Climbing activity.

<p><b>A)</b> Percentage of flies able to climb 8 cm in 10 seconds in wild type and atg7^−/− male flies fed 1 mM spermidine or not. 3 independent replicates were monitored (50 flies in each group and replicate). <b>B)</b> Percentage of flies able to climb 8 cm in 10 seconds in wild type and atg7^−/− female flies fed 1 mM spermidine or not. 3 independent replicates were monitored (50 flies in each group and replicate). *p<0.05; ***p<0.001.</p

Food intake, glycogen and protein contents.

<p><b>A)</b> Mean +/− SEM of food intake in wild type flies of both sexes fed 1 mM spermidine or not as measured by colorimetry (flies fed blue dye and dye intensity measured). 2 independent replicates were monitored (5 or 6 independent samples for each group in each replicate). <b>B, C)</b> Mean +/− SEM of glycogen (B) and protein (C) content in wild type or atg7^−/− flies of both sexes fed 1 mM spermidine or not as measured by colorimetry (anthrone reaction for B and Bradford reaction for C). 3 independent replicates were monitored (4 or 5 independent samples for each group in each replicate). **p<0.01; ***p<0.001.</p

Phospholipid profile of males positive survey.

<p><b>A–D)</b> Positive ion ES-MS survey scans (600–850 m/z) of total lipid extracts from male wild type (A, atg7^+/+) and atg7^−/− (B) flies as well as from spermidine-fed male wild type (C, atg7^+/+) and atg7^−/− (D) flies. Arrows indicate significant changes compared to normal flies untreated.</p

Phospholipid profile of females positive survey.

<p><b>A–D)</b> Positive ion ES-MS survey scans (600–1000 m/z) of total lipid extracts from female wild type (A, atg7^+/+) and atg7^−/− (B) flies as well as from spermidine-fed female wild type (C, atg7^+/+) and atg7^−/− (D) flies. Arrows indicate significant changes compared to normal flies untreated.</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

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

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

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

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