Three genetically distinct pathways modulating replicative life span. Based on evidence from many labs, it now seems clear that at least 3 genetically distinct pathways modulate RLS. Sir2 and Fob1 influence RLS largely through their role in the rDNA, although there is evidence for aging-related functions for Sir2 at other loci, including telomeres, as well as by modulating asymmetric inheritance of cytoplasmic damage. DR is mediated at least in part through reduced TOR/PKA/Sch9 signaling. Ubr2 and Rpn4 appear to influence RLS by modulating proteasome activity. The relationship of the retrograde response and mitochondrial dysfunction to these pathways remains unclear, although there is some evidence for altered Sir2 activity in one mitochondrial translation factor mutant. Dotted lines represent potential points of cross-talk between pathways that have been proposed.

(A) The standard 2% glucose (SDC) CLS assay. Overnight cultures are diluted (1:200) into 10 ml of fresh SDC medium (with flask to culture volume of 5:1) at 30°C with shaking (200 rpm) to ensure equal aeration of all cells. Cells grow logarithmically until they reach the mostly non-dividing high metabolism post-diauxic phase within 24 hours. Notably, aluminum foil, which reduces the level of oxygen in the flask, is used to cap the flask. The inoculation time point is time 0. Every two days, aliquots from the culture are diluted according to the estimated survival and plated on to YPD (Yeast Peptone Dextrose) plates. The YPD plates are incubated at 30°C for 2–3 days and viability in the flask is estimated by Colony Forming Unit (CFUs) counts. Viability at day 3, when the great majority of the cells stop dividing, is considered to be the initial survival (100%). Representative results of chronological survival of the wild type (DBY746), sch9Δ, tor1Δ and ras2Δ are shown.
(B) For extreme CR/starvation, cells from three-day-old SDC culture are washed three times with sterile distilled water, and resuspended in water. Cells are incubated in water at 30°C with shaking. Every 2–4 days, cells from the water cultures are washed to remove nutrients released from dead cells. For CR modeled by glucose reduction, overnight SDC cultures are diluted (1:200) into fresh SC medium supplemented with 0.5% instead of 2% glucose.
(C) Chronological survival in the presence of various carbon sources using the in situ viability assay. Day one SDC cultures are diluted and plated onto at least 10 agar plates (extreme calorie restriction) or tryptohpan drop-out (SC-TRP) plates (only trp auxotrophic cells). Plates are incubated at 30°C for the duration of the assay. Every two days, one plate from each set is retrieved and either tryptophan or the required nutrients are added to allow the growth and colony formation by the surviving cells.

Yeast Chronological Life Span Major Regulatory Pathways. The nutrient-sensing pathways controlled by Sch9, Tor, and Ras converge on the protein kinase Rim15. A major portion of the effect of CR on longevity appears to be mediated by the down-regulation of the Ras/AC/PKA and Tor/Sch9 pathways and consequent activation of the Rim15-controlled Msn2/4 and Gis1 stress-responsive transcription factors. Reduced Tor/Sch9 signaling (genetic mutations or rapamycin) also increases coupled mitochondrial respiration and membrane potential (Δψ m) during growth phase, which leads to an adaptive mitochondrial ROS signal. During stationary phase (i.e. chronological aging), Tor and Sch9 deficiencies and adaptive mitochondrial ROS signaling decrease ROS production and enhance cellular stress responses, culminating in life span extension.