The yeast osmostress response is carbon source dependent

Adaptation to altered osmotic conditions is a fundamental property of living cells and has been studied in detail in the yeast Saccharomyces cerevisiae. Yeast cells accumulate glycerol as compatible solute, controlled at different levels by the High Osmolarity Glycerol (HOG) response pathway. Up to now, essentially all osmostress studies in yeast have been performed with glucose as carbon and energy source, which is metabolised by glycolysis with glycerol as a by-product. Here we investigated the response of yeast to osmotic stress when yeast is respiring ethanol as carbon and energy source. Remarkably, yeast cells do not accumulate glycerol under these conditions and it appears that trehalose may partly take over the role as compatible solute. The HOG pathway is activated in very much the same way as during growth on glucose and is also required for osmotic adaptation. Slower volume recovery was observed in ethanol-grown cells as compared to glucose-grown cells. Dependence on key regulators as well as the global gene expression profile were similar in many ways to those previously observed in glucose-grown cells. However, there are indications that cells re-arrange redox-metabolism when respiration is hampered under osmostress, a feature that could not be observed in glucose-grown cells

Systems Level Analysis of the Yeast Osmo-Stat

Adaptation is an important property of living organisms enabling them to cope with environmental stress and maintaining homeostasis. Adaptation is mediated by signaling pathways responding to different stimuli. Those signaling pathways might communicate in order to orchestrate the cellular response to multiple simultaneous stimuli, a phenomenon called crosstalk. Here, we investigate possible mechanisms of crosstalk between the High Osmolarity Glycerol (HOG) and the Cell Wall Integrity (CWI) pathways in yeast, which mediate adaptation to hyper- and hypo-osmotic challenges, respectively. We combine ensemble modeling with experimental investigations to test in quantitative terms different hypotheses about the crosstalk of the HOG and the CWI pathways. Our analyses indicate that for the conditions studied i) the CWI pathway activation employs an adaptive mechanism with a variable volume-dependent threshold, in contrast to the HOG pathway, whose activation relies on a fixed volume-dependent threshold, ii) there is no or little direct crosstalk between the HOG and CWI pathways, and iii) its mainly the HOG alone mediating adaptation of cellular osmotic pressure for both hyper- as well as hypo-osmotic stress. Thus, by iteratively combining mathematical modeling with experimentation we achieved a better understanding of regulatory mechanisms of yeast osmo-homeostasis and formulated new hypotheses about osmo-sensing

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regard less of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7,gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, high-lighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the proteindisaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was nothindered when pro3-1 colocalized with a slowly resolved mis-folded protein. Finally, we observed using super-resolutionlight microscopy as well as immunogold labeling EM in which both showed an even distribution of the different mis-folded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compart-mentalization, allows for differential clearance of these mis-folding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing

Using reporters of different misfolded proteins reveals differential strategies in processing protein aggregates

The accumulation of misfolded proteins is a hallmark of aging and many neurodegenerative diseases, making it important to understand how the cellular machinery recognizes and processes such proteins. A key question in this respect is whether misfolded proteins are handled in a similar way regardless of their genetic origin. To approach this question, we compared how three different misfolded proteins, guk1-7, gus1-3, and pro3-1, are handled by the cell. We show that all three are nontoxic, even though highly overexpressed, highlighting their usefulness in analyzing the cellular response to misfolding in the absence of severe stress. We found significant differences between the aggregation and disaggregation behavior of the misfolded proteins. Specifically, gus1-3 formed some aggregates that did not efficiently recruit the protein disaggregase Hsp104 and did not colocalize with the other misfolded reporter proteins. Strikingly, while all three misfolded proteins generally coaggregated and colocalized to specific sites in the cell, disaggregation was notably different; the rate of aggregate clearance of pro3-1 was faster than that of the other misfolded proteins, and its clearance rate was not hindered when pro3-1 colocalized with a slowly resolved misfolded protein. Finally, we observed using super-resolution light microscopy as well as immunogold labeling EM in which both showed an even distribution of the different misfolded proteins within an inclusion, suggesting that misfolding characteristics and remodeling, rather than spatial compartmentalization, allows for differential clearance of these misfolding reporters residing in the same inclusion. Taken together, our results highlight how properties of misfolded proteins can significantly affect processing

Hitchhiking on vesicles: a way to harness age-related proteopathies?

The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies Central to proteopathies and leading to most age-related neurodegenerative disorders is a failure in protein quality control (PQC). To harness the toxicity of misfolded and damaged disease proteins, such proteins are either refolded, degraded by temporal PQC, or sequestered by spatial PQC into specific, organelle-associated, compartments within the cell. Here, we discuss the impact of vesicle trafficking pathways in general, and syntaxin 5 in particular, as key players in spatial PQC directing misfolded proteins to the surface of vacuole and mitochondria, which facilitates their clearance and detoxification. Since boosting vesicle trafficking genetically can positively impact on spatial PQC and make cells less sensitive to misfolded disease proteins, we speculate that regulators of such trafficking might serve as therapeutic targets for age-related neurological disorders

Rewiring yeast osmostress signalling through the MAPK network reveals essential and non-essential roles of Hog1 in osmoadaptation

Mitogen-activated protein kinases (MAPKs) have a number of targets which they regulate at transcriptional and post-translational levels to mediate specific responses. The yeast Hog1 MAPK is essential for cell survival under hyperosmotic conditions and it plays multiple roles in gene expression, metabolic regulation, signal fidelity and cell cycle regulation. Here we describe essential and non-essential roles of Hog1 using engineered yeast cells in which osmoadaptation was reconstituted in a Hog1-independent manner. We rewired Hog1-dependent osmotic stress-induced gene expression under the control of Fus3/Kss1 MAPKs, which are activated upon osmostress via crosstalk in hog1Δ cells. This approach revealed that osmotic up-regulation of only two Hog1-dependent glycerol biosynthesis genes, GPD1 and GPP2, is sufficient for successful osmoadaptation. Moreover, some of the previously described Hog1-dependent mechanisms appeared to be dispensable for osmoadaptation in the engineered cells. These results suggest that the number of essential MAPK functions may be significantly smaller than anticipated and that knockout approaches may lead to over-interpretation of phenotypic data

The Ashbya gossypiiEF-1α promoter of the ubiquitously used MX cassettes is toxic to Saccharomyces cerevisiae.

Protein overexpression based on introduction of multiple gene copies is well established. To improve purification or quantification, proteins are typically fused to peptide tags. In Saccharomyces cerevisiae, this has been hampered by multicopy toxicity of the TAP and GFP cassettes used in the global strain collections. Here, we show that this effect is due to the EF-1α promoter in the HIS3MX marker cassette rather than the tags per se. This promoter is frequently used in heterologous marker cassettes, including HIS3MX, KanMX, NatMX, PatMX and HphMX. Toxicity could be eliminated by promoter replacement or exclusion of the marker cassette. To our knowledge, this is the first report of toxicity caused by introduction of a heterologous promoter alone

The mitogen-activated protein kinase Slt2 modulates arsenite transport through the aquaglyceroporin Fps1.

Arsenite is widely present in nature; therefore, cells have evolved mechanisms to prevent arsenite influx and promote efflux. In yeast (Saccharomyces cerevisiae), the aquaglyceroporin Fps1 mediates arsenite influx and efflux. The mitogen-activated protein kinase (MAPK) Hog1 has previously been shown to restrict arsenite influx through Fps1. In this study, we show that another MAPK, Slt2, is transiently phosphorylated in response to arsenite influx. Our findings indicate that the protein kinase activity of Slt2 is required for its role in arsenite tolerance. While Hog1 prevents arsenite influx via phosphorylation of T231 at the N-terminal domain of Fps1, Slt2 promotes arsenite efflux through phosphorylation of S537 at the C terminus. Our data suggest that Slt2 physically interacts with Fps1 and that this interaction depends on phosphorylation of S537. We hypothesize that Hog1 and Slt2 may affect each other\u27s binding to Fps1, thereby controlling the opening and closing of the channel

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