Water-Transfer Slows Aging in <i>Saccharomyces cerevisiae</i>

<div><p>Transferring <i>Saccharomyces cerevisiae</i> cells to water is known to extend their lifespan. However, it is unclear whether this lifespan extension is due to slowing the aging process or merely keeping old yeast alive. Here we show that in water-transferred yeast, the toxicity of polyQ proteins is decreased and the aging biomarker 47Q aggregates at a reduced rate and to a lesser extent. These beneficial effects of water-transfer could not be reproduced by diluting the growth medium and depended on <i>de novo</i> protein synthesis and proteasomes levels. Interestingly, we found that upon water-transfer 27 proteins are downregulated, 4 proteins are upregulated and 81 proteins change their intracellular localization, hinting at an active genetic program enabling the lifespan extension. Furthermore, the aging-related deterioration of the heat shock response (HSR), the unfolded protein response (UPR) and the endoplasmic reticulum-associated protein degradation (ERAD), was largely prevented in water-transferred yeast, as the activities of these proteostatic network pathways remained nearly as robust as in young yeast. The characteristics of young yeast that are actively maintained upon water-transfer indicate that the extended lifespan is the outcome of slowing the rate of the aging process.</p></div

Without Rpn4, yeast survives poorly and is only partially rescued by water-transfer.

<p>BY4741 or Δ<i>rpn4</i> cells derived from them were grown in SC with 2% glucose. At 0.5–0.9 A₆₀₀ (logarithmic growth) cells were either transferred to water or maintained in SC with glucose. On the indicated days (A) 10-fold serial dilutions (starting with 7.5x10⁶ cells) were spotted on YPD plates, and (B) viability was monitored by PI staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148650#pone.0148650.g001" target="_blank">Fig 1B</a>. (C) Growth curves were plotted from 3 biological replicates and technical triplicates within each biological replicate. Data are presented as mean ± SE.</p

Survival in water and its effect on aggregation require continuous protein synthesis.

<p>(A) W303-1b cells expressing the 25Q were grown in SC-Ura supplemented with 2% fructose (Fru). Galactose (4%) was added either immediately (Gal 0) or on the indicated following days (Gal 1–9). On the indicated days afterwards, cells were collected and lysed, and proteins were dot-blotted onto nitrocellulose. Levels of 25Q and actin were determined by quantitative immunoblotting using rabbit anti-GFP and mouse anti-actin antibodies, respectively, as descried in Materials and Methods. (B-D) W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose induction. After 18 hours, cells were either kept in SC-Ura with galactose or in SC-Ura with galactose supplemented with CHX (20 μg/ml), or transferred to water or to water supplemented with CHX. On the indicated days, (B) Aggregation Index was calculated, (C) growth curves were plotted (mean ± SE of 2 biological replicates and technical triplicates within each biological replicate), and (D) CFU viability was tested by spotting on YPD plates. (E) W303-1b or BY4741 cells, logarithmically grown (0.5–0.9 A₆₀₀) in SC containing 2% glucose were either maintained in the same medium or transferred to water at logarithmic phase. Upon transfer (log) or on the following day (day 1), CHX (20 μg/ml) was added. Cells collected on the indicated days were tested for CFU viability on YPD plates. Shown is a representative experiment out of similar three.</p

Effects of water-transfer on aggregation and toxicity cannot be reproduced by medium dilution.

<p>W303-1b cells expressing the 47Q were logarithmically grown in SC-Ura under galactose induction. After 17 hours, cells were either maintained in SC-Ura with galactose, transferred to water, or one volume of their SC medium was diluted with the indicated volumes of water, without changing the cell concentration. On the indicated days, (A) Aggregation Index was calculated, (B) growth curves were plotted (mean ± SE of 3 biological replicates and technical triplicates within each biological replicate), and (C) CFU viability was tested by spotting on glucose or galactose plates.</p

The HSR, UPR and ERAD are compromised in aging yeast but maintained upon water-transfer.

<p>Logarithmically growing (log) BY4741 cells harboring either HSE2-<i>LacZ</i> (A), UPRE-<i>LacZ</i> (B), CPY*-HA (C,E) or 6myc-Hmg2 (D,F) plasmids were either maintained in SC-Ura with glucose or transferred to water. On the indicated days, (A) cells were either subjected to a 20 minutes heat shock at 42°C (HS) or maintained for 20 minutes at 30°C (control); (B) cells were incubated for 1 hour without (control) or with either tunicamycin (TM; 5 μg/ml) or dithiothreitol (DTT; 6 mM). HSR (A) and UPR (B) were monitored as β-galactosidase specific activity. (C-F) Cells were subjected to CHX chase at log phase (log) or after 2 days in either SC-Ura with glucose or water. At the indicated time points after CHX addition, cells were collected and lysed and proteins were resolved by SDS-PAGE. CPY*-HA (C,E) and 6myc-Hmg2 (D,F) were detected by immunoblotting (IB) and quantified using Odyssey Infrared Imaging System. Asterisk, instrument’s settings were adjusted to view the low levels of 6myc-Hmg2. (E) Quantified data from 3 biological replicates are presented as mean ± SE. (F) A representative experiment out of 2 biological replicates.</p