Autophagy is required for extension of yeast chronological life span by rapamycin

Abstract

Rapamycin is an antibiotic that stimulates autophagy in a wide variety of eukaryotes, including the budding yeast Saccharomyces cerevisiae. Low concentrations of rapamycin extend yeast chronological life span (CLS). We have recently shown that autophagy is required for chronological longevity in yeast, which is attributable in part to a role for autophagy in amino acid homeostasis. We report herein that low concentrations of rapamycin stimulate macroautophagy during chronological aging and extend CLS.

The nutrient-sensing target of rapamycin (TOR) pathway is implicated in the aging process in many organisms.^1-3 In yeast, the TOR signaling pathway has been implicated in both replicative and chronological aging as well as in life-span extension by calorie restriction.^3-6 In general, reduced signaling through the TOR pathway, achieved by either genetic or pharmacological means, results in increased longevity. Two underlying mechanisms, which are not mutually exclusive, are proposed: increased stress resistance and increased respiration.^4,5 The most well-known small molecule inhibitor of TOR signaling is rapamycin. Rapamycin (Sirolimus) is a macrolide antibiotic with anti-fungal and immunosuppressive properties that inhibits TOR kinase complexes, which leads to inhibition of translation and growth arrest.⁷ Inhibition of TOR signaling by rapamycin upregulates autophagy in many organisms, including yeast.⁸ However, inhibition of TOR signaling by rapamycin has an impact on many metabolic pathways other than autophagy.⁹ This raises the question: is autophagy required for extension of CLS by rapamycin treatment? To address this question, we have studied the effects of rapamycin on CLS in autophagy-deficient yeast and measured induction of macroautophagy by exposure to a low concentration of rapamycin that extends yeast CLS.

Autophagy is Required for Rapamycin to Extend CLS

If rapamycin extends CLS by upregulating autophagy, then rapamycin should not extend CLS in atg mutants. To test this prediction, life spans were measured in synthetic dextrose (SD) minimal medium containing concentrations of rapamycin (0.1–40 nM) that were well below the standard cytostatic dose (220 nM or 0.2 μg/ml). Four strains were compared: hoΔ, atg1Δ, atg7Δ and atg11Δ. The hoΔ strain has a “wild-type” capacity for autophagy, and is referred to as “WT” below. Atg1 is a protein serine/threonine kinase required for macroautophagy.¹⁰ Atg7 is an E1-type ubiquitin-activating enzyme that executes two steps required for autophagosome formation: conjugation of Atg12 (a ubiquitin-like modifier) to Atg5 and attachment of PE to Atg8.¹¹ An atg11Δ mutant was used as an additional control. ATG11 is not required for macroautophagy, but is required for the Cvt pathway and selective degradation of peroxisomes by microautophagy (pexophagy).¹²

Rapamycin at 10, 20 and 40 nM extended CLS in the WT and atg11Δ control strains as expected, but no extension was observed in atg1Δ or atg7Δ strains (Fig. 1B). Rapamycin at 0.1 or 1 nM final concentration did not extend CLS (Fig. 1A), although 0.33 nM (300 pg/ml) and 1.1 nM (1 ng/ml) rapamycin are reported to extend CLS in YPD.⁵ Cultures containing 10 nM or less rapamycin reached saturation after approximately 24 h of growth on day 1 of the CLS experiment. However, 20 nM and 40 nM rapamycin treatments slowed growth such that cultures achieved saturation on day 3 and 5 of the CLS experiment, respectively (data not shown). Similarly, treatment at 100 nM and 150 nM, which also extended CLS, delayed saturation until day 5 (data not shown). Importantly, however, the effects of rapamycin on growth rate were observed in both control and autophagy-deficient strains and there was no correlation between the time required to achieve saturation and chronological longevity.

Figure 1.

Autophagy is required for extension of chronological life span (CLS) by low concentrations of rapamycin. CLS was determined for yeast strains grown in liquid synthetic dextrose (SD) minimal medium containing essential supplements (H, K, L and uracil),¹⁵ 250 μg/ml G₄₁₈, 0.1% ethanol (solvent), and rapamycin at final concentrations ranging from 0–10 nM (A) or 0–40 nM (B). Strains in the BY4742 background contained the deletions hoΔ (WT), atg1Δ, atg7Δ or atg11Δ.¹⁶ Viability in terms of percent of colony forming units (CFU) observed on day 1 is plotted on a log scale over the 24 day time course of this experiment. Rapamycin was added on day zero to cultures containing a low cell density (OD₆₀₀ ∼ 0.01). Cultures were grown to saturation (OD₆₀₀ ∼ 1) and maintained at 30°C in a drum rotator at ∼15 rpm. For methodological details, see reference 13.

Autophagy and regulation of amino acid homeostasis are important factors in determining chronological longevity in yeast.¹³ Macroautophagy-deficient strains exhibit reduced chronological life span (CLS) in SD minimal medium compared to control strains. Increased availability of essential and nonessential branched side chain amino acids extends the CLS of macroautophagy-deficient strains by a mechanism that likely involves downregulation of the transcription factor Gcn4, which regulates general amino acid control. Furthermore, certain growth conditions, such as media containing low glucose or galactose, extend the CLS of autophagy-deficient yeast (unpublished results). These findings indicate that autophagy-deficient yeast are not necessarily short-lived relative to controls. Thus, the inability of rapamycin to extend CLS in autophagy-deficient strains is not simply due to the fact that these strains are inherently short-lived.

Rapamycin Promotes Autophagy during Chronological Aging

The observation that low concentrations of rapamycin extend CLS in autophagy-competent yeast raises the question: do low concentrations of rapamycin induce macroautophagy during chronological aging? To answer this question, a CLS experiment was done as described above (Fig. 1) and cells were collected and analyzed using a well-known assay for macroautophagy that is based on proteolytic conversion of a GFP-Atg8 fusion protein to GFP.¹⁴ During logarithmic growth, on day 0, macroautophagy is not induced, as expected (Fig. 2A). Macroautophagy is induced on day 1 of a CLS experiment, at which point the culture is saturated (Fig. 2A). However, during days 2–5, the level of macroautophagy was very low or not detectable (Fig. 2A). In contrast, growth in the presence of 10 nM rapamycin results in a prolongation of macroautophagic activity through day 12 of the CLS experiment (Fig. 2B). We conclude that a low concentration of rapamycin that is capable of extending CLS is also capable of upregulating macroautophagy during chronological aging.

Figure 2.

A low concentration of rapamycin induces macroautophagy. WT cells containing plasmid pCuGFPAUT7(416)¹⁷ were grown in liquid SD medium containing essential amino acids (H, K and L),¹⁵ 250 μg/ml G₄₁₈, and either no rapamycin (A) or 10 nM rapamycin (B). Cultures reached saturation (OD₆₀₀ ∼ 1) on day 1. Cells were collected on the indicated days and equivalent amounts of whole cell extracts (based on OD₆₀₀ cell density measurements) were analyzed by western blotting with the polyclonal anti-GFP antibody ab290 (Abcam, Inc.,). Macroautophagy-dependent proteolysis of the GFP-Atg8 fusion protein yields the GFP band, which is relatively stable to digestion by vacuolar proteinases. (B) shows days 3–12 to illustrate detection of the GFP band over a longer period of time than is evident in (A). Days 1 and 2 in (B) were comparable to lane 3 in (B); day 0 in (B) was equivalent to day 0 in (A) (data not shown).

Conclusions

Our findings suggest that upregulation of autophagy contributes to extension of CLS by treatment of yeast with low-dose rapamycin. It is becoming increasingly clear that modulation of autophagy contributes to chronological longevity via a number of different mechanisms, including nutrient recycling, organelle turnover and clearance of aberrant, damaged or toxic macromolecules. Small molecule interventions that upregulate autophagy offer the potential for enhancing health and life span, and an effective means for accomplishing this in vivo may be modulation of the TOR pathway.