Hog1 phosphorylation is delayed in severe hyper-osmotic stress.

<p>Western blot of Hog1 phosphorylation in wild type treated with 400mM and 800mM NaCl at time “0”. The upper blot was treated with antibody recognizing dually phosphorylated Hog1, the lower panel with an antibody that detects total Hog1.</p

The Hog1 nuclear import rate is similar for cells in different osmotic stress conditions.

<p><b>A</b>. FRAP (Fluorescence Recovery After Photobleaching) experiments on Hog1-GFP (Nrd1-mCherry as nuclear marker) in wild type cells to measure the rate of Hog1 nuclear import under two different osmostress conditions, 400mM and 800mM NaCl. The recovery curves, i.e. the mean intensity in a nuclear bleached region as a function of time, represent the average of the individual GFP-recovery curves for 15 cells. All measurements were performed after cells were treated with salt for 2.5 minutes. Subsequently, the area of the nucleus was bleached and the times on the x-axis represent the period after which the measurements were started. The recovery curves are fitted with a double exponential fit. <b>B</b>. Box plots for the fast and slow recovery half times from double exponential fits for 400 and 800 mM NaCl. The bottom and top of the boxes present the first and third quartiles. The diamond and dash line show the mean and median respectively. Whiskers indicate the variability of recovery half times outside the upper and lower quartiles. The data are consistent with two different mechanisms of Hog1 nuclear import under osmostress, a slow and probably passive mechanism as well as a fast and probably active mechanism. </p

Hog1 nuclear accumulation correlates with cell volume recovery dynamics.

<p><b>A</b>. Relative cell volume changes of cells treated with different concentrations of NaCl in wild type and the <i>gpd1∆ </i><i>gpd2∆</i> mutant. Colors indicate different salt concentrations and symbol sizes show the standard deviation for each time point. Ca. 60 cells were monitored. <b>B</b>. Relative cell volume (untreated cells = 1.0) as a function of NaCl concentration in wild type cells. Data represent the average of about 60 cells and the error bars indicate the standard deviation between cells. Cell volume was monitored over time and for each cell the lowest volume value at the relevant salt concentration was used to calculate the average. <b>C</b>. The time point at which Hog1 nuclear concentration reaches its maximum as a function of relative cell volume compression. Data on the y-axis represent the average time point of maximal Hog1 nuclear localization of about 60 cells and the vertical errors bars present the variation between the time points in which cells reach their Hog1 maximum localization. Data on the x-axis represent maximal relative cell volume reduction of those cells and the horizontal bars the standard deviation between cells. <b>D</b>. Changes of relative cell volume (left panels) and Hog1-GFP nuclear localization (right panels) upon treatment with 400mM and 800mM NaCl in wild type, <i>ptp2∆</i>, <i>ptp3∆</i>, and <i>fps1∆</i> mutants. Colors represent the different salt concentrations and symbol sizes indicate the standard deviation for each time point. Data represent about 60 cells for wild type and ca. 30 cells for each mutant.</p

Nuclear accumulation of Hog1 is delayed under severe hyperosmotic stress.

<p><b>A</b>. Scheme of the HOG signaling pathway. Upon hyperosmotic shock a branched cascade mediates dual phosphorylation and activation of Hog1. Phosphorylated Hog1 is then translocated into the nucleus where it associates with different DNA-binding proteins to mediate transcriptional regulation. <b>B</b>. Ratio of Hog1-GFP between nucleus and cytosol as a function of time in wild type cells. At time “0” the medium was adjusted to 400mM and 800mM NaCl, respectively. Data represent values for ca. 60 cells for each condition. <b>C</b>. Confocal time lapse images of the nuclear localization of Hog1 in 400mM and 800mM NaCl. Hog1 nuclear localization is delayed under severe osmotic condition (800mM NaCl). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080901#pone.0080901.s002" target="_blank">Figure S2</a> for control data including Nrd1-mCherry, which marks the nucleus. <b>D</b>. Mean ratio of nuclear versus cytosolic Hog1-GFP of about 60 cells as a function of time upon different stress levels ranging from 100mM to 1,000mM NaCl. Colors symbolize the different salt concentrations and symbol sizes correspond to the standard deviation for each time point as indicated.</p

Quantitative Analysis of Glycerol Accumulation, Glycolysis and Growth under Hyper Osmotic Stress

<div><p>We provide an integrated dynamic view on a eukaryotic osmolyte system, linking signaling with regulation of gene expression, metabolic control and growth. Adaptation to osmotic changes enables cells to adjust cellular activity and turgor pressure to an altered environment. The yeast <i>Saccharomyces cerevisiae</i> adapts to hyperosmotic stress by activating the HOG signaling cascade, which controls glycerol accumulation. The Hog1 kinase stimulates transcription of genes encoding enzymes required for glycerol production (Gpd1, Gpp2) and glycerol import (Stl1) and activates a regulatory enzyme in glycolysis (Pfk26/27). In addition, glycerol outflow is prevented by closure of the Fps1 glycerol facilitator. In order to better understand the contributions to glycerol accumulation of these different mechanisms and how redox and energy metabolism as well as biomass production are maintained under such conditions we collected an extensive dataset. Over a period of 180 min after hyperosmotic shock we monitored in wild type and different mutant cells the concentrations of key metabolites and proteins relevant for osmoadaptation. The dataset was used to parameterize an ODE model that reproduces the generated data very well. A detailed computational analysis using time-dependent response coefficients showed that Pfk26/27 contributes to rerouting glycolytic flux towards lower glycolysis. The transient growth arrest following hyperosmotic shock further adds to redirecting almost all glycolytic flux from biomass towards glycerol production. Osmoadaptation is robust to loss of individual adaptation pathways because of the existence and upregulation of alternative routes of glycerol accumulation. For instance, the Stl1 glycerol importer contributes to glycerol accumulation in a mutant with diminished glycerol production capacity. In addition, our observations suggest a role for trehalose accumulation in osmoadaptation and that Hog1 probably directly contributes to the regulation of the Fps1 glycerol facilitator. Taken together, we elucidated how different metabolic adaptation mechanisms cooperate and provide hypotheses for further experimental studies.</p></div

Contribution of glycerol accumulation mechanisms in different strains.

<p>(<b>A, B</b>) Absolute fluxes towards glycerol as well as relative contributions of specific mechanisms differ between wild type WT (<b>A</b>) and <i>gpd1Δ</i> (<b>B</b>). (<b>C</b>) Changes in relative contributions of Fps1, Gpd1, and other effects (basal glycerol production, uptake through Stl1, effects of volume change) over time are depicted for WT, <i>fps1</i>-<i>Δ1</i> and <i>hog1Δ</i>. Colors in (C) indicate time as shown on the x-axis in (A).</p

Effect of salt stress on growth rate.

<p><b>A</b>: <i>In vivo</i> doubling times (−: before, +: after addition of 0.4 M NaCl) strongly differ between strains. <b>B</b>: Model simulations of the flux towards biomass production (left) and glycerol production (right) in the different strains at 0, 20, and 90 minutes after osmotic upshift to 0.4 M NaCl indicate a link between insufficient glycerol accumulation and a prolonged decrease in growth rate.</p

Figure 2

<p>Time courses of (<b>A</b>) phosphorylated Hog1, (<b>B</b>) Gpd1, and (<b>C</b>) intracellular glycerol following hyperosmotic stress of 0.4 M NaCl at time point 0. The full dataset is provided in Datasets S1, S2, S3, S4, S5, S6, S7.</p

Model analysis with time-dependent response coefficients.

<p><b>A</b>: Model simulation for phosphorylated Hog1, intracellular glycerol, and abundance of open Fps1. <b>B</b>: Effect of small changes in Pfk26/27 activation on different model variables as expressed by normalized response coefficient indicates that Pfk26/27 contributes to a rerouting of flux towards lower glycolysis. <b>C</b>: Response of intracellular glycerol concentration to perturbations in Stl1 gene expression as measured by normalized response coefficients in different strains indicates a specific time- and context-dependent role of Stl1in osmoadaptation. <b>D</b>: Simulation of genetic perturbations of Stl1 results in time courses as expected from C: in wild type, deletion of STL1 affects intracellular glycerol levels only at later time points. In <i>gpd1Δ</i> background, the effect of additional deletion of STL1 is early and transient.</p

Yeast strains used in this study.

<p>*Strain YMR84 was kindly provided by Martijn Rep (Amsterdam) and contains a replacement of the GPD1 upstream region (−883 to +91) by the URA3 gene. The strains was generated using a PCR approach and does not express the GPD1 gene product.</p