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.

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), 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. 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.

Figure 2

A low concentration of rapamycin induces macroautophagy. WT cells containing plasmid pCuGFPAUT7(416) were grown in liquid SD medium containing essential amino acids (H, K and L), 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).