Longevity pathways and maintenance of the proteome: the role of autophagy and mitophagy during yeast ageing

Abstract

Ageing is a complex and multi-factorial process that results in the progressive accumulation of molecular alterations that disrupt different cellular functions. The budding yeast Saccharomyces cerevisiae is an important model organism that has significantly contributed to the identification of conserved molecular and cellular determinants of ageing. The nutrient-sensing pathways are well-recognized modulators of longevity from yeast to mammals, but their downstream effectors and outcomes on different features of ageing process are still poorly understood. A hypothesis that is attracting increased interest is that one of the major functions of these "longevity pathways" is to contribute to the maintenance of the proteome during ageing. In support of this hypothesis, evidence shows that TOR/Sch9 and Ras/PKA pathways are important regulators of autophagy that in turn are essential for healthy cellular ageing. It is also well known that mitochondria homeostasis and function regulate lifespan, but how mitochondrial dynamics, mitophagy and biogenesis are regulated during ageing remains to be elucidated. This review describes recent findings that shed light on how longevity pathways and metabolic status impact maintenance of the proteome in both yeast ageing paradigms. These findings demonstrate that yeast remain a powerful model system for elucidating these relationships and their influence on ageing regulation.

Keywords: ageing; autophagy; chronological lifespan; mitophagy; nutrient-sensing pathways; replicative lifespan; yeast.

Figure 1. FIGURE 1: SIR2 deletion does not have a major impact on CLS either in normal growth conditions or under caloric restriction.

Chronological lifespan of wild type (background BY4741) or sir2Δ cells submitted to (A) non-caloric restriction (non-CR) or (B) caloric restriction (CR) conditions. All experiments were performed in synthetic complete (SC) medium containing yeast nitrogen base and glucose, as a carbon source, supplemented with the appropriate amino acids and bases. The concentration of glucose used was 2% in non-CR conditions or 0.5% to promote CR. Cells were incubated at 26°C with shaking at 150 rpm. Cultures reached stationary phase 2 days later and this was considered day 0 of CLS. Survival was assessed by counting colony-forming units (CFUs) beginning at day 0 of CLS (when viability was considered to be 100%), and then again every 2-3 days until less than 0.1% of the cells in the culture were viable. The data represent mean ± SEM of three biological independent replicas. No statistical significance was obtained between the CLS curves presented both in panel A and in panel B, as determined by two-way ANOVA.

Figure 2. FIGURE 2: Autophagy regulation by nutrient-sensing pathways.

Tor1 is the main negative regulator of autophagy. Tor1 can directly exert its negative regulation through phosphorylation of Atg13 and Atg1 or through its downstream target, PP2A (serine/threonine protein phosphatase 2A), a negative regulator of autophagy. Atg1 and Atg13 could also be phosphorylated by PKA, at residues distinct from those targeted by Tor1. PKA and Sch9 pathways are also negative regulators of autophagy. Apparently, TOR and PKA pathways operate in parallel, whereas Sch9 acts in parallel with PKA and partially dependent on TOR. Regulation of autophagy by Sch9 and PKA can be mediated by the inhibition of Rim15 and the Msn2/4 transcription factors. Tor1 is also able to inhibit the activity of the metabolic sensor Snf1, which is a positive regulator of autophagy by still unknown mechanisms. Green arrows indicate interactions that induce autophagy, red bars indicate inhibition. See the text for details.

Figure 3. FIGURE 3: SIR2 deleted cells exhibit increased mitochondrial mass and altered mitochondria network.

Wild type and sir2Δ (background BY4741) cells expressing, GFP-Atg8 and mtDsRed were analyzed for mitophagy by confocal fluorescence microscopy. The cells were collected and analyzed at day 3 of chronological lifespan. Mitophagy was analyzed by the pattern of co-localization between GFP-Atg8 and mtDsRed, as exemplified. Images were acquired in a confocal Olympus FLUOVIEW microscope with an Olympus PLAPON 60X/oil objective, with a numerical aperture of 1.35. GFP and DsRed were excited with and argon laser and a helium-neon laser (GFP: 488 nm excitation; DsRed: 559 nm excitation). Background reduction was performed with appropriate saturation levels using software FV1000 (Olympus) and Adobe Photoshop CS. Image stacks for analysis were acquired with sequential steps of 0.25 to 0.5 µm per plane in the z-direction and a total thickness of 4-6 µm. The acquired stacks were rendered with FV1000 software. Scale bars: 5 µm.