An <i>oxa1</i> deletion does not influence the strains growth rate.

<p>(A) Growth curves of an <i>oxa1</i> deletion strain and the corresponding wild-type strain. Experiment was performed in triplicate. The y-axis is scaled logarithmically. (B) Size of isolated colonies of the indicated strains grown for 3 and 5 days on SCGlu plates, respectively. Diameters were evaluated by processing the photos with Metamorph Imaging (mean±SEM, <i>n</i> = 2).</p

Abrogation of respiration suppresses apoptosis and ROS production in a maximal proliferating solid cell population.

<p>(A) Isolated colonies were grown from single cells, manipulated onto agar plates. Clonogenic assay of cells removed from colonies of the wild-type strain, rho⁰ (no mitochondrial DNA) and two single gene-deletion strains (<i>mgm1</i> and <i>oxa1</i>), grown on SCGlu. After 3 days 500 cells of whole colonies, after 5 days 500 cells of the central colony-region were analysed (mean±SEM, <i>n</i> = 2). Statistical significance of p<0.006 compares colony forming units (cfu) of mutant strains to wild-type strain at respective time-points. (B) Cells from the whole colony (3 days) as well as from the central region (5 days) were stained for reactive oxygen species (ROS) with dihydroethidium and analysed by FACSAria flow cytometry (mean±SEM, <i>n</i> = 2; **p<0.01, ***p<0.001 compared to deletion strains at respective time-points). (C) Approximately 50 (2 days) and 10 (3 days) colonies grown on SCGlu plates were washed off and clonogenic assays were performed with the collected cells (mean±SEM, <i>n</i> = 5; **p<0.01, ***p<0.001). (D) Approximately 50 colonies grown on SCGlu plates for 2 days were stained for phosphatidylserine exposition (AnnV) and DNA breakage (TUNEL) and analyzed by FACSAria flow cytometry (mean±SEM, <i>n</i> = 3; **p<0.01, ***p<0.001). (E) Fluorescence microscopy from central and outer region of 5 days old wild-type colony cells, harbouring plasmid dsRED-MLS.</p

MetR specifically regulates induction of autophagy.

<p>MET⁺, Δ<i>met15</i> and Δ<i>met2</i> 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) <i>MET2</i> deletion strain (Δ<i>met2</i>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s003" target="_blank">Figure S3</a>.</p

Model of MetR-mediated longevity.

<p>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.</p

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

<p>Fluorescent microscopy of acidic vacuoles during chronological aging of MET+, Δ<i>met2</i>, and Δ<i>met2</i>/Δ<i>atg5</i> 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 Δ<i>met2</i> and Δ<i>met2</i>/Δ<i>atg5</i> 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s006" target="_blank">Figure S6</a>.</p

Methionine determines yeast chronological lifespan.

<p>(A) Chronological aging of methionine prototroph (MET⁺), semi-auxotroph (Δ<i>met15</i>) and auxotroph (Δ<i>met2</i>) 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 Δ<i>met15</i> 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) <i>MET2</i> deletion strain (Δ<i>met2</i>) 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 Δ<i>met15</i> reference strain) and mating type α wild type strain BY4742, as well as a methionine semi-auxotrophic variant thereof (BY4742 Δ<i>met15</i>), 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s001" target="_blank">Figure S1</a>.</p

Autophagy is crucial for MetR-mediated longevity.

<p>(A) Chronological aging of <i>MET2</i> deletion strains carrying an additional gene deletion (Δ<i>atg5</i>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen-1004347-g004" target="_blank">Figure 4E</a>) (C) (n = 4). (D) Chronological aging of MET⁺ strain deleted for <i>TOR1</i>. 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 <i>TOR1</i> and <i>ATG5</i> or <i>ATG5</i> alone. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 4–6). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s004" target="_blank">Figure S4</a>.</p

MetR is epistatic to other longevity treatments involving <i>TOR1</i> inhibition.

<p>(A and B) Chronological aging of <i>MET2</i> and <i>MET15</i> deletion strains deleted for <i>TOR1</i>. Cell survival of 500 cells plated at given time points, normalized to cell survival on day one (n = 6). Chronological aging of <i>MET2</i> (C) and <i>MET15</i> (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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen-1004347-g003" target="_blank">Figure 3C</a>) (E) (n = 4). See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004347#pgen.1004347.s005" target="_blank">Figure S5</a>.</p