Lifespan extension by methionine restriction requires autophagy-dependent vacuolar acidification

Abstract

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.

Figure 1. Methionine determines yeast chronological lifespan.

(A) Chronological aging of methionine prototroph (MET⁺), semi-auxotroph (Δmet15) and auxotroph (Δmet2) isogenic yeast strains in SCD media supplemented with all amino acids (aa). Cell survival was estimated as colony formation of 500 cells plated at given time points, normalized to cell survival on day one (n = 4). (B) Chronological aging of Δmet15 strain, in SCD media supplemented with all aa except for methionine which was added at given concentrations. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 4). (C) MET2 deletion strain (Δmet2) was grown to stationary phase in SCD (supplemented with all aa) and shifted to SCD media with different methionine concentrations. Cell survival of 500 cells plated at given time points, normalized to cell survival before the shift (n = 4). (D) Day 8 from experiment shown in C (n = 4). (E) Chronological aging of EUROSCARF BY4741 (also used above as Δmet15 reference strain) and mating type α wild type strain BY4742, as well as a methionine semi-auxotrophic variant thereof (BY4742 Δmet15), in SCD media supplemented with all aa. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 3). See also Figure S1.

Figure 2. MetR specifically regulates induction of autophagy.

MET⁺, Δmet15 and Δmet2 strains from chronological aging experiments were analyzed for vacuolar ALP activity (with a fluorescent plate reader) (A) (n = 6), and GFP-Atg8p processing (by Western-blot analysis) (B). (C) GFP-Atg8p localization was determined by using fluorescent microscopy (white arrows indicate vacuolar localization or autophagosome formation) and statistical analysis thereof (330–600 cells of each GFP-Atg8p expressing strain were evaluated from two independent samples) (D). (E) MET2 deletion strain (Δmet2) was grown to stationary phase in SCD (supplemented with all aa) and shifted to SCD media with given methionine concentrations. Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) (n = 6). (F) ALP assays of chronological aging of MET⁺ strain, in SCD media supplemented with all aa except for methionine which was added at given concentrations (n = 2). See also Figure S3.

Figure 3. Autophagy is crucial for MetR-mediated longevity.

(A) Chronological aging of MET2 deletion strains carrying an additional gene deletion (Δatg5) and MET⁺ strain in SCD media supplemented with all aa. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6). (B) Chronological aging of MET⁺ strain treated with indicated amounts of rapamycin (Rap). Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 3; p***). Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) and normalized to untreated controls at indicated time points (also compare to Figure 4E) (C) (n = 4). (D) Chronological aging of MET⁺ strain deleted for TOR1. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6; p***). (E) Chronological aging of the MET⁺ strain deleted for TOR1 and ATG5 or ATG5 alone. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 4–6). See also Figure S4.

Figure 4. MetR is epistatic to other longevity treatments involving TOR1 inhibition.

(A and B) Chronological aging of MET2 and MET15 deletion strains deleted for TOR1. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6). Chronological aging of MET2 (C) and MET15 (D) deletion strains treated with indicated amounts of rapamycin (Rap). Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 3). Autophagy was measured by means of ALP activity with a fluorescent plate reader (Tecan, Genios Pro) and normalized to untreated controls at indicated time points (also compare to Figure 3C) (E) (n = 4). See also Figure S5.

Figure 5. MetR enhancement of vacuolar acidification is autophagy-dependent and necessary for longevity.

Fluorescent microscopy of acidic vacuoles during chronological aging of MET+, Δmet2, and Δmet2/Δatg5 strains, by means of quinacrine accumulation and statistical analysis thereof. (>1000 cells of each strain from 3 to 5 independent samples at each time point were evaluated. Only cells with acidic vacuoles without an additionally stained cytoplasm were counted as positive, resulting in cell counts that represent cells which have a clearly intact pH-homeostasis. Positively counted cells are indicated by white arrowheads) (A and B). (C) Statistical analysis of fluorescent microscopy of acidic vacuoles by means of quinacrine accumulation. Strains Δmet2 and Δmet2/Δatg5 were grown to stationary phase under excess of methionine and shifted to media with the indicated amounts of methionine (>500 cells from each strain from 2 independent samples) and assayed for quinacrine accumulation after ∼20 hours (D) Chronological aging of the MET⁺ strain overexpressing Vma1p or Vph2p. Cell death was measured via propidium iodide staining of cells that have lost integrity and subsequent flow cytometry analysis (BD LSRFortessa) (n = 6 to 8). See also Figure S6.

Figure 6. Model of MetR-mediated longevity.

MetR specifically enhances autophagy, either by interfering upstream of TOR-pathway or presumably by impinging on (metabolic) pathways that potentially target autophagy directly, downstream of the TOR-pathway. MetR-specific vacuolar acidification is dependent on autophagy and elongates CLS. High levels of methionine inhibit autophagy induction during early phases of chronological aging, enhancing ROS and diminishing acidic vacuoles in a cell population, which leads to cell death.