Reduced Ssy1-Ptr3-Ssy5 (SPS) signaling extends replicative life span by enhancing NAD+ homeostasis in Saccharomyces cerevisiae

Abstract

Attenuated nutrient signaling extends the life span in yeast and higher eukaryotes; however, the mechanisms are not completely understood. Here we identify the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway as a novel longevity factor. A null mutation of SSY5 (ssy5Δ) increases replicative life span (RLS) by ∼50%. Our results demonstrate that several NAD(+) homeostasis factors play key roles in this life span extension. First, expression of the putative malate-pyruvate NADH shuttle increases in ssy5Δ cells, and deleting components of this shuttle, MAE1 and OAC1, largely abolishes RLS extension. Next, we show that Stp1, a transcription factor of the SPS pathway, directly binds to the promoter of MAE1 and OAC1 to regulate their expression. Additionally, deletion of SSY5 increases nicotinamide riboside (NR) levels and phosphate-responsive (PHO) signaling activity, suggesting that ssy5Δ increases NR salvaging. This increase contributes to NAD(+) homeostasis, partially ameliorating the NAD(+) deficiency and rescuing the short life span of the npt1Δ mutant. Moreover, we observed that vacuolar phosphatase, Pho8, is partially required for ssy5Δ-mediated NR increase and RLS extension. Together, our studies present evidence that supports SPS signaling is a novel NAD(+) homeostasis factor and ssy5Δ-mediated life span extension is likely due to concomitantly increased mitochondrial and vacuolar function. Our findings may contribute to understanding the molecular basis of NAD(+) metabolism, cellular life span, and diseases associated with NAD(+) deficiency and aging.

Keywords: NAD biosynthesis; cell metabolism; metabolic regulation; nicotinamide riboside salvage; yeast genetics; yeast metabolism.

FIGURE 1.

Yeast mutants with reduced SPS signaling show increased replicative life span. A, a simple model of the SPS nutrient-sensing pathway in S. cerevisiae. The left panel shows inactive SPS without amino acid stimulation. Upon activation by extracellular amino acids, SPS activates transcription factors Stp1 and Stp2 (right panel) to modulate (activate or inhibit) downstream gene expression. Pro, Ssy5 prodomain; Cat, Ssy5 catalytic domain; P, phosphorylation. B, deletions of various SPS pathway components extend RLS. The RLS of all mutants (except for stp2Δ) are significantly increased (p < 0.005) when compared with the WT. Δ: gene deletions. C, moderate CR further extends RLS in the SPS mutant ssy5Δ background (ssy5Δ versus ssy5Δ, CR; p < 0.05). CR, 0.5% glucose (versus 2% glucose in standard growth media). D, cdc25-10, a CR genetic mimic, further extends life span in the ssy5Δ background (ssy5Δ versus cdc25-10 ssy5Δ; p < 0.005). E, reduced amino acid uptake is not the main cause of RLS extension in ssy5Δ cells. Unlike ssy5Δ, deletion of SPS downstream amino acid permeases AGP1, BAP2, and BAP3 does not extend RLS. Data shown are representative of multiple independent experiments. Statistical analysis of RLS is determined by the Wilcoxon rank sum test.

FIGURE 2.

SPS signaling-mediated life span extension requires components of NADH shuttle systems. A, a simplified model of the putative malate-pyruvate NADH shuttle. Cellular NAD⁺/NADH ratio is regulated by groups of cytoplasmic (e.g. Pyc1/Pyc2 and Mdh2) and mitochondrial (e.g. Mae1) enzymes, which produce permeable or small metabolites that can be transported (via carrier proteins Dic1, Oac1, and Mpc1/Mpc2) across the inner membrane. As a result, the NAD⁺/NADH ratio and metabolites are balanced between the mitochondrial and cytoplasmic pools. B, gene expression of the malate-pyruvate shuttle components increases in the ssy5Δ mutant. Results show gene expression comparisons between WT and ssy5Δ cells determined by qPCR. MDH1 and AAT1 are components of another NADH shuttle, shown as a control. C, Mae1 and Oac1 protein levels increase in the ssy5Δ mutant. Results show Western blot analysis of HA-tagged Oac1 and Mae1 in both WT and ssy5Δ cells. D, deletions of both MAE1 and OAC1 largely abolish the life span extension by ssy5Δ (ssy5Δ versus ssy5Δmae1Δoac1Δ; p < 0.005). E, the levels and ratio of NAD⁺ and NADH are not changed by ssy5Δ. F, the level of nicotinamide riboside (NR, a NAD⁺ precursor) increases in ssy5Δ cells. Data shown are representative of multiple independent experiments. Error bars denote S.D. derived from triplicate samples. The p values were calculated using Student's t test (ns, not significant; *, p < 0.05; ***, p < 0.005) except for D (Wilcoxon rank sum test).

FIGURE 3.

NR production contributes to enhanced NAD⁺ homeostasis in ssy5Δ cells. A, overall gene expression of the NAD⁺ biosynthetic pathway remains largely similar in ssy5Δ cells compared with WT. Results show relative gene expression (normalized to ACT1) determined by qPCR. B, deletion of SSY5 increases gene expression of the phosphate sensing (PHO) pathway components. Results show gene expression comparisons between WT and ssy5Δ cells. C, deletion of SSY5 rescues the short RLS of the NAD⁺ biosynthesis-deficient npt1Δ mutant (npt1Δ versus ssy5Δnpt1Δ; p < 0.005). D, deletion of SSY5 increases both NAD⁺ and NADH levels in npt1Δ cells. E, deletion of SSY5 increases intracellular NR levels in npt1Δ cells. Data shown are representative of multiple independent experiments. Error bars denote S.D. derived from triplicate samples. The p values were calculated using Student's t test (ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.005) except for C (Wilcoxon rank sum test).

FIGURE 4.

Reduced SPS signaling increases the expression of specific PHO signaling and NADH shuttle components independent of PHO2 and PHO4. A, vacuolar Pho8 phosphatase activity is slightly increased in ssy5Δ cells. Results show Pho8 phosphatase activity using para-nitrophenyl phosphate as a substrate for cell lysate derived from WT and ssy5Δ cells. Pho8 activity is reflected by production rate of colorimetric para-nitrophenol (pNP), determined at A₄₀₀. B, deletion of PHO8 abolishes intracellular NR increase in ssy5Δ cells (left panel) but not released NR, which mostly originates from cytosolic phosphatases (right panel). C, deletion of PHO8 partially abolishes the life span extension in ssy5Δ cells (ssy5Δ versus ssy5Δpho8Δ; p < 0.05). D, deletions of both PHO2 and PHO4, transcription factors that regulate PHO pathway, do not alter the life span extension in ssy5Δ cells. E, ssy5Δ increases gene expression of PHO8 and the malate-pyruvate shuttle components independent of PHO2 and PHO4. Results show relative gene expression (normalized to ACT1) of WT, ssy5Δ, pho2Δpho4Δ, and ssy5Δpho2Δpho4Δ cells determined by qPCR. Data shown are representative of multiple independent experiments. Error bars denote S.D. derived from triplicate samples. The p values were calculated using Student's t test (ns, not significant; *, p < 0.05; ***, p < 0.005) except for C and D (Wilcoxon rank sum test).

FIGURE 5.

Stp1, a transcription factor of SPS signaling pathway, may directly bind to the promoter regions of NADH shuttle components. A, putative binding sites of Stp1 in the promoter regions of PHO8, OAC1, and MAE1 are shown in the left panel. DNA fragments used for ChIP analysis are shown in the right panel. # denotes fragments containing a putative Stp1 binding site. B, HA-tagged Stp1 (Stp1-HA) does not significantly bind to various promoter regions of PHO8 compared with AGP1 as positive control (+) and ACT1 as negative control (−), determined by qPCR. C, Stp1-HA significantly binds to OAC1-a^# but not other promoter regions of OAC1. D, Stp1-HA significantly binds to MAE1-d^# but not other promoter regions of MAE1. E, proposed model of how reduced SPS signaling leads to increased NAD⁺ homeostasis (via NR salvage and NADH shuttle), which works in concert with mitochondrial and vacuolar metabolism to maintain cell function and extend life span. For clarity, additional factors and interactions are not shown. Data shown are representative of multiple independent experiments. Error bars denote S.D. derived from triplicate samples. The p values were calculated using Student's t test (ns, not significant; ***, p < 0.005).