Genome-wide screen in Saccharomyces cerevisiae identifies vacuolar protein sorting, autophagy, biosynthetic, and tRNA methylation genes involved in life span regulation

Abstract

The study of the chronological life span of Saccharomyces cerevisiae, which measures the survival of populations of non-dividing yeast, has resulted in the identification of homologous genes and pathways that promote aging in organisms ranging from yeast to mammals. Using a competitive genome-wide approach, we performed a screen of a complete set of approximately 4,800 viable deletion mutants to identify genes that either increase or decrease chronological life span. Half of the putative short-/long-lived mutants retested from the primary screen were confirmed, demonstrating the utility of our approach. Deletion of genes involved in vacuolar protein sorting, autophagy, and mitochondrial function shortened life span, confirming that respiration and degradation processes are essential for long-term survival. Among the genes whose deletion significantly extended life span are ACB1, CKA2, and TRM9, implicated in fatty acid transport and biosynthesis, cell signaling, and tRNA methylation, respectively. Deletion of these genes conferred heat-shock resistance, supporting the link between life span extension and cellular protection observed in several model organisms. The high degree of conservation of these novel yeast longevity determinants in other species raises the possibility that their role in senescence might be conserved.

Figure 1. Screen of the yeast homozygous deletion collection for life span regulatory genes.

(A) CLS of the two pools used for the yeast deletion collection screen. The black arrows indicate when cell samples were collected for DNA extraction. The experiment was conducted by incubating the yeast pools in SDC medium throughout the experiment. (B) 10 aging profile clusters derived by K-means clustering analysis. The y-axis displays the log₂ fold ratio of tag intensity for each strain at each time point relative to the day 3 time point. Plots highlighted in red represent clusters classified as short-lived and the plot highlighted in green represents a cluster classified as long-lived. The dashed black line demarcates the boundary between short and long-lived strains. The red line is the centroid (average profile) for each cluster.

Figure 2. VPS genes are required for starvation/extreme CR–dependent life span extension and resistance to oxidative stress and acetic acid.

(A) CLS of wild type (BY4741) in SDC medium and under starvation/extreme CR. Starvation/extreme CR was obtained by transferring the yeast culture to water at day 3 (see Materials and Methods). The arrow indicates the time at which adaptive regrowth might be occurring. (B) CLS of wild type (BY4741) and of the vps36Δ, vps25Δ, vps8Δ, vps27Δ, and vps21Δ deletion mutants. Yeast cultures were transferred from medium to water at day 3. Data show mean±SEM of three experiments. (C) Resistance to H₂O₂ of the same mutants. After a 30 minute-exposure to 100–200 mM H₂O₂ in K-buffer, day 1–3 cells from SDC cultures were serially diluted and plated on YPD plates. D) Resistance to acetic acid of wild type cells, vps25Δ and vps27Δ mutants. Day 3 cultures were exposed to 300 mM of acetic acid for 3 hours before being serially diluted and plated onto YPD plates.

Figure 3. Novel long-lived mutants.

CLS of cultures under starvation/extreme CR of wild type (BY4741) and (A) acb1Δ, (B) cka2Δ, (C) trm9Δ, (D) ydr41cΔ, (E) aro7Δ, and (F) apd1Δ mutants. All figures show an average of 2–3 experiments except (F), which shows a representative experiment. The CFUs at day 3 before cells were transferred to water were: 131.3±5.1, 129.8±7.2, 111±8, 77.1±8.1, 169±2, 48.1±4.8, 152.1±3.3 (cells ×10⁶/mL±SEM) for wild type, acb1 Δ, cka2 Δ, trm9Δ, ydr41cΔ, aro7Δ, and apd1Δ, respectively.

Figure 4. Long-lived mutants are resistant to heat.

Day 3 chronologically aging wild type (BY4741) and the following mutants were serially diluted, plated onto YPD plates and heat-shocked at 55°C for 150–225 minutes: (A) acb1Δ, cka2Δ, cup9Δ, zta1Δ, apd1Δ, ydr417c Δ, (B) trm9Δ and aro7Δ; in the control panel (highest dilution factor) the arrows indicate the size of the trm9Δ colonies (row 2) in comparison with that of the wild type ones (row 1), and (C) cka1Δ, cka2Δ, ckb1Δ, ckb2Δ. (D) Day 5 cultures of wild type, acb1Δ, cka2Δ, ydr417cΔ, and sch9Δ were exposed to 500 mM acetic acid for 180 minutes, serially diluted, and plated onto YPD plates. (E) Day 5 cultures of wild type, trm9Δ, and aro7Δ exposed to 400 mM acetic acid for 180 minutes. (F) Day 3 cultures of wild type and acb1Δ transformed with either a centromeric plasmid carrying a wild type ACB1 gene or a control vector were serially diluted, plated onto YPD plates, and heat-shocked at 55°C for 240 minutes. The cultures used for the stress resistance experiments were in either SDC (wild type) or SDC-uracil (acb1Δ).

Figure 5. CK2 regulates life span in different genetic backgrounds.

(A) CLS of wild type (W303-1A) and cka2Δ. (B) CLS of DBY746 and of mutants lacking either Cka2 or Ckb2. (C) CLS of wild type DBY746 treated with increasing concentrations of TBBz (10–200µM) at day 2 and 5. DMSO was used as a vehicle. (A,B) show an average of 2–3 experiments. A representative experiment is shown in (C). All the survival studies were performed leaving the yeast cultures in medium until the end of the experiment.

Figure 6. Role of CK2 in the regulation of heat-shock resistance.

(A) Heat-shock resistance of two different cka2Δ isolates generated in the DBY746 genetic background monitored at day 1. (B) Heat-shock resistance of DBY746 and of two different cultures of the ckb2Δ mutant at day 1–3. (C) Heat-shock resistance of DBY746 yeast treated with 10, 50, 200 µM of TBBz at day 2.

Figure 7. Role of TRM9 and YDR417C in life span regulation and heat resistance.

CLS of DBY746 and mutants lacking either (A) Trm9, or (B) Ydr417c. (C) CLS of BY4741 and rpl12bΔ. (D) Day 3 heat-resistance of two different isolates of the trm9Δ mutant generated in the DBY746 background and of a ydr417cΔ mutant. In the control panel (highest dilution factor) the arrows indicate the colony size of the long-lived trm9Δ and ydr417cΔ mutants (row 2–4) as compared to that of the wild type (row 1). (E) Heat-resistance of BY4741 and of a mutant lacking Rpl12b at day 3. The survival studies in (A,B) were conducted incubating the cultures in SDC medium until the end of the experiment. The data shown represent an average of 2–3 experiments. Yeast cultures in (C) were transferred to water at day 3 (see Materials and Methods). A representative experiment is shown.

Figure 8. Efficiency of G1/G0-arrest of the novel long-lived mutants.

Budding index of BY4741 and mutants lacking either Acb1, Cka2, Trm9, Ydr417c, or Aro7 measured during exponential growth (OD₆₀₀ = 1) and chronological aging on day 1, 3, and 7. Data show mean±SEM. * p<0.01, trm9Δ vs WT. **p<0.05, p<0.001, p<0.01, acb1Δ vs WT in exponential phase, on day 1, and on day 3–7, respectively. ∧ p<0.001, p<0.01, aro7Δ vs WT, in exponential phase, day 7, and on day 3, respectively. # p<0.05, ydr417cΔ vs WT. The cultures used were incubated in SDC medium for the duration of the whole experiment.