles of NAD⁺ intermediates and biosynthetic enzymes”).

Snf3-Rgt2

PKA also phosphorylates and activates Rgt1 (restores glucose transport) ( Jouandot et al., 2011), a glucose-responsive transcription factor, which is part of the Snf3-Rgt2 glucose sensing pathway (Fig. 1). This leads to increased expression of hexose transporter (HXT) genes and therefore increased glucose transport ( Kim et al., 2006; Broach 2012; Ljungdahl and Daignan-Fornier, 2012; Conrad et al., 2014). Activation of Rgt1 by PKA requires the activity of Snf3-Rgt2 glucose-sensing pathway, which is composed of a low glucose sensor, Snf3 (sucrose non-fermenting), and its paralogous high glucose sensor, Rgt2 (restores glucose transport). Both are transporter-like proteins with only sensor function, localized to the plasma membrane. In the absence of glucose, the downstream transcription factor, Rgt1, complexes with the corepressor paralogs, Mth1 and Std1. This repression complex recruits the general corepressor complex, Ssn6-Tup1 (also called Cyc8-Tup1), to the HXT promoters and thus represses HXT gene expression. When glucose binds to Snf3 and Rgt2 sensors, corepressors Mth1 and Std1 are recruited to the plasma membrane where they are phosphorylated by Yck1 and Yck2 (Yck1/2; yeast casein kinase 1/2 homolog) ( Moriya and Johnston, 2004; Pasula et al., 2007). Yck1/2 are palmitoylated plasma membrane-bound serine/threonine protein kinases ( Moriya and Johnston, 2004; Pasula et al., 2007). Phosphorylation of Mth1 and Std1 by Yck1/2, leads to their ubiquitination by Grr1, an F-box component of a SCF ubiquitin-ligase complex, and subsequent proteasomal degradation ( Flick et al., 2003; Kim et al., 2006; Broach 2012). This frees Rgt1 for phosphorylation by PKA, after which phosphorylated Rgt1 binds to HXT promoters and promotes HXT gene expression, leading to increased glucose transport. Unlike some glucose sensing pathways, where reduced activity leads to increased life span, deletion of Snf3-Rgt2 pathway components has recently been shown to shorten both normal and CR mediated life span as well as decrease mitochondrial function ( Choi et al., 2015).

SNF1

SNF1 (sucrose non-fermenting 1) complex is another glucose sensing pathway that affects HXT gene expression (Fig. 1). In yeast, glucose depletion activates the SNF1 (serine/threonine kinase) complex, which plays an essential role in derepressing glucose-repressed genes ( Celenza and Carlson, 1986), and is required for growth on low-glucose or non-glucose carbon sources such as sucrose ( Celenza and Carlson, 1986). SNF1 is a yeast homolog of the mammalian AMP-activated kinase (AMPK), which is a heterotrimeric complex, including a catalytic α subunit and two regulatory subunits, β and γ. Yeast SNF1 complex includes the Snf1 kinase (α), an activating regulatory subunit Snf4 (γ), and one of the three β subunits (Gal83, Sip1 or Sip2) that determine SNF1 localization ( Hardie 2007). In mammalian cells, AMPK responds to energy depletion (increased AMP/ATP ratio) and regulates energy homeostasis ( Xiao et al., 2007). Unlike mammalian AMPK, yeast SNF1 is not activated by AMP binding, instead it responds directly to glucose level. In response to glucose depletion, activation of Snf1 results from direct phosphorylation of Thr201 in the activation loop of Snf1 ( McCartney and Schmidt, 2001; Rubenstein et al., 2008) by three redundant Snf1-activating kinases (Elm1, Tos3 and Sak1) ( Hong et al., 2003; Sutherland et al., 2003). Similar to AMPK, SNF1 is regulated primarily by phosphorylation status of its activation loop, however adenine nucleotide mediated allosteric stimulation also plays a role. It has been shown that ADP can enhance SNF1 (and AMPK) activity by binding to the regulatory γ subunit Snf4. Snf4 (when bound to ADP) exerts its regulation by preventing the access of the Reg1-Glc7 (Reg1: resistance to glucose repression, Glc7: glycogen) phosphatase to the activating phosphate on Snf1 (Thr210), thus prolonging Snf1 activation ( Mayer et al., 2011). It was also shown that ADP binding to the active loop of Snf1 ( Chandrashekarappa et al., 2013) could trigger a conformational change of SNF1 complex, rendering extended resistance to phosphatase activity. Therefore, SNF1/AMPK utilizes at least two different mechanisms for adenine nucleotides (ADP or AMP) to confer protection: through binding with the active site (as ligands) and through binding with the regulatory γ subunit Snf4.

SNF1 mediates derepression of genes for utilizing alternative (non-glucose) carbon sources by inactivation of the transcription repressors, Mig1/2 (multicopy inhibitor of GAL gene expression) ( Hahn and Young, 2011). Phosphorylation of Mig1 by SNF1 and Hxk2 prevent its nuclear localization, and thus Mig1 no longer represses its target genes. SNF1 also activates transcription factors responsible for inducing gluconeogenesis (Cat8, Sip4 and Rsd2) ( Roth et al., 2004), stress response (Hsf1 and Msn2) ( Sanz 2003; Hahn and Thiele, 2004; De Wever et al., 2005), and fatty acid metabolism (Adr1) ( Parua et al., 2010). Furthermore, SNF1 also controls gene expression by stimulating chromatin remodeling. For example during glucose depletion, activated SNF1 phosphorylates histone H3, leading to the recruitment of the SAGA complex and acetylation of histone H3 at the promoters of glucose transporter genes (HXT2 and HXT4) ( van Oevelen et al., 2006). Also, AMPK has been shown to affect yeast and worm life span ( Schulz et al., 2007). In yeast, increased activity of Snf1 was reported to reduce resistance to nutrient deprivation and shorten life span ( Ashrafi et al., 2000; Lin et al., 2003; Lu et al., 2011). It is reported that as cells age, Sip2, a regulatory subunit of SNF1 is less acetylated (controlled by NuA4 acetyltransferase and Rpd3 deacetylase) ( Lu et al., 2011). Sip2 acetylation enhances its interaction with Snf1 and inhibits Snf1 activity, leading to decreased phosphorylation of Sch9 by SNF1, and longer life span (see the subsection “Sch9”). In a recent study, SNF1 pathway components were shown to be required for life span extension due to increased proteasome activity ( Yao et al., 2015). Also, perturbation of Mig1 levels by either overexpression or reduction in catalytic activity had an adverse effect on proteasome-induced life span extension ( Yao et al., 2015).

HAP

HAP (Heme activator protein) refers to the Hap2/3/4/5 CCAAT binding complex ( Chodosh et al., 1988; Hahn and Guarente, 1988; McNabb et al., 1995), part of another glucose-regulated pathway (Fig. 1). HaP2/3/5 is a heme-activated, glucose-repressed global transcription factor complex, which is activated during respiratory growth on non-fermentable carbon sources ( Zitomer and Lowry, 1992; Gancedo 1998; Schüller 2003). Hap2/3/5 form a constitutively expressed DNA binding complex ( Hahn and Guarente, 1988; McNabb et al., 1995; McNabb and Pinto, 2005) with Hap4 as the activation component. Hap4 expression increases upon glucose depletion, and this increase alone induces gene expression of HAP targets ( Forsburg and Guarente, 1989; DeRisi et al., 1997; Lascaris et al., 2003). Hap1 does not complex with Hap2/3/4/5 and has a distinct function. Hap1 activity is strictly dependent and tightly regulated by heme. Heme plays key roles in oxygen sensing and utilization, inducing expression of many genes encoding respiratory functions, all of which are mediated by Hap1 ( Zitomer and Lowry, 1992; Mense and Zhang, 2006).

HAP may also be a target of SNF1 activation via Rds2 (regulator of drug sensitivity), a zinc cluster transcription factor that is hyperphosphorylated by Snf1 kinase, although HAP can induce its target genes independent of SNF1 activity ( Broach, 2012). After shifting from glucose to ethanol, Rds2 increases expression by binding to respiration gene promotors including Hap4 ( Soontorngun et al., 2007). Recently, a novel complex Hap2/3/5-Gln3 has been suggested to form. In this case, Gln3 (glutamine metabolism), a transcription factor under TOR pathway, provides the activation domain ( Hernández et al., 2011). This suggests competitive cross-talk between Gln3 and nitrogen source sensing versus glucose-depletion sensing of Hap4. In yeast, increased HAP activity extends life span ( Lin et al., 2002). HAP4 overexpression (and thus induction of HAP target genes) extends replicative life span and increases mitochondrial respiration, which may mimic CR-mediated life span extension ( Lin et al., 2002). Also, both CR- and amino acid starvation-induced retrograde response (RTG) and TCA cycle gene expression is in part due to upregulation of the HAP4 gene ( Wang et al., 2010).

Sch9

Sch9 is a serine/threonine kinase homolog of mammalian PKB (protein kinase B), S6 kinase/Akt. Sch9 has activating phosphorylations from various sensing pathways, including glucose, sphingolipid, and nitrogen sensors. Active Sch9 activates ribosome biogenesis, mass accumulation and cell growth ( Roosen et al., 2005; Smets et al., 2008; Conrad et al., 2014). Pkh1/2 (PKB-activating kinase homolog 1/2) are serine/threonine kinase homologs of mammalian 3-phosphoinositide-dependent protein kinase 1 (PDK1) ( Casamayor et al., 1999) that are activated by sphingolipids ( Jacinto and Lorberg, 2008; Broach, 2012). Activated Pkh1/2 phosphory-late Sch9, which is required for initial Sch9 activation ( Voordeckers et al., 2011).

Sch9 activation via glucose sensing pathways remains unclear. Sch9 and PKA have overlapping substrate specificity. In fact, overexpression of Sch9 can compensate for strains lacking PKA, but how Sch9 senses glucose is still unclear. Inactivated Sch9 does not reduce glucose-induced changes in transcription, whereas inactivation of PKA reduces it by 75%. Thus Sch9 may not be required for most glucose response; however in the absence of PKA, Sch9 is responsible for the remaining glucose response ( Zaman et al., 2009; Broach, 2012). Sch9 may also cross-regulate the PKA pathway, via localization and phosphorylation of the PKA regulatory subunit, Bcy1. In addition, Sch9 can be activated by another glucose responsive pathway, the AMPK homolog, SNF1, which phosphorylates and enhances the activity of Sch9 ( Lu et al., 2011). Not only can glucose-responsive pathways activate Sch9, but also in the presence of preferred nitrogen sources, Sch9 can be directly phosphorylated and activated by TOR. Sch9 is responsible for many cellular phosphorylation changes under TOR pathway ( Urban et al., 2007), including stress response via inhibitory phosphorylation and cytosolic sequestration of Rim15 ( Wanke et al., 2008) (see the subsection “PKA” for Rim15). Collectively, PKA, TOR and Sch9 regulate ribosome biogenesis, cell metabolism and stress response.

Mutations and deletion of Sch9 was shown to increase life span in both budding and fission yeast ( Fabrizio et al., 2001; Kaeberlein et al., 2005b; Zuin et al., 2010). In budding yeast, both replicative and chronological life span have been shown to increase with reduced Sch9 activity ( Fabrizio et al., 2001; Kaeberlein et al., 2005b; Longo and Fabrizio, 2012). Decreased Sch9 activity also led to increased stress resistance. Chronological life span increase by mutated Sch9 required stress-resistance transcription factors, Msn2/4 and Rim15 ( Fabrizio et al., 2001). (See the subsection “PKA” for Msn2/4 and Rim15). Reduced Sch9 may also increase chronological life span in a reduced TOR activity dependent manner ( Longo and Fabrizio, 2012).

TOR and nitrogen source sensing pathways

Yeast cells can sense nitrogen sources through the target of rapamycin (TOR) pathway. TOR is activated by preferred nitrogen sources such as glutamine, ammonium and certain amino acids. Although well studied, the exact mechanism of nitrogen source activation is still unclear. Reduction of TOR activity has been linked to increased yeast life span and CR ( Kaeberlein et al., 2005b; Wei et al., 2008). In this section, we introduce TOR, its complexes, TORC1 and TORC2, and its cross-talk with other pathways.

Yeast TOR complex I (TORC1) functions as a protein kinase complex and consists of either Tor1 or Tor2, Kog1 (homolog of mammalian raptor), Lst8 (contains WD-repeat; required for general amino acid permease, Gap1, transport to the cell surface) and Tco89 (Tor complex one; regulates global H3 K56 acetylation). Tor1 and Tor2 are 67% identical in sequence and are highly similar to mammalian TOR protein. They are founding members of the phosphatidyl inositol 3′ kinase related kinases (PIKK), but do not have lipid kinase activity ( Keith and Schreiber, 1995). TORC2 (TOR complex 2) complex contains exclusively Tor2, Avo1-3 (adheres voraciously to TOR2), Bit61 (binding partner of Tor2), and Lst8 ( Loewith et al., 2002; Wedaman et al., 2003). TORC1 is sensitive to rapamycin inhibition, whereas TORC2 is not ( Loewith et al., 2002). TORC2 mainly affects actin cytoskeleton organization via serine/threonine protein kinase Ypk1/2 (yeast protein kinase), lipid synthesis and cell survival ( Loewith et al., 2002; Kamada et al., 2005; Niles and Powers, 2014). Recent studies have also connected TORC2 to amino acid starvation induced autophagy ( Vlahakis et al., 2014; Vlahakis et al., 2014) and genome stability ( Shimada et al., 2013). In this section, we will focus on the downstream targets of TORC1.

Recent studies have shown that the lysosome/vacuole membrane is the main location for activation of TORC1 signaling ( Urban et al., 2007; Sturgill et al., 2008; Binda et al., 2009). In yeast, activity of TORC1 is regulated at the vacuolar membrane through interplay with the conserved EGO complex (EGOC), containing Ego1, Ego3, Gtr1, and Gtr2. Ego1, homologous to Ego3, is N-terminally myristoylated and palmitoylated, tethering EGOC to the vacuolar membrane ( Dubouloz et al., 2005; Binda et al., 2009; Zhang et al., 2012). Gtr1 and Gtr2 are Ras-family GTPases ( Sancak et al., 2008; Efeyan et al., 2012). Activated EGOC stimulates TORC1 via physical interaction in response to amino acids. In mammals, EGOC appears to trigger TORC1 relocalization from the cytoplasm to the vacuolar membrane, however, detailed mechanisms remain unclear. S. cerevisiae TORC1 seems to constantly associate with the vacuolar membrane regardless of the amino acid level ( Binda et al., 2009).

TORC1 controls various cellular processes such as nitrogen catabolite repression (NCR), amino acid transport, retrograde and stress response, and cell growth mainly by two major effector branches: Sch9, and the Tap42-PPase (Tap42: two A phosphatase associated protein; PPase: protein phosphatase) complex ( Loewith et al., 2002; Urban et al., 2007; Broach 2012; Conrad et al., 2014). When TORC1 is activated, it phosphorylates and activates Sch9, leading to increased ribosome biogenesis, mass accumulation and cell growth (see the subsection “Sch9”) ( Urban et al., 2007). Activated TORC1 also phosphorylates Tap42, which leads to binding of TORC1 to the Tap42-PPase complex, resulting in inhibition of this complex. TORC1 and Tap42-PPase work cooperatively to regulate the phosphorylation status of many downstream targets (Fig. 1): activated TORC1 phosphory-lates downstream targets and these phosphorylations are later removed by Tap42-PPase. TORC1 and Tap42-PPase also independently regulate different downstream targets by working with other kinases and phosphatases.

The main Tap42-PPase complex that regulates NCR, amino acid transport, retrograde and stress response is Tap42-PP2A (PP2A: protein phosphatase 2A) complex. When preferred nitrogen sources are present, inhibition of this complex by activated TORC1 prevents Tap42-PPA2 from accessing and dephosphorylating downstream targets. For example, Gln3 is a major transcription factor required for the induction of NCR genes in the absence of preferred nitrogen sources. When preferred nitrogen sources are present, Gln3 is heavily phosphorylated and kept in the cytoplasm via binding to its cytoplasmic sequestering partner, Ure2 ( Blinder et al., 1996), and thus NCR genes are not expressed. During nitrogen limitation, TORC1 is inactive, which frees up Tap42-PP2A to access Gln3, leading to reduced phosphorylation, nuclear localization of Gln3 and NCR gene expression ( Beck and Hall, 1999). The Tap42-PP2A complex also controls retrograde (RTG) response by regulating the nuclear localization of the Rtg1/3 transcription factors. Activated TORC1 phosphorylates the negative RTG regulator, Mks1. Phosphorylated Mks1 associates with 14-3-3 proteins (Bmh1/2) and this complex prevents nuclear localization of Rtg1/3. When TORC1 is inactive, Tap42-PP2A is activated, which dephosphorylates Mks1, leading to nuclear localization of Rtg1/3 and RTG gene expression. Rtg2 promotes Rtg1/3 nuclear localization by competing with 14-3-3 for binding with Mks1 (Mks1 is inactive when bound to Rtg2) ( Dilova et al., 2004; Conrad et al., 2014). TORC1 and Tap42-PP2A also regulate stress response by regulating the phosphorylation status of Rim15 and its nuclear localization together with 14-3-3 proteins (Bmh1/2) (see the subsection “PKA”). TORC1 controls specific amino acid transport mainly via Npr1 (nitrogen permease reactivator). Npr1 is a TORC1 responsive protein kinase that regulates plasma membrane sorting of amino acid permeases (AAPs) such as Gap1 and Tat2 (tryptophan amino acid transporter). Gap1 is the general AAP, which mediates amino acid transport during amino acid (or nitrogen) starvation. In contrast, many other AAPs (such as Tat2) are only expressed or stabilized at the plasma membrane when preferred amino acids are present (see the subsection “SPS”). Activated TORC1 phosphorylates Npr1, leading to degradation of Gap1 (via enhancing its endocytosis) and stabilization of Tat2 (by inhibiting its endocytosis). In response to nitrogen or amino acid starvation, dephosphorylation of Npr1 (possibly by Tap42-Sit4, Sit4: a type 2A-related PPase) leads to stabilization of Gap1 on the plasma membrane and enhances the degradation of Tat2 ( Broach 2012; Ljungdahl and Daignan-Fornier, 2012).

Amino acid sensing pathways

Yeast cells can sense amino acid sources through the GAAC (general amino acid control) and SPS (Ssy1-Ptr3-Ssy5) pathways (Fig. 1). Reduction of amino acid uptake has been shown to increase life span and linked to CR-mediated life span extension ( Jiang et al., 2000; Powers et al., 2006; Fabrizio and Longo, 2007). In this section we introduce GAAC and SPS pathways, and their cross-talk with other pathways.

GAAC

In response to amino acid starvation, the general amino acid control (GAAC) regulatory system induces enzymes across all amino acid biosynthesis pathways ( Marzluf 1997; Natarajan et al., 2001; Hinnebusch and Natarajan, 2002; Hinnebusch 2005; Ljungdahl and Daignan-Fornier, 2012). Amino acid starvation leads to uncharged tRNAs ( Zaborske et al., 2009; Zaborske et al., 2010). GAAC inhibits overall protein synthesis and activates amino acid biosynthesis by allosteric activation of Gcn2 (general control nonderepressible) kinase: binding of uncharged tRNAs to a histidyl-tRNA synthetase-like domain on Gcn2 ( Wek et al., 1989; Dong et al., 2000; Dever and Hinnebusch, 2005). Activated Gcn2 then phosphorylates Ser51 of the translation initiation factor, eIF2, leading to a reduction in the rate of ternary complex (TC: eIF2-GTP-Met-tRNAi, required for translation initiation) formation, which subsequently decreases overall protein synthesis ( Wek et al., 1989; Dong et al., 2000; Dever and Hinnebusch, 2005). Gcn2-activated amino acid biosynthesis gene expression is mediated by regulating the translation of Gcn4 (general control nonderepressible), a leucine-zipper transcription factor. Gcn4 is the effector of GAAC and is required for inducing the expression of amino acid biosynthesis genes ( Hinnebusch and Natarajan, 2002; Hinnebusch 2005). The mRNA of Gcn4 contains four small open reading frames in the 5′-untranslated region (uORF). When a scanning 40S ribosome with a TC encounters the first start codon of uORF1, the GTP bound to TC is hydrolyzed to GDP. Next, eIF2-GDP is released and the 60S ribosome is recruited to start translation. Translation terminates at the stop codon of uORF1, and 60S ribosome disassociates. The 40S ribosome continues to scan Gcn4 mRNA until it reacquires a TC to regain competency. When TC is high (no starvation), the 40C ribosome can reacquire TC and initiate translation at uORF2, uORF3 and then uORF4. Translation of uORF4 hinders the translation initiation at the start codon of Gcn4 resulting in no Gcn4 translation (and no induction of amino acid biosynthesis gene expression). When TC is low (under starvation conditions), after translation initiation at uORF1, the scanning 40S ribosome is more likely to reacquire TC after passing uORF2, uORF3 and uORF4, allowing translation initiation at the start codon of Gcn4. Gcn4 binds to promoters with the upstream activating sequence Gcn4 protein recognition element (UAS_GCRE) motif, GAGTCA, and activates amino acid biosynthesis genes ( Hinnebusch and Natarajan, 2002; Hinnebusch 2005; Ljungdahl and Daignan-Fornier, 2012). Starvation also decreases proteasomal degradation of Gcn4 ( Kornitzer et al., 1994; Ljungdahl and Daignan-Fornier, 2012).

GCN4 is also induced when NCR is derepressed (Fig. 1) ( Godard et al., 2007; Ljungdahl and Daignan-Fornier, 2012). As discussed in the section “TOR”, with non-preferred nitrogen sources, TORC1 no longer inhibits Tap42-PP2A, leading to increased NCR gene expression. When TORC1 is not active, Sit4, a type 2A-related PPase which is inhibited by active TORC1-Tap42-PP2A complex, dephosphorylates and activates Gcn2 ( Cherkasova and Hinnebusch, 2003; Conrad et al., 2014). Therefore, decreased TORC1 activity increases Gcn4 via Sit4 and Gcn2. Gcn4 has also been found to act with Gln3 for induction of genes during rapamycin treatment ( Staschke et al., 2010; Conrad et al., 2014). Snf1 has a complicated relationship with GAAC. During glucose depletion, which causes rapid translation inhibition (see details in the subsection “SNF1”), Snf1 also inhibits translation of Gcn4 and subsequent gene expression by Gcn4 ( Conrad et al., 2014). However, in the presence of glucose and low amino acid conditions, Snf1 stimulates and activates Gcn2, leading to Gcn4 translation whereas in amino acid rich and low glucose conditions, Snf1 instead inhibits Gcn2 by promoting phosphorylation or inhibiting dephosphorylation of Gcn2 by Sit4 and Glc7 ( Shirra et al., 2005; Shirra et al., 2008). Thus Snf1 both helps inhibit Gcn2 when GAAC is off, and helps stimulate Gcn2 when GAAC is on, depending on the presence of glucose.

SPS

Thus far in terms of amino acids and nitrogen sources we have discussed internal sensing pathways. The pathway responsible for external amino acid sensing is the SPS (Ssy1-Ptr3-Ssy5) amino acid nutrient-sensing pathway ( Abdel-Sater et al., 2011; Omnus et al., 2011). Ssy1 is a plasma membrane broad range amino acid permease-like sensor with no transport activity. Ssy1 is in complex with the phosphoprotein, Ptr3, and the serine protease, Ssy5. Upon formation, Ssy5 autocleaves into a catalytic domain and a prodomain, which remain associated until signaling is activated. In the presence of external amino acids, Ssy1 sends a signal via conformational changes that triggers subsequent phosphorylations of Ptr3 and Ssy5 by the kinases, Yck1 and Yck2 (also in the subsection “Snf3-Rgt2”) ( Liu et al., 2008; Omnus et al., 2011). Once phosphorylated, the prodomain and catalytic domain of Ssy5 disassociate. The phosphorylated Ssy5 prodomain is ubiquitinated by Grr1 and marked for proteasomal degradation. The Ssy5 catalytic domain then cleaves the N-terminal cytoplasmic retention domain of both SPS pathway transcription factors, Stp1 and Stp2 ( Andréasson et al., 2006; Omnus et al., 2011; Omnus and Ljungdahl, 2014). Without the cytoplasmic retention domain, Stp1 and Stp2 can bypass the surveillance of inner nuclear membrane ubiquitin ligase Asi (amino acid sensor-independent) complex components, Asi1, Asi2 and Asi3, entering the nucleus to modulate SPS pathway downstream gene expression such as increasing amino acid permease expression and decreasing amino acid biosynthesis ( Zargari et al., 2007; Foresti et al., 2014; Omnus and Ljungdahl, 2014). SPS amino acid sensing pathway and Snf3-Rgt2 glucose sensing pathway have much in common. Both require Yck1/2 phosphorylation and Grr1 ubiquitination for activation. Ssy1, Snf3 and Rgt2 are all transporter-like sensors with long-terminal tails and no transporter function ( Ljungdahl 2009; Rubio-Texeira et al., 2010; Broach 2012; Ljungdahl and Daignan-Fornier, 2012; Conrad et al., 2014). Both sensors also directly activate their transcription factors to induce changes in gene expression targets. Recently in yeast, decreased SPS pathway activity was shown to extend replicative life span ( Tsang et al., 2015), which was further increased by CR. SPS-induced life span extension required components of the malate-pyruvate NADH shuttle and partially required Pho8, a vacuolar phosphatase. Decreased SPS activity also increased phosphate sensing (PHO) pathway expression, thus active SPS likely has a repressive effect on PHO pathway components ( Tsang et al., 2015) (see the section “Phosphate sensing pathway” for cross-talk).

Phosphate sensing pathway

In yeast, inorganic phosphate (Pi) is sensed by the phosphate-responsive signaling (PHO) pathway. Recently, PHO pathway was linked to life span: Pho8, a target of PHO pathway, was partially required for reduced SPS-mediated life span extension ( Tsang et al., 2015). In this section we will discuss Pi sensing and response by the PHO pathway and cross-talk with other pathways.

Pi sensing, acquisition, and storage are mainly mediated by the PHO pathway that controls Pi transporters, regulatory factors, and effectors ( Lenburg and O’Shea, 1996; Persson et al., 2003; Auesukaree et al., 2004). When Pi is highly available, PHO signaling downstream targets are repressed by the cyclin-dependent kinase complex, Pho80-Pho85 (Pho80: cyclin, Pho85: cyclin dependent kinase), which phosphorylates and inactivates the major transcription factor, Pho4, by preventing its nuclear localization. Upon Pi limitation, the synthesis of inositol heptakisphosphate (IP7) increases ( Auesukaree et al., 2005; Lee et al., 2007). IP7 was shown to allosterically bind to the tertiary complex of Pho81 (a cyclin-dependent kinase inhibitor)-Pho80-Pho85, inducing a conformational change of Pho81 that prevents phosphorylation of Pho4 by Pho85 ( Lee et al., 2008). Unphosphorylated Pho4 accumulates in the nucleus, and along with transcription factor, Pho2, activates PHO-responsive genes whose expression products include high affinity Pi transporters (Pho84 and Pho89), repressible phosphatases (Pho5 and Pho8), and factors for phosphate mobilization from vacuolar polyphosphate storage.

Pi sensing also affects the cell cycle: Cln3 (G₁ cyclin) is a target for phosphorylation by Pho80-Pho85 ( Menoyo et al., 2013). Under Pi starvation, Pho80-Pho85 is inactive and Cln3 is dephosphorylated, which leads to proteasomal degradation and cell cycle arrest. Repletion of Pi requires and activates Pho80-Pho85 to phosphorylate and activate Cln3. This allows cells to progress over start in G₁ and re-enter the cell cycle ( Menoyo et al., 2013).

Activation of PHO pathway also concomitantly increases salvaging of nicotinamide riboside (NR), a precursor to NAD⁺, since certain phosphatases such as Pho5 and Pho8 can utilize NAD⁺ intermediate NMN as a substrate, producing NR while removing Pi ( Lu et al., 2011). It is not yet fully understood how cells sense the level of available Pi to elicit proper metabolic responses. Interestingly, the pho84Δ mutant has constitutive expression of PHO5 ( Ueda and Oshima, 1975; Bun-Ya et al., 1991). The defect in Pi transport caused by PHO84 mutations resulted in a low level of intracellular Pi, leading to constitutive derepression of the PHO pathway and its downstream genes ( Wykoff and O’Shea, 2001; Auesukaree et al., 2004). In addition to Pi availability, some studies show that the PHO signaling pathway also responds to changes in intermediate metabolites of the purine synthesis pathway in an IP7-independent manner ( Gauthier et al., 2008; Pinson et al., 2009).

Phosphate signaling has also been linked to glucose sensing pathways. For example, after prolonged Pi starvation, repletion of Pi to cells leads to PKA activation-dependent degradation of phosphate transporter, Pho84 ( Giots et al., 2003; Mouillon and Persson, 2005; Lundh et al., 2009; Popova et al., 2010; Samyn et al., 2012). These studies support a role for Pho84 as a Pi transceptor, a sensor and transporter of Pi. Also, other components in the PHO pathway have been associated with glucose metabolism ( Kang et al., 2014; Kang et al., 2014). Phosphate sensing PHO pathway and glucose sensing PKA pathway also have similar effects on the transcription factor, Rim15 (see the subsection “PKA”). Both PKA and Pho80-Pho85 complex phosphorylate and inhibit Rim15. Phosphorylated Rim15 is anchored to the cytoplasm by 14-3-3 (Bmh1/2) proteins ( Wanke et al., 2005). Active Rim15 increases expression of stress response and post diauxic shift genes via Msn2/4 and Gis1, respectively ( Lee et al., 2013). Active Msn2/4 also increases Pnc1 activity (see the subsection “PKA”), which increases genome stability and NAD⁺ metabolism. Thus PHO pathway may regulate NAD⁺ metabolism (see the subsection “Other nutrient sensing pathways and NAD⁺ metabolism”) via increased NR salvaging, as previously mentioned, and via this Rim15-Msn2/4-Pnc1 pathway.

As previously mentioned in the subsection “SPS”, another point of cross-talk is between phosphate sensing PHO pathway and extracellular amino acid sensing SPS pathway. Decreased SPS pathway activity increases PHO pathway activity: increased overall expression of PHO pathway, including the PHO pathway transcription factor, Pho4, and slightly increased activity of repressible vacuolar phosphatase, Pho8. Also, expression of Pho8, increases under reduced SPS activity, independent of the canonical PHO transcription factors Pho2 and Pho4. Full SPS-mediated life span extension also required Pho8 ( Tsang et al., 2015). Thus components of PHO pathway may also be activated under other conditions or sensor pathways.

Factors contributing to life span extension

For decades the budding yeast, Saccharomyces cerevisiae, has been a model organism for uncovering genes that regulate life span. Studies in model systems including yeast, demonstrate that single gene mutations can alter longevity ( Jazwinski 2000; Kenyon 2001; Dilova et al., 2007; Longo and Fabrizio, 2012). Additionally, CR extends life span in a variety of species, further supporting conservation of longevity-regulating pathways ( Weindruch and Walford, 1998). CR also delayed or reduced the incidence of many age-associated diseases such as cancer and diabetes ( Weindruch and Walford, 1998; Guarente, 2013). Like CR, mitigated nutrient sensing activity generally leads to life span extension. S. cerevisiae has contributed considerably to aging and CR studies, and helped pave the way for further research in metazoans. Although the molecular mechanisms underlying life span extension still remain unclear, as more longevity factors are identified, it is evident that aging is modulated by the complex interplay of signaling pathways at both the cellular and organismal level.

Yeast life span is studied in two ways: replicative life span (RLS) and chronological life span (CLS). RLS measures total cell divisions an individual yeast cell undergoes before senescence (division potential) ( Mortimer and Johnston, 1959; Jazwinski, 1990), whereas CLS measures the time cells remain viable at a non-dividing state (post-mitotic survival) ( Fabrizio and Longo, 2003; Longo and Fabrizio, 2012). The mother-daughter cell asymmetry in S. cerevisiae can be easily observed under the microscope, which allowed development of the RLS assay ( Mortimer and Johnston, 1959). Although RLS and CLS define two very distinct forms of longevity, many longevity factors appear to regulate both CLS and RLS.

Generally, reduced nutrient signaling can increase life span, for example CR. In yeast, moderate CR is imposed by reducing the glucose in rich media from 2% to 0.5% ( Lin et al., 2000; Easlon et al., 2008; Wei et al., 2008). Under this CR condition, growth rate remains robust and both RLS and CLS extend. Moderate CR shunts carbon metabolism from fermentation toward the mitochondrial TCA cycle, with a concomitant increase in respiration ( Lin et al., 2002). In yeast, moderate CR is suggested to extend life span through reducing the activities of conserved nutrient-sensing pathways such as the PKA, Sch9 and TOR kinase pathways ( Lin et al., 2000; Fabrizio et al., 2001; Kaeberlein et al., 2005b). Decreased activity of the glucose sensing PKA pathway extends both RLS and CLS ( Sun et al., 1994; Lin et al., 2000; Fabrizio et al., 2001). Also, deleting the nutrient responsive Sch9 (sch9∆) and nitrogen source sensing Tor1 (tor1∆) kinases extend both RLS and CLS ( Fabrizio et al., 2001; Longo 2003; Kaeberlein et al., 2005b), supporting that reduced nutrient sensing activity can extend life span. Notably, mutants of the PKA, TOR and Sch9 pathways have been suggested to genetically mimic CR. However, some nutrient sensing pathways can modulate life span independent of the CR pathway. For example, deleting components of the amino acid sensing SPS pathway extends RLS and CR further extends the long life span of these SPS mutants ( Tsang et al., 2015).

In addition to nutrient sensing pathways, other longevity factors were identified in yeast, some of which have also been linked to CR. These factors include proteins that modulate mitochondrial function ( Barros et al., 2004; Bonawitz et al., 2007; Scheckhuber et al., 2007; Veatch et al., 2009; Ocampo et al., 2012; Erjavec et al., 2013), stress response/hormesis/mitohormesis ( Bonawitz et al., 2007; Mesquita et al., 2010; Pan 2011; Pan et al., 2011; Longo and Fabrizio, 2012; Ocampo et al., 2012), NAD⁺-dependent deacetylase Sir2 family activity ( Imai and Guarente, 2014) (discussed later), asymmetric partitioning of damaged proteins ( Erjavec and Nyström, 2007; Erjavec et al., 2007, 2008), genome stability ( Weinberger et al., 2007; Andersen et al., 2008; Unal et al., 2011), homeostasis of NAD⁺ and other metabolic factors ( Lin et al., 2000; Anderson et al., 2002; Belenky et al., 2007; Matecic et al., 2010; Kato and Lin, 2014a) (see the section “NAD⁺ biosynthetic and signaling pathways in yeast”), vacuolar function ( Fabrizio et al., 2010; Hughes and Gottschling, 2012), ribosome biogenesis ( Steffen et al., 2012), cell size/hypertrophy ( Biliński and Bartosz, 2006; Yang et al., 2011; Biliński et al., 2012), and regulation of proteostasis ( Kruegel et al., 2011; Delaney et al., 2013; Schleit et al., 2013; Yao et al., 2015). Additionally, acetic acid released into the growth medium as a metabolic byproduct during chronological aging was suggested to be the major factor that shortens CLS. Although pH neutralization was shown to offset acetic acid induced toxicity and protect both CLS and RLS ( Burtner et al., 2009; Murakami et al., 2011; Murakami et al., 2012), other studies showed that acetic acid and pH are not the only determinants of CLS ( Longo and Fabrizio, 2012; Wu et al., 2013).

Variations in CR protocols such as amino acid limitation and/or further carbon source reduction/substitution have also been implemented ( Jiang et al., 2000; Kaeberlein et al., 2005a; Fabrizio and Longo, 2007; Longo and Fabrizio, 2012). In yeast, the role of Sir2 in CR is highly debated: RLS extension by moderate CR (0.5% glucose) ( Lin et al., 2000; Kaeberlein et al., 2002; Lamming et al., 2005; Easlon et al., 2007; Smith et al., 2007; Easlon et al., 2008; Wei et al., 2008) requires both NAD⁺ and Sir2 ( Lin et al., 2000), whereas RLS extension by more severe CR (0.05% glucose) does not require Sir2 ( Kaeberlein et al., 2004; Easlon et al., 2007). In fact, without the Sir2 family, severe CR further extends RLS ( Easlon et al., 2007). Akin to Sir2 in CLS: under nutrient deprivation, deleting SIR2 further extends CLS in certain genetic backgrounds ( Fabrizio et al., 2005). Cells likely respond differently to each CR regimen. Perhaps severe CR additionally triggers gluconeogenesis, which is activated upon glucose starvation. Gluconeogenesis may also be induced in nutrient deprived sir2∆ mutants that further extend CLS ( Fabrizio et al., 2005). A key gluconeogenesis enzyme, Pck1, is a Sir2 substrate, thus in sir2∆ mutant, Pck1 remains active and promotes gluconeogenesis ( Lin et al., 2009). Yet, age-enhanced gluconeogenesis has been shown to shorten CLS and homologues of Sir2, Hst3 and Hst4, downregulate age-enhanced gluconeogenesis ( Hachinohe et al., 2013). Moreover, human Sirt1 has been reported to deacetylate PGC1-α, which induces gluconeogenesis ( Rodgers et al., 2005). Together, these studies demonstrate the complex roles of Sir2 in CR, a complexity typical of metabolic longevity factors.

NAD⁺ biosynthetic and signaling pathways in yeast

Pyridine nucleotides are in a myriad of cellular processes. In yeast, factors in the NAD⁺ biosynthetic pathways have several roles. As previously mentioned, Sir2 and moderate CR require the longevity factor, NAD⁺ ( Lin et al., 2000). Also, NAD⁺ metabolism is required for normal life span as well as life span extension by reduced SPS amino acid signaling activity ( Tsang et al., 2015). NAD⁺ intermediates can be signaling molecules, and high intracellular concentrations can inhibit certain cellular processes. In this section we discuss the NAD⁺ biosynthetic pathways in yeast and their roles in signaling.

Overview of NAD⁺ biosynthesis

Cellular NAD⁺ is synthesized from several precursor molecules. Eukaryotes utilize tryptophan to synthesis NAD⁺de novo and also salvage NAD⁺ intermediates such as nicotinamide (Nam), nicotinic acid (NA) and nicotinamide riboside (NR). In S. cerevisiae, NAD⁺ is synthesized from two key intermediates, nicotinic acid mononucleotide (NaMN) and nicotinamide mononucleotide (NMN), via the de novo and NA/Nam/NR salvaging pathways. As shown in Fig. 2, quinolinic acid (QA) is mainly derived from tryptophan via the de novo pathway: five enzymatic reactions (Bna2, Bna7, Bna1, Bna4, Bna5) and a spontaneous cyclization ( Bender 1983; Wogulis et al., 2008). QA is then converted into NaMN by phosphoribosyltransferase, Bna6, transfer of the phosphoribose moiety of phosphoribosyl pyrophosphate (PRPP) ( Panozzo et al., 2002). De novo synthesis and NA/Nam salvaging pathways converge at NaMN. As part of NA/NAM salvage, another phosphoribosyltransferase, Npt1, converts NA into NaMN ( Preiss and Handler, 1958a , 1958b). NA is mainly salvaged from Nam by Pnc1 deamidase, but the NA transporter, Tna1, also imports both NA and QA ( Llorente and Dujon, 2000; Ohashi et al., 2013). Next, NaMN adenylyltransferases, (NaMNATs), Nma1 and Nma2, convert NaMN into nicotinic acid adenine dinucleotide (NaAD or deamido-NAD⁺) by transfer of the AMP moiety from ATP. Amidation of NaAD by Qns1, a glutamine-dependent NAD⁺ synthetase, completes NAD⁺ synthesis ( Bieganowski et al., 2003).

In the shortest branch of NR salvaging, NR is phosphorylated by Nrk1 to produce NMN ( Bieganowski and Brenner, 2004; Belenky et al., 2007) and is subsequently adenylylated to NAD⁺ by NMN adenylyltransferases (NMNATs), Nma1, Nma2 and Pof1 ( Natalini et al., 1986; Emanuelli et al., 1999; Garavaglia et al., 2002; Emanuelli et al., 2003; Magni et al., 2004; Kato and Lin, 2014). Nrk1 can also convert nicotinic acid riboside (NaR), a deamidated form of NR, to NaMN ( Belenky et al., 2007). Unlike higher eukaryotes including mammals, yeasts do not possess Nam phosphorybosyltransferase (Nampt), the enzyme that converts Nam to NMN. Instead, Nam is deamidated to NA by Pnc1, and shunted into NA/NAM salvaging ( Ghislain et al., 2002). The newer, longer branch of NR salvaging connects NR to NA/Nam salvaging, and includes nucleosidases Pnp1, Urh1, and Meu1, which convert NR to Nam, or NaR to NA ( Belenky et al., 2007). Furthermore, the nucleotidases, Isn1 and Sdt1 ( Bogan et al., 2009), and phosphatases, Pho5 and Pho8 ( Lu et al., 2009; Lu et al., 2011), contribute to NR metabolism by converting NMN to NR.

Yeast cells produce NAD⁺ predominantly from the NA/Nam salvage pathway during exponential growth ( Sporty et al., 2009), due to niacin (NA and Nam)-rich standard media and very efficient NA transport by Tna1 ( Llorente and Dujon, 2000). Also, NR is an efficient NAD⁺ precursor: contributing up to ~50% of the NAD⁺ pool and supporting NAD⁺-dependent reactions ( Bieganowski and Brenner, 2004; Belenky et al., 2007). Although standard media is NR-free, NR is an endogenous metabolite that is dynamically released into and retrieved from the environment ( Lu et al., 2009). Extracellular NR is imported by the high-affinity NR transporter, Nrt1 ( Belenky et al., 2008). Perhaps a flexible NR/NAD⁺ pool facilitates prompt adjustments in response to metabolic stresses ( Lu and Lin, 2010). Indeed, NR salvaging activity was essential for maintaining NAD⁺ homeostasis, cellular fitness and life span ( Bogan et al., 2009; Lu et al., 2009).

Additional roles of NAD⁺ intermediates and biosynthetic enzymes

Intermediates of NAD⁺ biosynthesis can also function as signaling molecules. Intracellular concentrations of many NAD⁺ intermediates are maintained at low levels, characteristic of signaling molecules ( Evans et al., 2010), or compartmentalized (see the subsection “Compartmentalization of NAD⁺ metabolism”). We previously discovered that low NaMN level is associated with activation of the PHO pathway ( Lu et al., 2011). PHO-regulated Pho5 activity increased specifically in npt1Δ mutant (Npt1 is the major NaMN producer), suggesting that depletion of NaMN leads to PHO activation ( Lu et al., 2011). Activated PHO pathway leads to increased transport and mobilization of Pi with concomitant increase in NR levels and thus boosts NAD⁺ metabolism (see the section “Phosphate sensing pathway”). Moreover, nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic ADP-ribose (cADPR), function as signaling molecules to regulate calcium homeostasis in a variety of organisms ( Clapper et al., 1987; Guse and Lee, 2008). CD38, an ADP-ribosylcyclase, has been shown to produce NAADP and cADPR from NADP⁺ and NAD⁺, respectively ( Graeff et al., 2006; Li et al., 2013). Other NAD⁺ intermediates, such as NR and NMN, may also function as signaling molecules to regulate NAD⁺ homeostasis or cellular processes ( Kato and Lin, 2014). We recently showed that most NR is vacuolar and NMN may be sequestered by Pof1 binding, leading to lower levels of free NR and NMN ( Kato and Lin, 2014b).

Additionally, it may be crucial to maintain low levels of intracellular NAD⁺ intermediates since high concentrations can inhibit certain cellular processes. For example in bacteria, NMN is a product and inhibitor of NAD⁺-dependent DNA ligase, thus NMN deamidase is suggested to maintain low levels of intracellular NMN ( Cheng and Roth, 1995). In yeast, we recently showed that NMN is constantly converted into NR, which is more mobile and readily excreted, re-assimilated or sent to storage ( Kato and Lin, 2014b). The NAD⁺ intermediate, QA, was also shown to be produced and excreted like NR ( Ohashi et al., 2013). Additionally, Nam is a product and inhibitor of NAD⁺-dependent deacetylase, Sir2, thus Nam clearance is critical for Sir2 activity and life span ( Gallo et al., 2004) (see the subsection “Sir2 and NAD⁺ metabolism”). Nam can be cleared by enzymes of the NA/NAM salvage pathways: in yeast by Pnc1, a Nam deamidase; in mammals by Nampt, a Nam phosphoribosyltransferase ( Bitterman et al., 2002; Revollo et al., 2004). Nam is also methylated by the Nam methyltransferase, Nnt1, a homolog of human NNMT (N-nicotinamide methyltransferase) ( Anderson et al., 2003; Lin et al., 2004), a modification that induces a hormetic response to protect against oxidative damage ( Schmeisser et al., 2013). A recent study in C. elegans suggests that methylated Nam can induce a transient hormetic response, which leads to life span extension ( Schmeisser et al., 2013). NADH was also reported to competitively inhibit Sir2 ( Lin et al., 2004) (see the subsection “Sir2 and NAD⁺ metabolism”). Thus, balancing the concentrations of pyridine nucleotides and their metabolites may be critical for regulating many cellular processes. Moreover, specific NAD⁺ synthesis enzymes, such as NMNATs, have been shown to play roles in other cellular processes. Salmonella NadR is a multi-functional NMNAT with a role in transcriptional regulation ( Grose et al., 2005). Drosophila NMNAT was shown to possess chaperone activity which helps protect axons ( Zhai et al., 2008). In S. cerevisiae, Nma1 and Nma2 were shown to alleviate proteotoxicity in yeast models of proteinopathies ( Ocampo et al., 2013). Overall, these studies demonstrate that specific NAD⁺ intermediates as well as NAD⁺ biosynthesis enzymes also play roles in other cellular processes, further highlighting the importance of NAD⁺ homeostasis factors in maintaininFg cellular function. The next section further discusses the regulation of NAD⁺ metabolism and cross-talk with other signaling pathways.

Regulation of NAD⁺ metabolism

Sir2 and NAD⁺ metabolism

The Sir2 family proteins, sirtuins, are highly conserved from bacteria to humans ( Brachmann et al., 1995; Frye, 2000), with a conserved core domain required for their enzymatic activity as NAD⁺-dependent protein deacetylases or ADP-ribosylases ( North and Verdin, 2004). Sir2 was first identified in the Sir1/2/3/4 silencing complexes in yeast ( Ivy et al., 1986; Rine and Herskowitz, 1987), which are required to maintain and repress three repetitive genomic regions: telomeres, the cryptic mating type loci (HML and HMR), and the rDNA loci ( Hecht et al., 1996; Smith and Boeke, 1997; Strahl-Bolsinger et al., 1997; Moazed 2001). Each locus employs unique DNA binding factors that recruit specific Sir proteins to deacetylate histones, compacting chromatin configuration, thus mediating silencing. Among these Sir proteins, only Sir2 is required for silencing of all three regions, and the NAD⁺-dependence of Sir2 activity links cellular metabolic status to Sir2 complex-mediated transcriptional silencing ( Imai et al., 2000; Landry et al., 2000; Smith et al., 2000). Sir2 homologs in yeast include Hst1, Hst2, Hst3 and Hst4 (homologous to Sir two), of which nuclear Hst1 is most homologous to Sir2 and when overexpressed suppresses silencing defects at the mating type loci of Sir2 deletion mutant ( Brachmann et al., 1995). Hst1 may also mediate Sir complex-independent transcriptional regulation by interacting with the transcription regulator, Sum1 ( Xie et al., 1999; Sutton et al., 2001). Although Hst1 is most homologous, Hst2 is the most abundant Sir2 homolog in yeast, accounting for the bulk of intracellular NAD⁺-dependent deacetylase activity ( Smith et al., 2000). Hst2 appears to shuttle between the nucleus and cytoplasm to regulate rDNA and telomere silencing ( Smith et al., 2000; Perrod et al., 2001; Halme et al., 2004; Lamming et al., 2005; Wilson et al., 2006). Nuclear Hst3 and Hst4 have also been linked to genome stability by regulating rDNA gene silencing and controlling histone H3 (K56) acetylation ( Brachmann et al., 1995; Celic et al., 2006; Hachinohe et al., 2011).

Key Sir2 regulators include NAD⁺ and its derivatives, thus NAD⁺ levels affect Sir2 activity and its downstream events. Sir2-mediated processes are affected by mutations that cause deficiency in NAD⁺ production (see the subsection “Overview of NAD⁺ biosynthesis”). Deleting the NA phosphoribosyltransferase, NPT1, gene significantly reduces NAD⁺ levels, abolishes Sir2-mediated silencing ( Smith et al., 2000) and CR-mediated life span extension (which requires both Sir2 and NAD⁺) ( Lin et al., 2000). Nam, a product of Sir2-mediated deacetylation, is also a potent non-competitive inhibitor of Sir2 ( Bitterman et al., 2002; Sauve and Schramm, 2003; Schmidt et al., 2004). Preventing Nam accumulation is important for Sir2 function since increasing Nam concentration in growth media or blocking Nam clearance by deleting the nicotinamidase, PNC1 ( Ghislain et al., 2002), reduced Sir2-mediated silencing and shortened life span ( Bitterman et al., 2002; Gallo et al., 2004). Also, CR has been suggested to activate Sir2 by decreasing Nam accumulation ( Bitterman et al., 2002). Interestingly, the NAD⁺ intermediate, NMN, could also inhibit Sir2 ( Schmidt et al., 2004), therefore if NAD⁺ synthesis is not required, cells may maintain low levels of NMN (see the subsection “Additional roles of NAD⁺ intermediates and biosynthetic enzymes”). Another NAD⁺ derivative, NADH, was reported to competitively inhibit Sir2: lowering the NADH level by genetic modifications increased Sir2-mediated silencing and life span ( Lin et al., 2004). However, NADH is a weak Sir2 inhibitor, and reported cellular NADH levels appear too low to inhibit Sir2 activity in vivo ( Schmidt et al., 2004). Perhaps intracellular compartmentalization of NAD⁺, NADH and/or specific protein- protein interactions create locally high NADH levels (low NAD⁺/NADH ratio) which inhibit Sir2 in vivo, and when NADH levels are lowered, (higher NAD⁺/NADH ratio) Sir2 is activated. It has been suggested that Sir2 affinity/sensitivity toward its substrates and inhibitors varies when Sir2 is in complex with different interacting partners ( Tanny et al., 2004).

Although regulated by NAD⁺ and its derivatives, Sir2 and Hst1 also regulate NAD⁺ metabolism. It was reported that de novo NAD⁺ synthesis is repressed by Hst1 and that HST1 deletion raises the steady-state level of NAD⁺ ( Bedalov et al., 2003). Hst1 has also been shown to repress the expression of NR transporter, Nrt1 ( Li et al., 2013) and our previous studies suggested that Sir2 modulates the flux of NR salvage, since NR release increases in the sir2∆ mutant ( Lu et al., 2009). Perhaps the Sir2 family preserves the NAD⁺ pool by producing Nam in deacetylation reactions, thus supporting NA/Nam salvaging (Fig. 2) ( Anderson et al., 2002) and decreasing flux into the NR branch. Indeed, deleting SIR2 further decreased the NAD⁺ pool in cells lacking NR assimilation activity ( Lu et al., 2009). Still, the mechanisms remain unclear and we await future studies determining the molecular and biochemical roles of the Sir2 family in NAD⁺ metabolism.

Compartmentalization of NAD⁺ metabolism

In yeast, NAD⁺ synthesis occurs mainly in the cytosol and it is unclear how NAD⁺ and its derivatives are transported into or from various intracellular compartments, such as the mitochondria and vacuole. This section discusses how compartmentalization of NAD⁺ metabolism can regulate NAD⁺ homeostasis and cellular functions.

Mitochondrial compartmentalization of NAD⁺(H)

In yeast, it is unclear how NAD⁺ and its derivatives are transported into the mitochondria. When mitochondrial activity is high, NAD⁺ is required for many of its reactions, but the mitochondrial inner membrane is not permeable to NAD⁺ or NADH. NAD⁺ may be transported into the mitochondria by the NAD⁺ carrier proteins, Ndt1 and Ndt2 ( Todisco et al., 2006), and other NAD⁺ intermediate transporters may also exist. Our knowledge of mitochondrial NAD⁺(H) homeostasis derives mostly from studying the NADH shuttle systems: groups of enzymes and carriers that move small or permeable redox equivalents of NAD⁺ and NADH across the mitochondrial membrane to balance the NAD⁺/NADH ratio between the mitochondrial and the cytosolic/nuclear pools ( Bakker et al., 2001; Houtkooper et al., 2010). We previously showed that the CR-mediated respiration-induced increase in the mitochondrial NAD⁺/NADH ratio (see the subsection “Sir2 and NAD⁺ metabolism” for NAD⁺/NADH ratio) can be transmitted to the cytosol by the malate-aspartate shuttle ( Easlon et al., 2008). This balancing of NAD⁺/NADH ratio between mitochondrial and cytosolic pools was required for CR, since deleting NADH shuttle components abolished CR-mediated life span extension ( Easlon et al., 2008). Another pathway that requires NADH shuttles for life span extension is SPS signaling. In this system, a different NADH shuttle, the malate-pyruvate shuttle, plays a key role in reduced SPS-induced life span extension, (whereas the malate-aspartate shuttle is required for CR) ( Easlon et al., 2008). Interestingly, although CR increases NAD⁺/NADH ratio ( Easlon et al., 2008), reduced SPS pathway does not. Although detailed molecular mechanisms remain uncharacterized, these studies suggest that increased flux between the mitochondrial and cytoplasmic NAD⁺(H) pools due to NADH shuttles may confer metabolic flexibility to support various life span extending changes ( Tsang et al., 2015). In support, rapid transmission of NAD⁺/NADH may generally be important since overexpressing NADH shuttle components alone extended life span ( Easlon et al., 2008). Also in mammals, impairments of mitochondrial metabolism and NADH shuttles have been implicated in age-associated diseases such as diabetes ( Eto et al., 1999; Haigis et al., 2006). In summary, NADH shuttles play important roles in mitochondrial metabolism and metabolic fitness by regulating NAD⁺ homeostasis. Identifying additional mitochondrial NAD⁺ homeostasis factors will provide insight into the role of mitochondrial compartmentalization in NAD⁺ homeostasis.

Vacuolar and cytosolic compartmentalization of NMN and NR

The vacuole plays an important role in NR and NAD⁺ homeostasis. Vacuolar and mitochondrial function are also linked. We previously showed the vacuole is not only a major source of NR production, but also an intracellular NR storage compartment ( Lu et al., 2011). There are two major intracellular pools of NR in yeast: vacuolar pool and cytosolic pool. Released NR level in the growth medium reflects the dynamic cytosolic NR pool and deleting cytosolic ISN1 and SDT1 (nucleotidases that produce NR) greatly decreases NR release ( Bogan et al., 2009; Lu et al., 2011; Kato and Lin, 2014b). Whereas intracellular NR level reflects the stored (vacuolar) pool and deleting vacuolar PHO8 largely decreases intracellular NR level ( Bogan et al., 2009; Lu et al., 2011; Kato and Lin, 2014b). Pho8 produces NR from NMN ( Lu et al., 2011), requires a vacuolar peptidase for activation ( Noda and Klionsky, 2008), and is canonically activated by the PHO pathway. However, we recently showed PHO-independent activation of Pho8 in mutants with reduced SPS signaling activity ( Tsang et al., 2015). Supporting a critical role for the vacuole in NR homeostasis, our previous genetic screens identified 31 mutants which had increased NR release that were deficient in either vacuolar ATP synthase (v-ATPase) assembly ( Lu et al., 2011), (maintains vacuolar matrix acidity) or potentially related vesicle-mediated transport. Thus the vacuole and vesicle-mediated transport may play a role in NR metabolism and homeostasis. Previously, the v-ATPase was also shown to play essential roles in preserving mitochondrial function in yeast ( Hughes and Gottschling, 2012). Overexpression of v-ATPase proteins preserved vacuolar acidity and amino acid import, and extended RLS ( Hughes and Gottschling, 2012). This RLS was suggested to extend by preserving vacuolar function, allowing proper transport of metabolites between the cytoplasm and vacuole and increasing metabolic flexibility since compromised vacuolar function led to mitochondrial fragmentation and malfunction ( Hughes and Gottschling, 2012). Another pathway that links the importance of vacuolar and mitochondrial function to NR/NAD⁺ homeostasis and life span extension is amino acid sensing SPS signaling ( Tsang et al., 2015). (See the subsection “Other nutrient sensing pathways and NAD⁺ metabolism”) Thus vacuolar and mitochondrial function are also linked and proper metabolism within one compartment may affect the other.

If the vacuole is a major NR production and storage compartment, how is vacuolar NR converted to NAD⁺ in the cytosol? We have previously identified Fun26, as a putative NR transporter on the vacuolar membrane ( Vickers et al., 2000; Wiederhold et al., 2009; Lu et al., 2011). Recently Boswell-Casteel et al. showed that Fun26 is a high affinity transporter with broad range nucleoside and nucleobase substrate affinity, potentially including the nucleoside, NR ( Boswell-Casteel et al., 2014). Fun26 is a yeast homolog of the human equilibrative nucleoside transporter (hENT) family, which mediates bi-directional transport of specific nucleosides ( Baldwin et al., 2005; Endo et al., 2007; Young et al., 2008). In yeast, the Fun26 protein was reported to have a minor role in NR transport relative to plasma membrane NR transporter, Nrt1 ( Belenky et al., 2008). However, deleting FUN26 causes a similarly significant increase in NR release as that of nrt1∆ mutant, and unlike the nrt1∆ mutant, the fun26∆ mutant also had increased intracellular NR accumulation ( Lu et al., 2011). Therefore the NR balancing function of Fun26 may be key to cytosolic NAD⁺ synthesis from the NR pool produced and stored in the vacuole. Currently in higher eukaryotes, cellular NR uptake and transport mechanisms are still unknown, however there are four hENT transporters related to Fun26 ( Young et al., 2008). Since yeast Fun26 balances NR pools, perhaps plasma membrane, hENT1 and hENT2 and lysosomal membrane, hENT3, also participate in NR homeostasis and NAD⁺ metabolism in humans. Another potential NAD⁺ homeostasis compartment is the peroxisome: peroxisomal Nudix hydrolase, Npy1, a NADH pyrophosphatase was shown to produce NMN(H) from NADH ( AbdelRaheim et al., 2001) and contribute to NAD⁺ homeo-stasis ( Kato and Lin, 2014a). Perhaps Npy1 produces NMN(H) as a storage form of NAD⁺ that can be quickly salvaged via NR salvaging (see the subsection “Overview of NAD⁺ biosynthesis”). Future studies on novel NAD⁺ homeostasis factors will further our understanding of how compartmentalization regulates NAD⁺ metabolism and cell functions.

Signaling pathways that regulate and/or cross-talk with NAD⁺ metabolism

We previously introduced the PHO, SPS, and PKA pathways and NAD⁺ metabolism. In this section we will discuss regulation of or cross-talk with NAD⁺ metabolism by these and other pathways (Fig. 3).

The PHO pathway and NAD⁺ metabolism

Our previous studies linked the regulation of NAD⁺ metabolism and phosphate signaling ( Lu et al., 2011). In S. cerevisiae, phosphate homeostasis is regulated by the PHO pathway, which monitors and responds to changes in Pi availability ( Lenburg and O’Shea, 1996; Auesukaree et al., 2004) (see the section “Phosphate sensing pathway”). We previously reported that NR production positively correlates with PHO activation ( Lu et al., 2011): intracellular NR increased under active PHO pathway (i.e., low Pi growth media or deletion of PHO84), and decreased in cells defective in PHO signaling (i.e., pho4∆ mutant). Increased NR production under high PHO activity is mostly due to Pho8, since deletion of PHO8 gene largely decreases intracellular NR level ( Lu et al., 2011). Our studies also demonstrated that uptake of extracellular NMN requires prior conversion to NR by periplasmic Pho5, a PHO-regulated phosphatase ( Lu and Lin, 2010). The cytosolic nucleotidase, Sdt1, which also produces NR from NMN ( Bogan et al., 2009), has been suggested to also be under PHO regulation ( Xu et al., 2013). Altogether, these studies provide evidence for PHO regulation of NR homeostasis and NAD⁺ metabolism in yeast. Yet in mammals, the method by which cells uptake NAD⁺ precursors remains unclear. Mammalian cells do uptake some forms of NAD⁺, since supplementing NAD⁺ precursors and intermediates was shown to replenish NAD⁺ level and ameliorate defects from abnormal NAD⁺ metabolism in mammalian models ( Sasaki et al., 2006; Bogan and Brenner, 2008; Ramsey et al., 2008; Imai and Guarente, 2014). In addition, NR has been suggested to circulate in the peripheral bloodstream in mammals ( Schmidt-Brauns et al., 2001). Perhaps in mammals, large impermeable NAD⁺ precursors are converted to NR and other smaller NAD⁺ intermediates prior to cell assimilation. To date, phosphate signaling in mammalian cells also remains unclear; and, perhaps NAD⁺ metabolism is also linked to phosphate signaling in higher eukaryotes.

In addition to regulating NAD⁺ metabolism, PHO pathway perhaps responds to rate of NAD⁺ metabolism since decreased intracellular NaMN level may activate PHO pathway ( Lu et al., 2011) (see the subsection “Additional roles of NAD⁺ intermediates and biosynthetic enzymes”). The cross-regulation of PHO signaling and NAD⁺ metabolism is unclear, but coupling these pathways may be metabolically advantageous in certain conditions. For example, the Pi moiety of various NAD⁺ derivatives are potential targets for Pi scavenging during Pi limitation. Moreover, as a key intermediate for NAD⁺ biosynthesis, low level NaMN might signal impaired NAD⁺ biosynthesis and activate alternative NAD⁺ salvage routes. Since NR-mediated NAD⁺ synthesis requires Pi (in the form of ATP), coordinated activation of the PHO pathway is expected to support NAD⁺ synthesis and homeostasis.

Other nutrient sensing pathways and NAD⁺ metabolism

In the previous section, we discussed the PHO pathway and NAD⁺ metabolism. Here we will discuss additional nutrient sensing pathways that may regulate and/or cross-talk with NAD⁺ metabolism: the SPS, PKA, TOR and Sch9 pathways. In yeast, mutations that reduce SPS amino acid sensing pathway extend life span via increasing NADH shuttle activity and NR homeostasis, both of which contribute to NAD⁺ homeostasis. Components of the mitochondrial malate-pyruvate NADH shuttle play an important role in this life span extension ( Tsang et al., 2015) (see the subsection “Mitochondrial compartmentalization of NAD⁺(H) ” for NADH shuttle). Expression of the malate-pyruvate NADH shuttle components is increased in a low SPS activity mutant, ssy5∆, and the SPS transcription factor, Stp1, binds to the promoters of shuttle components, MAE1 and OAC1. Moreover, deleting MAE1 and OAC1 significantly reduced ssy5∆-induced life span extension. NAD⁺ homeostasis of ssy5∆ is also enhanced by increasing NR salvaging. Deletion of SSY5 increases NR levels, which partially restores the NAD⁺ pool and life span of the short-lived and NAD⁺-depleted npt1∆ mutant. The main source of increased NR in ssy5∆ cells is Pho8, which is required for full life span extension by ssy5∆. Interestingly, ssy5∆-induced PHO8 expression is independent of the canonical PHO pathway and PHO8 expression likely involves yet-to-be identified transcription factors since Stp1 does not appear to directly bind to PHO8 promoter ( Tsang et al., 2015). Overall, these studies suggest SPS signaling is a regulator of NAD⁺ homeostasis. Enhanced NAD⁺ homeostasis due to reduced SPS signaling may concomitantly increase mitochondrial and vacuolar function, all contributing to life span extension.

Other pathways that may play a role in NAD⁺ metabolism include PKA, Sch9, and TOR. In the section “Phosphate sensing pathway”, we discussed the interaction of PKA with PHO signaling. Interestingly, all these pathways may converge on Rim15 to regulate stress response transcription factors, Msn2 and Msn4 ( Carroll et al., 2001; Swinnen et al., 2006; Wei et al., 2008). Msn2 and Msn4 have been shown to regulate expression of the NA/NAM salvaging enzyme, PNC1 ( Medvedik et al., 2007). Various mild stresses were observed to increase PNC1 expression: glucose or amino acid restriction, heat or salt stresses, and mRNA mistranslation ( Anderson et al., 2003). Given that mild stress can mediate life span extension ( Shama et al., 1998; Kaeberlein et al., 2002; Anderson et al., 2003; Li et al., 2011) Pnc1 may be a key factor connecting stress signals to the regulation of Sir2 ( Lamming et al., 2004) and NAD⁺ metabolism. Interestingly, Nampt in higher eukaryotes, is also responsive to stresses and may also connect stress to NAD⁺ metabolism ( Jia et al., 2004; van der Veer et al., 2005). Complex cross-talk between these pathways may allow cells to quickly adapt to environmental changes.

Conclusions and perspectives

Integral to all living organisms, a multitude of nutrient sensing mechanisms allows fine control of cell activity. The budding yeast, Saccharomyces cerevisiae, has been used to study nutrient sensing pathways, their cross-talk and cross-regulation. Factors that regulate nutrient sensing can also modulate longevity. Reduced activity of nutrient sensing pathways such as PKA, TOR and Sch9 have been linked to increased life span in yeast and higher eukaryotes ( Lin et al., 2000; Fabrizio et al., 2001; Kaeberlein et al., 2005b; Wei et al., 2008; Wang et al., 2009; Longo and Fabrizio, 2012). Recently in yeast, reduced activity of amino acid sensing SPS pathway was also linked to increased life span ( Tsang et al., 2015). Metabolic factors such as NAD⁺ metabolism have also been proposed to play an important role in yeast life span extension.

Pyridine nucleotides are in a myriad of cellular processes and therefore, it is essential to maintain NAD⁺ homeostasis: aberrant NAD⁺ metabolism has been implicated in numerous metabolic and age-associated diseases ( Imai and Guarente, 2014). Multiple NAD⁺ synthesis pathways suggest that NAD⁺ metabolism is dynamic and flexible, ensuring cells can adapt to many environmental changes. And due to this complex nature, factors regulating NAD⁺ metabolism and homeostasis are not completely understood, although recent studies have identified novel regulators of NAD⁺ homeostasis. For example, the mitochondria and vacuole were shown to play important roles in the biosynthesis and metabolism of pyridine nucleotides such as NAD⁺(H) and NADP⁺(H) ( Sies 1982; Bakker et al., 2001; Kato and Lin, 2014a; Lewis et al., 2014). And, generally compartmentalization may be important for cellular control: NAD⁺ intermediates can be signaling molecules, and high intracellular concentrations can inhibit certain cellular processes. Recent studies support that compartmentalization of pyridine metabolites is pivotal for regulating NAD⁺ homeostasis and balancing their concentrations is critical for the regulation of many cellular processes. Future studies to understand the multiple roles of NAD⁺, NAD⁺ intermediates, as well as novel factors regulating these processes are highly anticipated.

To maintain adequate levels of pyridine nucleotides, changes in NAD⁺ precursor levels must be sensed. NAD⁺ metabolism is likely tightly regulated in concert with other nutrient sensing pathways. Recently, NAD⁺ metabolism was linked to the PHO and SPS pathways in yeast. PHO pathway activation increases NR production and mobilization ( Lu et al., 2011). To extend life span, reduced SPS signaling required increased NAD⁺ and NR homeostasis ( Tsang et al., 2015). Also, cross-regulation of PHO, PKA, TOR and Sch9 pathways has been reported to potentially affect NAD⁺ metabolism ( Kato and Lin, 2014a); however, detailed mechanisms remain unclear. Most likely, the complex interplay between nutrient signaling pathways confers essential metabolic flexibility in different growth conditions. Future studies to identify additional signaling factors that regulate cross-talk between NAD⁺ homeostasis, longevity regulating, and nutrient signaling pathways are highly anticipated. Overall, these studies contribute to our understanding of how nutrient signaling pathways cross-talk with and regulate NAD⁺ homeostasis pathways, and may also provide further insight into the molecular mechanisms of diseases related to defects in NAD⁺ metabolism.

References

[1] Abdel-Sater F, Jean C, Merhi A, Vissers S, André B (2011). Amino acid signaling in yeast: activation of Ssy5 protease is associated with its phosphorylation-induced ubiquitylation. J Biol Chem, 286(14): 12006–12015

[2] AbdelRaheim S R, Cartwright J L, Gasmi L, McLennan A G (2001). The NADH diphosphatase encoded by the Saccharomyces cerevisiae NPY1 nudix hydrolase gene is located in peroxisomes. Arch Biochem Biophys, 388(1): 18–24

[3] Andersen M P, Nelson Z W, Hetrick E D, Gottschling D E (2008). A genetic screen for increased loss of heterozygosity in Saccharomyces cerevisiae. Genetics, 179(3): 1179–1195

[4] Anderson R M, Bitterman K J, Wood J G, Medvedik O, Cohen H, Lin S S, Manchester J K, Gordon J I, Sinclair D A (2002). Manipulation of a nuclear NAD⁺ salvage pathway delays aging without altering steady-state NAD⁺ levels. J Biol Chem, 277(21): 18881–18890

[5] Anderson R M, Bitterman K J, Wood J G, Medvedik O, Sinclair D A (2003). Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature, 423(6936): 181–185

[6] Andréasson C, Heessen S, Ljungdahl P O (2006). Regulation of transcription factor latency by receptor-activated proteolysis. Genes Dev, 20(12): 1563–1568

[7] Ashrafi K, Lin S S, Manchester J K, Gordon J I (2000). Sip2p and its partner snf1p kinase affect aging in S. cerevisiae. Genes Dev, 14(15): 1872–1885

[8] Auesukaree C, Homma T, Tochio H, Shirakawa M, Kaneko Y, Harashima S (2004). Intracellular phosphate serves as a signal for the regulation of the PHO pathway in Saccharomyces cerevisiae. J Biol Chem, 279(17): 17289–17294

[9] Auesukaree C, Tochio H, Shirakawa M, Kaneko Y, Harashima S (2005). Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae. J Biol Chem, 280(26): 25127–25133

[10] Bakker B M, Overkamp K M, Kötter P, Luttik M A, Pronk J T, van Dijken J P, Pronk J T, and the van Maris AJ, and the van Dijken J P (2001). Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol Rev, 25(1): 15–37

[11] Baldwin S A, Yao S Y, Hyde R J, Ng A M, Foppolo S, Barnes K, Ritzel M W, Cass C E, Young J D (2005). Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes. J Biol Chem, 280(16): 15880–15887

[12] Barros M H, Bandy B, Tahara E B, Kowaltowski A J (2004). Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J Biol Chem, 279(48): 49883–49888

[13] Beck T, Hall M N (1999). The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature, 402(6762): 689–692

[14] Bedalov A, Hirao M, Posakony J, Nelson M, Simon J A (2003). NAD⁺-dependent deacetylase Hst1p controls biosynthesis and cellular NAD⁺ levels in Saccharomyces cerevisiae. Mol Cell Biol, 23(19): 7044–7054

[15] Belenky P, Racette F G, Bogan K L, McClure J M, Smith J S, Brenner C (2007). Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD⁺. Cell, 129(3): 473–484

[16] Belenky P A, Moga T G, Brenner C (2008). Saccharomyces cerevisiae YOR071C encodes the high affinity nicotinamide riboside transporter Nrt1. J Biol Chem, 283(13): 8075–8079

[17] Bender D A (1983). Biochemistry of tryptophan in health and disease. Mol Aspects Med, 6(2): 101–197

[18] Bieganowski P, Brenner C (2004). Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD⁺ in fungi and humans. Cell, 117(4): 495–502

[19] Bieganowski P, Pace H C, Brenner C (2003). Eukaryotic NAD⁺ synthetase Qns1 contains an essential, obligate intramolecular thiol glutamine amidotransferase domain related to nitrilase. J Biol Chem, 278(35): 33049–33055

[20] Biliński T, Bartosz G (2006). Hypothesis: cell volume limits cell divisions. Acta Biochim Pol, 53(4): 833–835

[21] Biliński T, Zadrąg-Tęcza R, Bartosz G (2012). Hypertrophy hypothesis as an alternative explanation of the phenomenon of replicative aging of yeast. FEMS Yeast Res, 12(1): 97–101

[22] Binda M, Péli-Gulli M P, Bonfils G, Panchaud N, Urban J, Sturgill T W, Loewith R, De Virgilio C (2009). The Vam6 GEF controls TORC1 by activating the EGO complex. Mol Cell, 35(5): 563–573

[23] Bitterman K J, Anderson R M, Cohen H Y, Latorre-Esteves M, Sinclair D A (2002). Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1. J Biol Chem, 277(47): 45099–45107

[24] Blinder D, Coschigano P W, Magasanik B (1996). Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae. J Bacteriol, 178(15): 4734–4736

[25] Bogan K L, Brenner C (2008). Nicotinic acid, nicotinamide, and nicotinamide riboside: a molecular evaluation of NAD⁺ precursor vitamins in human nutrition. Annu Rev Nutr, 28(1): 115–130

[26] Bogan K L, Evans C, Belenky P, Song P, Burant C F, Kennedy R, Brenner C (2009). Identification of Isn1 and Sdt1 as glucose- and vitamin-regulated nicotinamide mononucleotide and nicotinic acid mononucleotide [corrected] 5′-nucleotidases responsible for production of nicotinamide riboside and nicotinic acid riboside. J Biol Chem, 284(50): 34861–34869

[27] Bonawitz N D, Chatenay-Lapointe M, Pan Y, Shadel G S (2007). Reduced TOR signaling extends chronological life span via increased respiration and upregulation of mitochondrial gene expression. Cell Metab, 5(4): 265–277

[28] Boswell-Casteel R C, Johnson J M, Duggan K D, Roe-Žurž Z, Schmitz H, Burleson C, Hays F A (2014). FUN26 (function unknown now 26) protein from Saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter. J Biol Chem, 289(35): 24440–24451

[29] Brachmann C B, Sherman J M, Devine S E, Cameron E E, Pillus L, Boeke J D (1995). The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability. Genes Dev, 9(23): 2888–2902

[30] Broach J R (2012). Nutritional control of growth and development in yeast. Genetics, 192(1): 73–105

[31] Bun-Ya M, Nishimura M, Harashima S, Oshima Y (1991). The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol Cell Biol, 11(6): 3229–3238

[32] Burtner C R, Murakami C J, Kennedy B K, Kaeberlein M (2009). A molecular mechanism of chronological aging in yeast. Cell Cycle, 8(8): 1256–1270

[33] Carroll A S, Bishop A C, DeRisi J L, Shokat K M, O’Shea E K (2001). Chemical inhibition of the Pho85 cyclin-dependent kinase reveals a role in the environmental stress response. Proc Natl Acad Sci USA, 98(22): 12578–12583

[34] Casamayor A, Torrance P D, Kobayashi T, Thorner J, Alessi D R (1999). Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast. Curr Biol, 9(4): 186–197

[35] Celenza J L, Carlson M (1986). A yeast gene that is essential for release from glucose repression encodes a protein kinase. Science, 233(4769): 1175–1180

[36] Celic I, Masumoto H, Griffith W P, Meluh P, Cotter R J, Boeke J D, Verreault A (2006). The sirtuins hst3 and Hst4p preserve genome integrity by controlling histone h3 lysine 56 deacetylation. Curr Biol, 16(13): 1280–1289

[37] Chandrashekarappa D G, McCartney R R, Schmidt M C (2013). Ligand binding to the AMP-activated protein kinase active site mediates protection of the activation loop from dephosphorylation. J Biol Chem, 288(1): 89–98

[38] Cheng W, Roth J (1995). Isolation of NAD cycle mutants defective in nicotinamide mononucleotide deamidase in Salmonella typhimurium. J Bacteriol, 177(23): 6711–6717

[39] Cherkasova V A, Hinnebusch A G (2003). Translational control by TOR and TAP42 through dephosphorylation of eIF2alpha kinase GCN2. Genes Dev, 17(7): 859–872

[40] Chodosh L A, Olesen J, Hahn S, Baldwin A S, Guarente L, Sharp P A (1988). A yeast and a human CCAAT-binding protein have heterologous subunits that are functionally interchangeable. Cell, 53(1): 25–35

[41] Choi K M, Kwon Y Y, Lee C K (2015). Disruption of Snf3/Rgt2 glucose sensors decreases lifespan and caloric restriction effectiveness through Mth1/Std1 by adjusting mitochondrial efficiency in yeast. FEBS Lett, 589(3): 349–357

[42] Clapper D L, Walseth T F, Dargie P J, Lee H C (1987). Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. J Biol Chem, 262(20): 9561–9568

[43] Conrad M, Schothorst J, Kankipati H N, Van Zeebroeck G, Rubio-Texeira M, Thevelein J M (2014). Nutrient sensing and signaling in the yeast Saccharomyces cerevisiae. FEMS Microbiol Rev, 38(2): 254–299

[44] De Wever V, Reiter W, Ballarini A, Ammerer G, Brocard C (2005). A dual role for PP1 in shaping the Msn2-dependent transcriptional response to glucose starvation. EMBO J, 24(23): 4115–4123

[45] Delaney J R, Ahmed U, Chou A, Sim S, Carr D, Murakami C J, Schleit J, Sutphin G L, An E H, Castanza A, Fletcher M, Higgins S, Jelic M, Klum S, Muller B, Peng Z J, Rai D, Ros V, Singh M, Wende H V, Kennedy B K, Kaeberlein M (2013). Stress profiling of longevity mutants identifies Afg3 as a mitochondrial determinant of cytoplasmic mRNA translation and aging. Aging Cell, 12(1): 156–166

[46] DeRisi J L, Iyer V R, Brown P O (1997). Exploring the metabolic and genetic control of gene expression on a genomic scale. Science, 278(5338): 680–686

[47] Dever T E, Hinnebusch A G (2005). GCN2 whets the appetite for amino acids. Mol Cell, 18(2): 141–142

[48] Dilova I, Aronova S, Chen J C, Powers T (2004). Tor signaling and nutrient-based signals converge on Mks1p phosphorylation to regulate expression of Rtg1.Rtg3p-dependent target genes. J Biol Chem, 279(45): 46527–46535

[49] Dilova I, Easlon E, Lin S J (2007). Calorie restriction and the nutrient sensing signaling pathways. Cell Mol Life Sci, 64(6): 752–767

[50] Dohlman H G, Thorner J W (2001). Regulation of G protein-initiated signal transduction in yeast: paradigms and principles. Annu Rev Biochem, 70(1): 703–754

[51] Dong J, Qiu H, Garcia-Barrio M, Anderson J, Hinnebusch A G (2000). Uncharged tRNA activates GCN2 by displacing the protein kinase moiety from a bipartite tRNA-binding domain. Mol Cell, 6(2): 269–279

[52] Dubouloz F, Deloche O, Wanke V, Cameroni E, De Virgilio C (2005). The TOR and EGO protein complexes orchestrate microautophagy in yeast. Mol Cell, 19(1): 15–26

[53] Easlon E, Tsang F, Dilova I, Wang C, Lu S P, Skinner C, Lin S J (2007). The dihydrolipoamide acetyltransferase is a novel metabolic longevity factor and is required for calorie restriction-mediated life span extension. J Biol Chem, 282(9): 6161–6171

[54] Easlon E, Tsang F, Skinner C, Wang C, Lin S J (2008). The malate-aspartate NADH shuttle components are novel metabolic longevity regulators required for calorie restriction-mediated life span extension in yeast. Genes Dev, 22(7): 931–944

[55] Efeyan A, Zoncu R, Sabatini D M (2012). Amino acids and mTORC1: from lysosomes to disease. Trends Mol Med, 18(9): 524–533

[56] Emanuelli M, Amici A, Carnevali F, Pierella F, Raffaelli N, Magni G (2003). Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae. Protein Expr Purif, 27(2): 357–364

[57] Emanuelli M, Carnevali F, Lorenzi M, Raffaelli N, Amici A, Ruggieri S, Magni G (1999). Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme. FEBS Lett, 455(1–2): 13–17

[58] Endo Y, Obata T, Murata D, Ito M, Sakamoto K, Fukushima M, Yamasaki Y, Yamada Y, Natsume N, Sasaki T (2007). Cellular localization and functional characterization of the equilibrative nucleoside transporters of antitumor nucleosides. Cancer Sci, 98(10): 1633–1637

[59] Erjavec N, Bayot A, Gareil M, Camougrand N, Nystrom T, Friguet B, Bulteau A L (2013). Deletion of the mitochondrial Pim1/Lon protease in yeast results in accelerated aging and impairment of the proteasome. Free Radic Biol Med, 56: 9–16

[60] Erjavec N, Cvijovic M, Klipp E, Nyström T (2008). Selective benefits of damage partitioning in unicellular systems and its effects on aging. Proc Natl Acad Sci USA, 105(48): 18764–18769

[61] Erjavec N, Larsson L, Grantham J, Nyström T (2007). Accelerated aging and failure to segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p. Genes Dev, 21(19): 2410–2421

[62] Erjavec N, Nyström T (2007). Sir2p-dependent protein segregation gives rise to a superior reactive oxygen species management in the progeny of Saccharomyces cerevisiae. Proc Natl Acad Sci USA, 104(26): 10877–10881

[63] Eto K, Tsubamoto Y, Terauchi Y, Sugiyama T, Kishimoto T, Takahashi N, Yamauchi N, Kubota N, Murayama S, Aizawa T, Akanuma Y, Aizawa S, Kasai H, Yazaki Y, Kadowaki T (1999). Role of NADH shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion. Science, 283(5404): 981–985

[64] Evans C, Bogan K L, Song P, Burant C F, Kennedy R T, Brenner C (2010). NAD⁺ metabolite levels as a function of vitamins and calorie restriction: evidence for different mechanisms of longevity. BMC Chem Biol, 10(1): 2

[65] Fabrizio P, Gattazzo C, Battistella L, Wei M, Cheng C, McGrew K, Longo V D (2005). Sir2 blocks extreme life-span extension. Cell, 123(4): 655–667

[66] Fabrizio P, Hoon S, Shamalnasab M, Galbani A, Wei M, Giaever G, Nislow C, Longo V D (2010). Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation. PLoS Genet, 6(7): e1001024

[67] Fabrizio P, Longo V D (2003). The chronological life span of Saccharomyces cerevisiae. Aging Cell, 2(2): 73–81

[68] Fabrizio P, Longo V D (2007). The chronological life span of Saccharomyces cerevisiae. Methods Mol Biol, 371: 89–95

[69] Fabrizio P, Pozza F, Pletcher S D, Gendron C M, Longo V D (2001). Regulation of longevity and stress resistance by Sch9 in yeast. Science, 292(5515): 288–290

[70] Flick K M, Spielewoy N, Kalashnikova T I, Guaderrama M, Zhu Q, Chang H C, Wittenberg C (2003). Grr1-dependent inactivation of Mth1 mediates glucose-induced dissociation of Rgt1 from HXT gene promoters. Mol Biol Cell, 14(8): 3230–3241

[71] Foresti O, Rodriguez-Vaello V, Funaya C, Carvalho P (2014). Quality control of inner nuclear membrane proteins by the Asi complex. Science, 346(6210): 751–755

[72] Forsburg S L, Guarente L (1989). Identification and characterization of HAP4: a third component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev, 3(8): 1166–1178

[73] Frye R A (2000). Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins. Biochem Biophys Res Commun, 273(2): 793–798

[74] Gallo C M, Smith D L Jr, Smith J S (2004). Nicotinamide clearance by Pnc1 directly regulates Sir2-mediated silencing and longevity. Mol Cell Biol, 24(3): 1301–1312

[75] Gancedo J M (1998). Yeast carbon catabolite repression. Microbiol Mol Biol Rev, 62(2): 334–361

[76] Garavaglia S, D’Angelo I, Emanuelli M, Carnevali F, Pierella F, Magni G, Rizzi M (2002). Structure of human NMN adenylyltransferase. A key nuclear enzyme for NAD homeostasis. J Biol Chem, 277(10): 8524–8530

[77] Gauthier S, Coulpier F, Jourdren L, Merle M, Beck S, Konrad M, Daignan-Fornier B, Pinson B (2008). Co-regulation of yeast purine and phosphate pathways in response to adenylic nucleotide variations. Mol Microbiol, 68(6): 1583–1594

[78] Ghislain M, Talla E, François J M (2002). Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1. Yeast, 19(3): 215–224

[79] Giots F, Donaton M C, Thevelein J M (2003). Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase A pathway in the yeast Saccharomyces cerevisiae. Mol Microbiol, 47(4): 1163–1181

[80] Godard P, Urrestarazu A, Vissers S, Kontos K, Bontempi G, van Helden J, André B (2007). Effect of 21 different nitrogen sources on global gene expression in the yeast Saccharomyces cerevisiae. Mol Cell Biol, 27(8): 3065–3086

[81] Görner W, Durchschlag E, Martinez-Pastor M T, Estruch F, Ammerer G, Hamilton B, Ruis H, Schüller C (1998). Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev, 12(4): 586–597

[82] Görner W, Durchschlag E, Wolf J, Brown E L, Ammerer G, Ruis H, Schüller C (2002). Acute glucose starvation activates the nuclear localization signal of a stress-specific yeast transcription factor. EMBO J, 21(1–2): 135–144

[83] Graeff R, Liu Q, Kriksunov I A, Hao Q, Lee H C (2006). Acidic residues at the active sites of CD38 and ADP-ribosyl cyclase determine nicotinic acid adenine dinucleotide phosphate (NAADP) synthesis and hydrolysis activities. J Biol Chem, 281(39): 28951–28957

[84] Grose J H, Bergthorsson U, Roth J R (2005). Regulation of NAD synthesis by the trifunctional NadR protein of Salmonella enterica. J Bacteriol, 187(8): 2774–2782

[85] Guarente L (2013). Introduction: sirtuins in aging and diseases. Methods Mol Biol, 1077: 3–10

[86] Guse A H, Lee H C (2008). NAADP: a universal Ca²⁺ trigger. Sci Signal, 1(44): re10

[87] Hachinohe M, Hanaoka F, Masumoto H (2011). Hst3 and Hst4 histone deacetylases regulate replicative lifespan by preventing genome instability in Saccharomyces cerevisiae. Genes Cells, 16(4): 467–477

[88] Hachinohe M, Yamane M, Akazawa D, Ohsawa K, Ohno M, Terashita Y, Masumoto H (2013). A reduction in age-enhanced gluconeogenesis extends lifespan. PLoS ONE, 8(1): e54011

[89] Hahn J S, Thiele D J (2004). Activation of the Saccharomyces cerevisiae heat shock transcription factor under glucose starvation conditions by Snf1 protein kinase. J Biol Chem, 279(7): 5169–5176

[90] Hahn S, Guarente L (1988). Yeast HAP2 and HAP3: transcriptional activators in a heteromeric complex. Science, 240(4850): 317–321

[91] Hahn S, Young E T (2011). Transcriptional regulation in Saccharomyces cerevisiae: transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators. Genetics, 189(3): 705–736

[92] Haigis M C, Mostoslavsky R, Haigis K M, Fahie K, Christodoulou D C, Murphy A J, Valenzuela D M, Yancopoulos G D, Karow M, Blander G, Wolberger C, Prolla T A, Weindruch R, Alt F W, Guarente L (2006). SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells. Cell, 126(5): 941–954

[93] Halme A, Bumgarner S, Styles C, Fink G R (2004). Genetic and epigenetic regulation of the FLO gene family generates cell-surface variation in yeast. Cell, 116(3): 405–415

[94] Hardie D G (2007). AMP-activated/SNF1 protein kinases: conserved guardians of cellular energy. Nat Rev Mol Cell Biol, 8(10): 774–785

[95] Hecht A, Strahl-Bolsinger S, Grunstein M (1996). Spreading of transcriptional repressor SIR3 from telomeric heterochromatin. Nature, 383(6595): 92–96

[96] Hernández H, Aranda C, López G, Riego L, González A (2011). Hap2-3-5-Gln3 determine transcriptional activation of GDH1 and ASN1 under repressive nitrogen conditions in the yeast Saccharomyces cerevisiae. Microbiology, 157(Pt 3): 879–889

[97] Hinnebusch A G (2005). Translational regulation of GCN4 and the general amino acid control of yeast. Annu Rev Microbiol, 59(1): 407–450

[98] Hinnebusch A G, Natarajan K (2002). Gcn4p, a master regulator of gene expression, is controlled at multiple levels by diverse signals of starvation and stress. Eukaryot Cell, 1(1): 22–32

[99] Hong S P, Leiper F C, Woods A, Carling D, Carlson M (2003). Activation of yeast Snf1 and mammalian AMP-activated protein kinase by upstream kinases. Proc Natl Acad Sci USA, 100(15): 8839–8843

[100] Houtkooper R H, Cantó C, Wanders R J, Auwerx J (2010). The secret life of NAD⁺: an old metabolite controlling new metabolic signaling pathways. Endocr Rev, 31(2): 194–223

[101] Hughes A L, Gottschling D E (2012). An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast. Nature, 492(7428): 261–265

[102] Imai S, Armstrong C M, Kaeberlein M, Guarente L (2000). Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature, 403(6771): 795–800

[103] Imai S I, Guarente L (2014). NAD and sirtuins in aging and disease. Trends Cell Biol.

[104] Ivy J M, Klar A J, Hicks J B (1986). Cloning and characterization of four SIR genes of Saccharomyces cerevisiae. Mol Cell Biol, 6: 688–702

[105] Jacinto E, Lorberg A (2008). TOR regulation of AGC kinases in yeast and mammals. Biochem J, 410(1): 19–37

[106] Jazwinski S M (1990). An experimental system for the molecular analysis of the aging process: the budding yeast Saccharomyces cerevisiae. J Gerontol, 45(3): B68–B74

[107] Jazwinski S M (2000). Aging and longevity genes. Acta Biochim Pol, 47(2): 269–279

[108] Jia S H, Li Y, Parodo J, Kapus A, Fan L, Rotstein O D, Marshall J C (2004). Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis. J Clin Invest, 113(9): 1318–1327

[109] Jiang J C, Jaruga E, Repnevskaya M V, Jazwinski S M (2000). An intervention resembling caloric restriction prolongs life span and retards aging in yeast. FASEB J, 14(14): 2135–2137

[110] Jouandot D 2nd, Roy A, Kim J H (2011). Functional dissection of the glucose signaling pathways that regulate the yeast glucose transporter gene (HXT) repressor Rgt1. J Cell Biochem, 112(11): 3268–3275

[111] Kaeberlein M, Andalis A A, Fink G R, Guarente L (2002). High osmolarity extends life span in Saccharomyces cerevisiae by a mechanism related to calorie restriction. Mol Cell Biol, 22(22): 8056–8066

[112] Kaeberlein M, Hu D, Kerr E O, Tsuchiya M, Westman E A, Dang N, Fields S, Kennedy B K (2005a). Increased life span due to calorie restriction in respiratory-deficient yeast. PLoS Genet, 1(5): e69

[113] Kaeberlein M, Kirkland K T, Fields S, Kennedy B K (2004). Sir2-independent life span extension by calorie restriction in yeast. PLoS Biol, 2(9): E296

[114] Kaeberlein M, Powers R W 3rd, Steffen K K, Westman E A, Hu D, Dang N, Kerr E O, Kirkland K T, Fields S, Kennedy B K (2005b). Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science, 310(5751): 1193–1196

[115] Kamada Y, Fujioka Y, Suzuki N N, Inagaki F, Wullschleger S, Loewith R, Hall M N, Ohsumi Y (2005). Tor2 directly phosphorylates the AGC kinase Ypk2 to regulate actin polarization. Mol Cell Biol, 25(16): 7239–7248

[116] Kang H J, Jeong S J, Kim K N, Baek I J, Chang M, Kang C M, Park Y S, Yun C W (2014). A novel protein, Pho92, has a conserved YTH domain and regulates phosphate metabolism by decreasing the mRNA stability of PHO4 in Saccharomyces cerevisiae. Biochem J, 457(3): 391–400

[117] Kato M, Lin S J (2014a). Regulation of NAD⁺ metabolism, signaling and compartmentalization in the yeast Saccharomyces cerevisiae. DNA Repair (Amst), 23: 49–58

[118] Kato M, Lin S J (2014b). YCL047C/POF1 is a novel nicotinamide mononucleotide adenylyltransferase (NMNAT) in Saccharomyces cerevisiae. J Biol Chem, 289(22): 15577–15587

[119] Keith C T, Schreiber S L (1995). PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science, 270(5233): 50–51

[120] Kenyon C (2001). A conserved regulatory system for aging. Cell, 105(2): 165–168

[121] Kim J H, Brachet V, Moriya H, Johnston M (2006). Integration of transcriptional and posttranslational regulation in a glucose signal transduction pathway in Saccharomyces cerevisiae. Eukaryot Cell, 5(1): 167–173

[122] Kim J H, Johnston M (2006). Two glucose-sensing pathways converge on Rgt1 to regulate expression of glucose transporter genes in Saccharomyces cerevisiae. J Biol Chem, 281(36): 26144–26149

[123] Kornitzer D, Raboy B, Kulka R G, Fink G R (1994). Regulated degradation of the transcription factor Gcn4. EMBO J, 13(24): 6021–6030

[124] Kruegel U, Robison B, Dange T, Kahlert G, Delaney J R, Kotireddy S, Tsuchiya M, Tsuchiyama S, Murakami C J, Schleit J, Sutphin G, Carr D, Tar K, Dittmar G, Kaeberlein M, Kennedy B K, Schmidt M (2011). Elevated proteasome capacity extends replicative lifespan in Saccharomyces cerevisiae. PLoS Genet, 7(9): e1002253

[125] Lamming D W, Latorre-Esteves M, Medvedik O, Wong S N, Tsang F A, Wang C, Lin S J, Sinclair D A (2005). HST2 mediates SIR2-independent life-span extension by calorie restriction. Science, 309(5742): 1861–1864

[126] Lamming D W, Wood J G, Sinclair D A (2004). Small molecules that regulate lifespan: evidence for xenohormesis. Mol Microbiol, 53(4): 1003–1009

[127] Landry J, Sutton A, Tafrov S T, Heller R C, Stebbins J, Pillus L, Sternglanz R (2000). The silencing protein SIR2 and its homologs are NAD-dependent protein deacetylases. Proc Natl Acad Sci USA, 97(11): 5807–5811

[128] Lascaris R, Bussemaker H J, Boorsma A, Piper M, van der Spek H, Grivell L, Blom J (2003). Hap4p overexpression in glucose-grown Saccharomyces cerevisiae induces cells to enter a novel metabolic state. Genome Biol, 4(1): R3

[129] Lee P, Kim M S, Paik S M, Choi S H, Cho B R, Hahn J S (2013). Rim15-dependent activation of Hsf1 and Msn2/4 transcription factors by direct phosphorylation in Saccharomyces cerevisiae. FEBS Lett, 587(22): 3648–3655

[130] Lee Y S, Huang K, Quiocho F A, O’Shea E K (2008). Molecular basis of cyclin-CDK-CKI regulation by reversible binding of an inositol pyrophosphate. Nat Chem Biol, 4(1): 25–32

[131] Lee Y S, Mulugu S, York J D, O’Shea E K (2007). Regulation of a cyclin-CDK-CDK inhibitor complex by inositol pyrophosphates. Science, 316(5821): 109–112

[132] Lenburg M E, O’Shea E K (1996). Signaling phosphate starvation. Trends Biochem Sci, 21(10): 383–387

[133] Lewis C A, Parker S J, Fiske B P, McCloskey D, Gui D Y, Green C R, Vokes N I, Feist A M, Vander Heiden M G, Metallo C M (2014). Tracing compartmentalized NADPH metabolism in the cytosol and mitochondria of mammalian cells. Mol Cell, 55(2): 253–263

[134] Li B, Skinner C, Castello P R, Kato M, Easlon E, Xie L, Li T, Lu S P, Wang C, Tsang F, Poyton R O, Lin S J (2011). Identification of potential calorie restriction-mimicking yeast mutants with increased mitochondrial respiratory chain and nitric oxide levels. J Aging Res, 2011: 673185

[135] Li M, Valsakumar V, Poorey K, Bekiranov S, Smith J S (2013). Genome-wide analysis of functional sirtuin chromatin targets in yeast. Genome Biol, 14(5): R48

[136] Li P L, Zhang Y, Abais J M, Ritter J K, Zhang F (2013). Cyclic ADP-ribose and NAADP in vascular regulation and diseases. Messenger (Los Angel), 2(2): 63–85

[137] Lin S J, Defossez P A, Guarente L (2000). Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science, 289(5487): 2126–2128

[138] Lin S J, Ford E, Haigis M, Liszt G, Guarente L (2004). Calorie restriction extends yeast life span by lowering the level of NADH. Genes Dev, 18(1): 12–16

[139] Lin S J, Kaeberlein M, Andalis A A, Sturtz L A, Defossez P A, Culotta V C, Fink G R, Guarente L (2002). Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature, 418(6895): 344–348

[140] Lin S S, Manchester J K, Gordon J I (2003). Sip2, an N-myristoylated beta subunit of Snf1 kinase, regulates aging in Saccharomyces cerevisiae by affecting cellular histone kinase activity, recombination at rDNA loci, and silencing. J Biol Chem, 278(15): 13390–13397

[141] Lin Y Y, Lu J Y, Zhang J, Walter W, Dang W, Wan J, Tao S C, Qian J, Zhao Y, Boeke J D, Berger S L, Zhu H (2009). Protein acetylation microarray reveals that NuA4 controls key metabolic target regulating gluconeogenesis. Cell, 136(6): 1073–1084

[142] Liu Z, Thornton J, Spírek M, Butow R A (2008). Activation of the SPS amino acid-sensing pathway in Saccharomyces cerevisiae correlates with the phosphorylation state of a sensor component, Ptr3. Mol Cell Biol, 28(2): 551–563

[143] Ljungdahl P O (2009). Amino-acid-induced signalling via the SPS-sensing pathway in yeast. Biochem Soc Trans, 37(Pt 1): 242–247

[144] Ljungdahl P O, Daignan-Fornier B (2012). Regulation of amino acid, nucleotide, and phosphate metabolism in Saccharomyces cerevisiae. Genetics, 190(3): 885–929

[145] Llorente B, Dujon B (2000). Transcriptional regulation of the Saccharomyces cerevisiae DAL5 gene family and identification of the high affinity nicotinic acid permease TNA1 (YGR260w). FEBS Lett, 475(3): 237–241

[146] Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo J L, Bonenfant D, Oppliger W, Jenoe P, Hall M N (2002). Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol Cell, 10(3): 457–468

[147] Longo V D (2003). The Ras and Sch9 pathways regulate stress resistance and longevity. Exp Gerontol, 38(7): 807–811

[148] Longo V D, Fabrizio P (2012). Chronological aging in Saccharomyces cerevisiae. Subcell Biochem, 57: 101–121

[149] Lu J Y, Lin Y Y, Sheu J C, Wu J T, Lee F J, Chen Y, Lin M I, Chiang F T, Tai T Y, Berger S L, Zhao Y, Tsai K S, Zhu H, Chuang L M, Boeke J D (2011). Acetylation of yeast AMPK controls intrinsic aging independently of caloric restriction. Cell, 146(6): 969–979

[150] Lu S P, Kato M, Lin S J (2009). Assimilation of endogenous nicotinamide riboside is essential for calorie restriction-mediated life span extension in Saccharomyces cerevisiae. J Biol Chem, 284(25): 17110–17119

[151] Lu S P, Lin S J (2010). Regulation of yeast sirtuins by NAD(+) metabolism and calorie restriction. Biochim Biophys Acta, 1804(8): 1567–1575

[152] Lu S P, Lin S J (2011). Phosphate-responsive signaling pathway is a novel component of NAD⁺ metabolism in Saccharomyces cerevisiae. J Biol Chem, 286(16): 14271–14281

[153] Lundh F, Mouillon J M, Samyn D, Stadler K, Popova Y, Lagerstedt J O, Thevelein J M, Persson B L (2009). Molecular mechanisms controlling phosphate-induced downregulation of the yeast Pho84 phosphate transporter. Biochemistry, 48(21): 4497–4505

[154] Magni G, Amici A, Emanuelli M, Orsomando G, Raffaelli N, Ruggieri S (2004). Structure and function of nicotinamide mononucleotide adenylyltransferase. Curr Med Chem, 11(7): 873–885

[155] Marzluf G A (1997). Molecular genetics of sulfur assimilation in filamentous fungi and yeast. Annu Rev Microbiol, 51(1): 73–96

[156] Matecic M, Smith D L, Pan X, Maqani N, Bekiranov S, Boeke J D, Smith J S (2010). A microarray-based genetic screen for yeast chronological aging factors. PLoS Genet, 6(4): e1000921

[157] Mayer F V, Heath R, Underwood E, Sanders M J, Carmena D, McCartney R R, Leiper F C, Xiao B, Jing C, Walker P A, Haire L F, Ogrodowicz R, Martin S R, Schmidt M C, Gamblin S J, Carling D (2011). ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase. Cell Metab, 14(5): 707–714

[158] McCartney R R, Schmidt M C (2001). Regulation of Snf1 kinase. ACTIVATION REQUIRES PHOSPHORYLATION OF THREONINE 210 BY AN UPSTREAM KINASE AS WELL AS A DISTINCT STEP MEDIATED BY THE SNF4 SUBUNIT. J Biol Chem, 276(39): 36460–36466

[159] McNabb D S, Pinto I (2005). Assembly of the Hap2p/Hap3p/Hap4p/Hap5p-DNA complex in Saccharomyces cerevisiae. Eukaryot Cell, 4(11): 1829–1839

[160] McNabb D S, Xing Y, Guarente L (1995). Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding. Genes Dev, 9(1): 47–58

[161] Medvedik O, Lamming D W, Kim K D, Sinclair D A (2007). MSN2 and MSN4 link calorie restriction and TOR to sirtuin-mediated lifespan extension in Saccharomyces cerevisiae. PLoS Biol, 5(10): e261

[162] Menoyo S, Ricco N, Bru S, Hernández-Ortega S, Escoté X, Aldea M, Clotet J (2013). Phosphate-activated cyclin-dependent kinase stabilizes G1 cyclin to trigger cell cycle entry. Mol Cell Biol, 33(7): 1273–1284

[163] Mense S M, Zhang L (2006). Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases. Cell Res, 16(8): 681–692

[164] Mesquita A, Weinberger M, Silva A, Sampaio-Marques B, Almeida B, Leão C, Costa V, Rodrigues F, Burhans W C, Ludovico P (2010). Caloric restriction or catalase inactivation extends yeast chronological lifespan by inducing H2O2 and superoxide dismutase activity. Proc Natl Acad Sci USA, 107(34): 15123–15128

[165] Moazed D (2001). Common themes in mechanisms of gene silencing. Mol Cell, 8(3): 489–498

[166] Moriya H, Johnston M (2004). Glucose sensing and signaling in Saccharomyces cerevisiae through the Rgt2 glucose sensor and casein kinase I. Proc Natl Acad Sci USA, 101(6): 1572–1577

[167] Mortimer R K, Johnston J R (1959). Life span of individual yeast cells. Nature, 183(4677): 1751–1752

[168] Mouillon J M, Persson B L (2005). Inhibition of the protein kinase A alters the degradation of the high-affinity phosphate transporter Pho84 in Saccharomyces cerevisiae. Curr Genet, 48(4): 226–234

[169] Murakami C, Delaney J R, Chou A, Carr D, Schleit J, Sutphin G L, An E H, Castanza A S, Fletcher M, Goswami S, Higgins S, Holmberg M, Hui J, Jelic M, Jeong K S, Kim J R, Klum S, Liao E, Lin M S, Lo W, Miller H, Moller R, Peng Z J, Pollard T, Pradeep P, Pruett D, Rai D, Ros V, Schuster A, Singh M, Spector B L, Vander Wende H, Wang A M, Wasko B M, Olsen B, Kaeberlein M (2012). pH neutralization protects against reduction in replicative lifespan following chronological aging in yeast. Cell Cycle, 11(16): 3087–3096

[170] Murakami C J, Wall V, Basisty N, Kaeberlein M (2011). Composition and acidification of the culture medium influences chronological aging similarly in vineyard and laboratory yeast. PLoS ONE, 6(9): e24530

[171] Natalini P, Ruggieri S, Raffaelli N, Magni G (1986). Nicotinamide mononucleotide adenylyltransferase. Molecular and enzymatic properties of the homogeneous enzyme from baker’s yeast. Biochemistry, 25(12): 3725–3729

[172] Natarajan K, Meyer M R, Jackson B M, Slade D, Roberts C, Hinnebusch A G, Marton M J (2001). Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol Cell Biol, 21(13): 4347–4368

[173] Niles B J, Powers T (2014). TOR complex 2-Ypk1 signaling regulates actin polarization via reactive oxygen species. Mol Biol Cell, 25(24): 3962–3972

[174] Noda T, Klionsky D J (2008). The quantitative Pho8Delta60 assay of nonspecific autophagy. Methods Enzymol, 451: 33–42

[175] North B J, Verdin E (2004). Sirtuins: Sir2-related NAD-dependent protein deacetylases. Genome Biol, 5(5): 224

[176] Ocampo A, Liu J, Barrientos A (2013). NAD⁺ salvage pathway proteins suppress proteotoxicity in yeast models of neurodegeneration by promoting the clearance of misfolded/oligomerized proteins. Hum Mol Genet, 22(9): 1699–1708

[177] Ocampo A, Liu J, Schroeder E A, Shadel G S, Barrientos A (2012). Mitochondrial respiratory thresholds regulate yeast chronological life span and its extension by caloric restriction. Cell Metab, 16(1): 55–67

[178] Ohashi K, Kawai S, Murata K (2013). Secretion of quinolinic acid, an intermediate in the kynurenine pathway, for utilization in NAD⁺ biosynthesis in the yeast Saccharomyces cerevisiae. Eukaryot Cell, 12(5): 648–653

[179] Omnus D J, Ljungdahl P O (2014). Latency of transcription factor Stp1 depends on a modular regulatory motif that functions as cytoplasmic retention determinant and nuclear degron. Mol Biol Cell, 25(23): 3823–3833

[180] Omnus D J, Pfirrmann T, Andréasson C, Ljungdahl P O (2011). A phosphodegron controls nutrient-induced proteasomal activation of the signaling protease Ssy5. Mol Biol Cell, 22(15): 2754–2765

[181] Overton M C, Chinault S L, Blumer K J (2005). Oligomerization of G-protein-coupled receptors: lessons from the yeast Saccharomyces cerevisiae. Eukaryot Cell, 4(12): 1963–1970

[182] Pan Y (2011). Mitochondria, reactive oxygen species, and chronological aging: a message from yeast. Exp Gerontol, 46(11): 847–852

[183] Pan Y, Schroeder E A, Ocampo A, Barrientos A, Shadel G S (2011). Regulation of yeast chronological life span by TORC1 via adaptive mitochondrial ROS signaling. Cell Metab, 13(6): 668–678

[184] Panozzo C, Nawara M, Suski C, Kucharczyka R, Skoneczny M, Bécam A M, Rytka J, Herbert C J (2002). Aerobic and anaerobic NAD⁺ metabolism in Saccharomyces cerevisiae. FEBS Lett, 517(1–3): 97–102

[185] Parua P K, Ratnakumar S, Braun K A, Dombek K M, Arms E, Ryan P M, Young E T (2010). 14-3-3 (Bmh) proteins inhibit transcription activation by Adr1 through direct binding to its regulatory domain. Mol Cell Biol, 30(22): 5273–5283

[186] Pasula S, Jouandot D 2nd, Kim J H (2007). Biochemical evidence for glucose-independent induction of HXT expression in Saccharomyces cerevisiae. FEBS Lett, 581(17): 3230–3234

[187] Peeters T, Louwet W, Geladé R, Nauwelaers D, Thevelein J M, Versele M (2006). Kelch-repeat proteins interacting with the Gα protein Gpa2 bypass adenylate cyclase for direct regulation of protein kinase A in yeast. Proc Natl Acad Sci USA, 103(35): 13034–13039

[188] Perrod S, Cockell M M, Laroche T, Renauld H, Ducrest A L, Bonnard C, Gasser S M (2001). A cytosolic NAD-dependent deacetylase, Hst2p, can modulate nucleolar and telomeric silencing in yeast. EMBO J, 20(1–2): 197–209

[189] Persson B L, Lagerstedt J O, Pratt J R, Pattison-Granberg J, Lundh K, Shokrollahzadeh S, Lundh F (2003). Regulation of phosphate acquisition in Saccharomyces cerevisiae. Curr Genet, 43(4): 225–244

[190] Pinson B, Vaur S, Sagot I, Coulpier F, Lemoine S, Daignan-Fornier B (2009). Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways. Genes Dev, 23(12): 1399–1407

[191] Popova Y, Thayumanavan P, Lonati E, Agrochão M, Thevelein J M (2010). Transport and signaling through the phosphate-binding site of the yeast Pho84 phosphate transceptor. Proc Natl Acad Sci USA, 107(7): 2890–2895

[192] Powers R W 3rd, Kaeberlein M, Caldwell S D, Kennedy B K, Fields S (2006). Extension of chronological life span in yeast by decreased TOR pathway signaling. Genes Dev, 20(2): 174–184

[193] Preiss J, Handler P (1958a). Biosynthesis of diphosphopyridine nucleotide. I. Identification of intermediates. J Biol Chem, 233(2): 488–492

[194] Preiss J, Handler P (1958b). Biosynthesis of diphosphopyridine nucleotide. II. Enzymatic aspects. J Biol Chem, 233(2): 493–500

[195] Ramsey K M, Mills K F, Satoh A, Imai S (2008). Age-associated loss of Sirt1-mediated enhancement of glucose-stimulated insulin secretion in beta cell-specific Sirt1-overexpressing (BESTO) mice. Aging Cell, 7(1): 78–88

[196] Revollo J R, Grimm A A, Imai S (2004). The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyl transferase regulates Sir2 activity in mammalian cells. J Biol Chem, 279(49): 50754–50763

[197] Rine J, Herskowitz I (1987). Four genes responsible for a position effect on expression from HML and HMR in Saccharomyces cerevisiae. Genetics, 116(1): 9–22

[198] Rodgers J T, Lerin C, Haas W, Gygi S P, Spiegelman B M, Puigserver P (2005). Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature, 434(7029): 113–118

[199] Rolland F, De Winde J H, Lemaire K, Boles E, Thevelein J M, Winderickx J (2000). Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process. Mol Microbiol, 38(2): 348–358

[200] Roosen J, Engelen K, Marchal K, Mathys J, Griffioen G, Cameroni E, Thevelein J M, De Virgilio C, De Moor B, Winderickx J (2005). PKA and Sch9 control a molecular switch important for the proper adaptation to nutrient availability. Mol Microbiol, 55(3): 862–880

[201] Roth S, Kumme J, Schüller H J (2004). Transcriptional activators Cat8 and Sip4 discriminate between sequence variants of the carbon source-responsive promoter element in the yeast Saccharomyces cerevisiae. Curr Genet, 45(3): 121–128

[202] Rubenstein E M, McCartney R R, Zhang C, Shokat K M, Shirra M K, Arndt K M, Schmidt M C (2008). Access denied: Snf1 activation loop phosphorylation is controlled by availability of the phosphorylated threonine 210 to the PP1 phosphatase. J Biol Chem, 283(1): 222–230

[203] Rubio-Texeira M, Van Zeebroeck G, Voordeckers K, Thevelein J M (2010). Saccharomyces cerevisiae plasma membrane nutrient sensors and their role in PKA signaling. FEMS Yeast Res, 10(2): 134–149

[204] Samyn D R, Ruiz-Pávon L, Andersson M R, Popova Y, Thevelein J M, Persson B L (2012). Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H(+) transceptor and its effect on signalling to the PKA and PHO pathways. Biochem J, 445(3): 413–422

[205] Sancak Y, Peterson T R, Shaul Y D, Lindquist R A, Thoreen C C, Bar-Peled L, Sabatini D M (2008). The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science, 320(5882): 1496–1501

[206] Sanz P (2003). Snf1 protein kinase: a key player in the response to cellular stress in yeast. Biochem Soc Trans, 31(Pt 1): 178–181

[207] Sasaki Y, Araki T, Milbrandt J (2006). Stimulation of nicotinamide adenine dinucleotide biosynthetic pathways delays axonal degeneration after axotomy. J Neurosci, 26(33): 8484–8491

[208] Sauve A A, Schramm V L (2003). Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry. Biochemistry, 42(31): 9249–9256

[209] Scheckhuber C Q, Erjavec N, Tinazli A, Hamann A, Nyström T, Osiewacz H D (2007). Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models. Nat Cell Biol, 9(1): 99–105

[210] Schleit J, Johnson S C, Bennett C F, Simko M, Trongtham N, Castanza A, Hsieh E J, Moller R M, Wasko B M, Delaney J R, Sutphin G L, Carr D, Murakami C J, Tocchi A, Xian B, Chen W, Yu T, Goswami S, Higgins S, Jeong K S, Kim J R, Klum S, Liao E, Lin M S, Lo W, Miller H, Olsen B, Peng Z J, Pollard T, Pradeep P, Pruett D, Rai D, Ros V, Singh M, Spector B L, Wende H V, An E H, Fletcher M, Jelic M, Rabinovitch P S, Maccoss M J, Han J D, Kennedy B K, Kaeberlein M (2013). Molecular mechanisms underlying genotype-dependent responses to dietary restriction. Aging Cell, 12(6): 1050–1061

[211] Schmeisser K, Mansfeld J, Kuhlow D, Weimer S, Priebe S, Heiland I, Birringer M, Groth M, Segref A, Kanfi Y, Price N L, Schmeisser S, Schuster S, Pfeiffer A F, Guthke R, Platzer M, Hoppe T, Cohen H Y, Zarse K, Sinclair D A, Ristow M, Klum S, Liao E, Lin M S, Lo W, Miller H, Olsen B, Peng Z J, Pollard T, Pradeep P, Pruett D, Rai D, Ros V, Singh M, Spector B L, Wende H V, An E H, Fletcher M, Jelic M, Rabinovitch P S, Maccoss M J, Han J D, Kennedy B K, Kaeberlein M (2013). Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide. Nat Chem Biol, 9(11): 693–700

[212] Schmidt M T, Smith B C, Jackson M D, Denu J M (2004). Coenzyme specificity of Sir2 protein deacetylases: implications for physiological regulation. J Biol Chem, 279(38): 40122–40129

[213] Schmidt-Brauns J, Herbert M, Kemmer G, Kraiss A, Schlör S, Reidl J (2001). Is a NAD pyrophosphatase activity necessary for Haemophilus influenzae type b multiplication in the blood stream? Int J Med Microbiol, 291(3): 219–225

[214] Schüller H J (2003). Transcriptional control of nonfermentative metabolism in the yeast Saccharomyces cerevisiae. Curr Genet, 43(3): 139–160

[215] Schulz T J, Zarse K, Voigt A, Urban N, Birringer M, Ristow M (2007). Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress. Cell Metab, 6(4): 280–293

[216] Shama S, Lai C Y, Antoniazzi J M, Jiang J C, Jazwinski S M (1998). Heat stress-induced life span extension in yeast. Exp Cell Res, 245(2): 379–388

[217] Shimada K, Filipuzzi I, Stahl M, Helliwell S B, Studer C, Hoepfner D, Seeber A, Loewith R, Movva N R, Gasser S M (2013). TORC2 signaling pathway guarantees genome stability in the face of DNA strand breaks. Mol Cell, 51(6): 829–839

[218] Shirra M K, McCartney R R, Zhang C, Shokat K M, Schmidt M C, Arndt K M (2008). A chemical genomics study identifies Snf1 as a repressor of GCN4 translation. J Biol Chem, 283(51): 35889–35898

[219] Shirra M K, Rogers S E, Alexander D E, Arndt K M (2005). The Snf1 protein kinase and Sit4 protein phosphatase have opposing functions in regulating TATA-binding protein association with the Saccharomyces cerevisiae INO1 promoter. Genetics, 169(4): 1957–1972

[220] Sies H (1982). Metabolic Compartmentation. Orlando, FL, Academic Press

[221] Smets B, De Snijder P, Engelen K, Joossens E, Ghillebert R, Thevissen K, Marchal K, Winderickx J (2008). Genome-wide expression analysis reveals TORC1-dependent and-independent functions of Sch9. FEMS Yeast Res, 8(8): 1276–1288

[222] Smith D L Jr, McClure J M, Matecic M, Smith J S (2007). Calorie restriction extends the chronological lifespan of Saccharomyces cerevisiae independently of the Sirtuins. Aging Cell, 6(5): 649–662

[223] Smith J S, Boeke J D (1997). An unusual form of transcriptional silencing in yeast ribosomal DNA. Genes Dev, 11(2): 241–254

[224] Smith J S, Brachmann C B, Celic I, Kenna M A, Muhammad S, Starai V J, Avalos J L, Escalante-Semerena J C, Grubmeyer C, Wolberger C, Boeke J D (2000). A phylogenetically conserved NAD⁺-dependent protein deacetylase activity in the Sir2 protein family. Proc Natl Acad Sci USA, 97(12): 6658–6663

[225] Soontorngun N, Larochelle M, Drouin S, Robert F, Turcotte B (2007). Regulation of gluconeogenesis in Saccharomyces cerevisiae is mediated by activator and repressor functions of Rds2. Mol Cell Biol, 27(22): 7895–7905

[226] Sporty J, Lin S J, Kato M, Ognibene T, Stewart B, Turteltaub K, Bench G (2009). Quantitation of NAD⁺ biosynthesis from the salvage pathway in Saccharomyces cerevisiae. Yeast, 26(7): 363–369

[227] Staschke K A, Dey S, Zaborske J M, Palam L R, McClintick J N, Pan T, Edenberg H J, Wek R C (2010). Integration of general amino acid control and target of rapamycin (TOR) regulatory pathways in nitrogen assimilation in yeast. J Biol Chem, 285(22): 16893–16911

[228] Steffen K K, McCormick M A, Pham K M, MacKay V L, Delaney J R, Murakami C J, Kaeberlein M, Kennedy B K (2012). Ribosome deficiency protects against ER stress in Saccharomyces cerevisiae. Genetics, 191(1): 107–118

[229] Strahl-Bolsinger S, Hecht A, Luo K, Grunstein M (1997). SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast. Genes Dev, 11(1): 83–93

[230] Sturgill T W, Cohen A, Diefenbacher M, Trautwein M, Martin D E, Hall M N (2008). TOR1 and TOR2 have distinct locations in live cells. Eukaryot Cell, 7(10): 1819–1830

[231] Sun J, Kale S P, Childress A M, Pinswasdi C, Jazwinski S M (1994). Divergent roles of RAS1 and RAS2 in yeast longevity. J Biol Chem, 269(28): 18638–18645

[232] Sutherland C M, Hawley S A, McCartney R R, Leech A, Stark M J, Schmidt M C, Hardie D G (2003). Elm1p is one of three upstream kinases for the Saccharomyces cerevisiae SNF1 complex. Curr Biol, 13(15): 1299–1305

[233] Sutton A, Heller R C, Landry J, Choy J S, Sirko A, Sternglanz R (2001). A novel form of transcriptional silencing by Sum1-1 requires Hst1 and the origin recognition complex. Mol Cell Biol, 21(10): 3514–3522

[234] Swinnen E, Wanke V, Roosen J, Smets B, Dubouloz F, Pedruzzi I, Cameroni E, De Virgilio C, Winderickx J (2006). Rim15 and the crossroads of nutrient signalling pathways in Saccharomyces cerevisiae. Cell Div, 1(1): 3

[235] Tanny J C, Kirkpatrick D S, Gerber S A, Gygi S P, Moazed D (2004). Budding yeast silencing complexes and regulation of Sir2 activity by protein-protein interactions. Mol Cell Biol, 24(16): 6931–6946

[236] Thevelein J M, Cauwenberg L, Colombo S, Donation M, Dumortier F, Kraakman L, Lemaire K, Ma P, Nauwelaers D, Rolland F, Teunissen A, Versele M, Wera S, Winderickx J, Wera S, Winderickx J, De Winde J H, Van Dijck P (2000). Nutrient-induced signal transduction through the protein kinase A pathway and its role in the control of metabolism, stress resistance, and growth in yeast. Enzyme Microb Technol, 26(9–10): 819–825

[237] Todisco S, Agrimi G, Castegna A, Palmieri F (2006). Identification of the mitochondrial NAD⁺ transporter in Saccharomyces cerevisiae. J Biol Chem, 281(3): 1524–1531

[238] Tsang F, James C, Kato M, Myers V, Ilyas I, Tsang M, Lin S J (2015). Reduced Ssy1-Ptr3-Ssy5 (SPS) signaling extends replicative life span by enhancing NAD⁺ homeostasis in Saccharomyces cerevisiae. J Biol Chem, 290(20):12753–12764

[239] Ueda Y, Oshima Y (1975). A constitutive mutation, phoT, of the repressible acid phosphatase synthesis with inability to transport inorganic phosphate in Saccharomyces cerevisiae. Mol Gen Genet, 136: 255–259

[240] Unal E, Kinde B, Amon A (2011). Gametogenesis eliminates age-induced cellular damage and resets life span in yeast. Science, 332(6037): 1554–1557

[241] Urban J, Soulard A, Huber A, Lippman S, Mukhopadhyay D, Deloche O, Wanke V, Anrather D, Ammerer G, Riezman H, Broach J R, De Virgilio C, Hall M N, Loewith R (2007). Sch9 is a major target of TORC1 in Saccharomyces cerevisiae. Mol Cell, 26(5): 663–674

[242] van der Veer E, Nong Z, O’Neil C, Urquhart B, Freeman D, Pickering J G (2005). Pre-B-cell colony-enhancing factor regulates NAD⁺-dependent protein deacetylase activity and promotes vascular smooth muscle cell maturation. Circ Res, 97(1): 25–34

[243] van Oevelen C J, van Teeffelen H A, van Werven F J, Timmers H T (2006). Snf1p-dependent Spt-Ada-Gcn5-acetyltransferase (SAGA) recruitment and chromatin remodeling activities on the HXT2 and HXT4 promoters. J Biol Chem, 281(7): 4523–4531

[244] Veatch J R, McMurray M A, Nelson Z W, Gottschling D E (2009). Mitochondrial dysfunction leads to nuclear genome instability via an iron-sulfur cluster defect. Cell, 137(7): 1247–1258

[245] Vickers M F, Yao S Y, Baldwin S A, Young J D, Cass C E (2000). Nucleoside transporter proteins of Saccharomyces cerevisiae. Demonstration of a transporter (FUI1) with high uridine selectivity in plasma membranes and a transporter (FUN26) with broad nucleoside selectivity in intracellular membranes. J Biol Chem, 275(34): 25931–25938

[246] Vlahakis A, Graef M, Nunnari J, Powers T (2014). TOR complex 2-Ypk1 signaling is an essential positive regulator of the general amino acid control response and autophagy. Proc Natl Acad Sci USA, 111(29): 10586–10591

[247] Vlahakis A, Powers T (2014). A role for TOR complex 2 signaling in promoting autophagy. Autophagy, 10(11): 2085–2086

[248] Voordeckers K, Kimpe M, Haesendonckx S, Louwet W, Versele M, Thevelein J M (2011). Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9. J Biol Chem, 286(25): 22017–22027

[249] Wang C, Skinner C, Easlon E, Lin S J (2009). Deleting the 14-3-3 protein Bmh1 extends life span in Saccharomyces cerevisiae by increasing stress response. Genetics, 183(4): 1373–1384

[250] Wang J, Jiang J C, Jazwinski S M (2010). Gene regulatory changes in yeast during life extension by nutrient limitation. Exp Gerontol, 45(7–8): 621–631

[251] Wanke V, Cameroni E, Uotila A, Piccolis M, Urban J, Loewith R, De Virgilio C (2008). Caffeine extends yeast lifespan by targeting TORC1. Mol Microbiol, 69(1): 277–285

[252] Wanke V, Pedruzzi I, Cameroni E, Dubouloz F, De Virgilio C (2005). Regulation of G0 entry by the Pho80-Pho85 cyclin-CDK complex. EMBO J, 24(24): 4271–4278

[253] Wedaman K P, Reinke A, Anderson S, Yates J 3rd, McCaffery J M, Powers T (2003). Tor kinases are in distinct membrane-associated protein complexes in Saccharomyces cerevisiae. Mol Biol Cell, 14(3): 1204–1220

[254] Wei M, Fabrizio P, Hu J, Ge H, Cheng C, Li L, Longo V D (2008). Life span extension by calorie restriction depends on Rim15 and transcription factors downstream of Ras/PKA, Tor, and Sch9. PLoS Genet, 4(1): e13

[255] Weinberger M, Feng L, Paul A, Smith D L Jr, Hontz R D, Smith J S, Vujcic M, Singh K K, Huberman J A, Burhans W C (2007). DNA replication stress is a determinant of chronological lifespan in budding yeast. PLoS ONE, 2(8): e748

[256] Weindruch W, Walford R L (1998). The retardation of aging and diseases by dietary restriction. Springfield, Illinois, USA, Charles C. Thomas

[257] Wek R C, Jackson B M, Hinnebusch A G (1989). Juxtaposition of domains homologous to protein kinases and histidyl-tRNA synthetases in GCN2 protein suggests a mechanism for coupling GCN4 expression to amino acid availability. Proc Natl Acad Sci USA, 86(12): 4579–4583

[258] Wiederhold E, Gandhi T, Permentier H P, Breitling R, Poolman B, Slotboom D J (2009). The yeast vacuolar membrane proteome. Mol Cell Proteomics, 8(2): 380–392

[259] Wilson J M, Le V Q, Zimmerman C, Marmorstein R, Pillus L (2006). Nuclear export modulates the cytoplasmic Sir2 homologue Hst2. EMBO Rep, 7(12): 1247–1251

[260] Wogulis M, Chew E R, Donohoue P D, Wilson D K (2008). Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence. Biochemistry, 47(6): 1608–1621

[261] Wu Z, Liu S Q, Huang D (2013). Dietary restriction depends on nutrient composition to extend chronological lifespan in budding yeast Saccharomyces cerevisiae. PLoS ONE, 8(5): e64448

[262] Wykoff D D, O’Shea E K (2001). Phosphate transport and sensing in Saccharomyces cerevisiae. Genetics, 159(4): 1491–1499

[263] Xiao B, Heath R, Saiu P, Leiper F C, Leone P, Jing C, Walker P A, Haire L, Eccleston J F, Davis C T, Martin S R, Carling D, Gamblin S J (2007). Structural basis for AMP binding to mammalian AMP-activated protein kinase. Nature, 449(7161): 496–500

[264] Xie J, Pierce M, Gailus-Durner V, Wagner M, Winter E, Vershon A K (1999). Sum1 and Hst1 repress middle sporulation-specific gene expression during mitosis in Saccharomyces cerevisiae. EMBO J, 18(22): 6448–6454

[265] Xu Y F, Létisse F, Absalan F, Lu W, Kuznetsova E, Brown G, Caudy A A, Yakunin A F, Broach J R, Rabinowitz J D (2013). Nucleotide degradation and ribose salvage in yeast. Mol Syst Biol, 9(1): 665

[266] Yang J, Dungrawala H, Hua H, Manukyan A, Abraham L, Lane W, Mead H, Wright J, Schneider B L (2011). Cell size and growth rate are major determinants of replicative lifespan. Cell Cycle, 10(1): 144–155

[267] Yao Y, Tsuchiyama S, Yang C, Bulteau A L, He C, Robison B, Tsuchiya M, Miller D, Briones V, Tar K, Potrero A, Friguet B, Kennedy B K, Schmidt M (2015). Proteasomes, Sir2, and Hxk2 form an interconnected aging network that impinges on the AMPK/Snf1-regulated transcriptional repressor Mig1. PLoS Genet, 11(1): e1004968

[268] Young J D, Yao S Y, Sun L, Cass C E, Baldwin S A (2008). Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins. Xenobiotica, 38(7–8): 995–1021

[269] Zaborske J M, Narasimhan J, Jiang L, Wek S A, Dittmar K A, Freimoser F, Pan T, Wek R C (2009). Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p. J Biol Chem, 284(37): 25254–25267

[270] Zaborske J M, Wu X, Wek R C, Pan T (2010). Selective control of amino acid metabolism by the GCN2 eIF2 kinase pathway in Saccharomyces cerevisiae. BMC Biochem, 11(1): 29

[271] Zaman S, Lippman S I, Schneper L, Slonim N, Broach J R (2009). Glucose regulates transcription in yeast through a network of signaling pathways. Mol Syst Biol, 5: 245

[272] Zargari A, Boban M, Heessen S, Andréasson C, Thyberg J, Ljungdahl P O (2007). Inner nuclear membrane proteins Asi1, Asi2, and Asi3 function in concert to maintain the latent properties of transcription factors Stp1 and Stp2. J Biol Chem, 282(1): 594–605

[273] Zhai R G, Zhang F, Hiesinger P R, Cao Y, Haueter C M, Bellen H J (2008). NAD synthase NMNAT acts as a chaperone to protect against neurodegeneration. Nature, 452(7189): 887–891

[274] Zhang T, Péli-Gulli M P, Yang H, De Virgilio C, Ding J (2012). Ego3 functions as a homodimer to mediate the interaction between Gtr1-Gtr2 and Ego1 in the ego complex to activate TORC1. Structure, 20(12): 2151–2160

[275] Zitomer R S, Lowry C V (1992). Regulation of gene expression by oxygen in Saccharomyces cerevisiae. Microbiol Rev, 56(1): 1–11

[276] Zuin A, Carmona M, Morales-Ivorra I, Gabrielli N, Vivancos A P, Ayté J, Hidalgo E (2010). Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway. EMBO J, 29(5): 981–991

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg