(A–D) Day 3 wild type (DBY746) and cells lacking Tor1, Sch9, or Ras2 were challenged with heat shock (55°C: A, 105 min; B, 75 min; C, 150 min; and D, 120 min) or oxidative stresses (H₂O₂, 100 mM for 60 min; or menadione, 250 µM for 30 min). (E) Mutation frequency over time measured as Can^r mutants per million cells. The average of four experiments is shown. Error bars represent SEM. (F) Chronological survival in minimal complete medium (SDC) of wild type, tor1Δ, and mutants overexpressing either SCH9 or constitutively active Ras2 (ras2^Val19). (G) Chronological survival of wild type and mutants lacking Tor1, Sch9, Ras2 or combinations. The data represent average of at least 4 experiments. Error bars show SEM. For mean life span calculated from non-linear curve fitting see Table S1. (H) Longevity regulatory pathways in yeast. The nutrient sensing pathways controlled by Sch9, Tor, and Ras converge on the protein kinase Rim15. In turn, the stress response transcription factors Msn2, Msn4, and Gis1 transactivate stress response genes and enhance cellular protection, which lead to life span extension. Pro-longevity effects of CR are partially mediated by Sch9, Tor, and Ras, and may also require additional yet-to-be indentified mechanism(s).

(A) Schematic representation of glycerol metabolism. For illustration purpose, genes up- or down-regulated more than 20%, compared to wild type (DBY746) in all three long-lived mutants, are labeled in red or green, respectively. (B) Fold change in expression levels of glycerol biosynthetic genes in sch9Δ, tor1Δ, and ras2Δ mutants compared to wild type at day 2.5 (for complete microarray data, see Table S2 and Table S6). Data were expressed in fold change, WT = 1. Gene expression levels that are 20% higher or lower than wild type cells are marked in red or green. ¹ Predicted PDS motif: AWAGGGAT; ² predicted STRE motif: ARGGGG; ³ predicted STRE motif: AGGGG. (C) Real time quantitative PCR analysis of GPD1 mRNA level sch9Δ and sch9Δ gis1Δ cells at day 2.5. Data represent mean and SEM, n = 4. * p<0.05, t-test, two-tailed, sch9Δ vs. WT.

(A) Glycerol concentration in the medium. Data represent mean and SEM of 4 cultures analyzed. * p<0.05, ** p<0.01, unpaired t-test, two tailed, sch9Δ vs. rhr2Δ sch9Δ. (B) Life span of wild type (DBY746), sch9Δ, rhr2Δ, and Sch9-deficient mutants lacking Rhr2. Glycerol (1%, final concentration) was added to the one day-old rhr2Δ sch9Δ culture. Data represent mean and SEM of 4–5 cultures analyzed. (C) Day3 cells were exposed to heat shock (55°C for 105 min) or H₂O₂ (150 mM for 60 min). Strains shown are wild type (DBY746), rhr2Δ, sch9Δ, and rhr2Δ sch9Δ. (D) Ethanol concentration in the medium. Data represent mean and SEM, n = 3. Ethanol in sch9Δ culture at day 5 was at the lower detection limit of the assay. (E) Life span of wild type (BY4741), sch9Δ, and Sch9-deficient mutants lacking Gpd1, Gpd2, or Rhr2. Data represent mean and SEM of 3 experiments. (F) Heat shock (55°C) and oxidative stress (H₂O₂, 500 mM, 60 min) resistance of day 3 mutants lacking glycerol biosynthesis genes (in the BY4741 genetic background).

The metabolic changes in long lived mutants generate a calorie restriction-like environment, which contributes to enhanced stress resistance and extended life span.

(A) Venn diagram of genes up- or down-regulated more than 2-fold in the tor1Δ, sch9Δ, and ras2Δ mutants, at day 2.5, compared to wild type cells (DBY746). Microarray analyses were carried out in triplicates. The number of up- or down-regulated genes was shown in red or green, respectively. For the significance of overlapping in up- and down-regulated genes see Table S3. (B) Life span of mutants with deletions of genes most upregulated in long-lived mutants in the sch9Δ background. Data represent mean and SEM of pair matched, pooled experiments. For mean life span calculated from non-linear curve fitting see Table S1.

(A) Intracellular glycerol contents of wild type (DBY746) and cells lacking Sch9 were measured on day 1 and day 3. Data represent mean and SEM of 8 cultures analyzed. (B) Glycerol concentration in the medium of wild type and sch9Δ cultures. Data represent mean and SEM of 5–7 cultures analyzed. ** p<0.01, unpaired t-test, two-tailed, sch9Δ vs. WT. (C–D) Glycerol and ethanol concentrations in the medium of wild type (C) and sch9Δ (D) cultures. Data represent mean and SEM of 3–5 cultures analyzed. (E) Nile red staining of neutral lipids of day 1 wild type and sch9Δ mutants. Nile red staining is shown on the right, and phase contrast left. Bar, 10 µm.

(A) Day 3 wild type (DBY746) and sch9Δ mutants expressing bacterial heat-sensitive luciferase were subject to heat stress (42°C for 60 min). Data represent mean and SEM, n = 3. * p<0.05, unpaired t-test, two tailed. (B) Recovery of luciferase activity after heat stress (42°C for 60 min) in wild type cells pre-treated with glycerol (with concentrations indicated) for 30 min. Data represent mean and SEM, n = 3. (C) Day 3 wild type and sch9Δ mutants grown in SDC were washed 3 times with water and exposed to high concentrations of NaCl with or without glycerol for 24 hours. The cells were then washed 3 times to remove the salt, serially diluted, and spotted on to YPD plate. (D) Chronological survival of wild type cells grown in SDC supplemented with glycerol. Data represent mean and SEM, n = 3. (E) Chronological survival in the presence of various carbon sources using the in situ viability assay. Day 1 SDC wild type cultures were diluted and plated onto SC-Trp plates (no carbon source, CS), SC-Trp plates supplemented with glucose (2%, Glc), ethanol (0.8%, EtOH), or glycerol (3%, Gly), or agar plates (extreme CR). Data represent mean and SEM, n = 3–8. (F, G) One-day old SDC wild type cells were switched to water and incubated for 4 hours. The STRE- (F) and PDS-lacZ (G) activities were measured 2 and 4 after the addition of glucose, glycerol, or ethanol (final, 0.8%) and shown as the percentage of CR. Data shown are mean and SEM of four independent samples assayed. *, p<0.05; **, p<0.01; ***, p<0.001, Tukey's multiple comparison test, carbon source added vs. CR. (H) Chronological survival of wild type and msn2Δ msn4Δ gis1Δ mutants grown in normal (SC+2% glucose), reduced glucose (SC+1% glucose), or glucose/glycerol (SC+1%+1%) medium. Data represent mean and SEM of 4 cultures analyzed. (I) Day 3 wild type cells grown in SDC medium were washed three times with water and incubated in water (extreme CR/starvation) with or without glycerol (0.1% or 1%). Plot shows a representative experiment (mean of duplicates) repeated three times with similar results. (J) Yeast grown in SDC was sampled (1 ml) at indicated time points. [1,2,3-³H] Glycerol (ARC, Inc) was added to the aliquot and incubated at 30°C with shaking for 24 hours. Cells were then washed three times with water. The cellular [³H]-content was determined by scintillation counting (Wallac 1410, Pharmacia) and normalized to cell number (viability by CFU). Data represent mean and SEM of 4 cultures analyzed.