The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability.

The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase) to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc) homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

Comparative physiological and transcriptional analysis of weak organic acid stress in Bacillus subtilis.

The advent of 'omics' techniques bears significant potential for the assessment of the microbiological stability of foods. This requires the integration of molecular data with their implication for cellular physiology. Here we performed a comparative physiological and transcriptional analysis of Bacillus subtilis stressed with three different weak organic acids: the commonly used food preservatives sorbic- and acetic-acid, plus the well-known uncoupler carbonyl cyanide-m-chlorophenyl hydrazone (CCCP). The concentration of each compound needed to cause a similar reduction of the growth rate negatively correlated with their membrane solubility, and positively with the concentration of undissociated acid. Intracellular acidification was demonstrated by expressing a pH-sensitive GFP derivative. The largest drop in intracellular pH was observed in CCCP-stressed cells and was accompanied by the transcriptional induction of the general stress response (GSR) and SigM regulon, responses known to be induced by acidification. The GSR was induced by acetate, but not by sorbate in mildly-stressed cells. Microarray analysis further revealed that all three acids activate transcriptional programs normally seen upon nutrient limitation and cause diverse responses indicative of an adaptation of the cell envelope. Based on the responses observed and the utilized pH measurements, the inhibitory effect of sorbic acid seems to be more focused on the cell membrane than that of acetic acid or CCCP.

Metabolic phenotypes of Saccharomyces cerevisiae mutants with altered trehalose 6-phosphate dynamics.

In Saccharomyces cerevisiae, synthesis of T6P (trehalose 6-phosphate) is essential for growth on most fermentable carbon sources. In the present study, the metabolic response to glucose was analysed in mutants with different capacities to accumulate T6P. A mutant carrying a deletion in the T6P synthase encoding gene, TPS1, which had no measurable T6P, exhibited impaired ethanol production, showed diminished plasma membrane H⁺-ATPase activation, and became rapidly depleted of nearly all adenine nucleotides which were irreversibly converted into inosine. Deletion of the AMP deaminase encoding gene, AMD1, in the tps1 strain prevented inosine formation, but did not rescue energy balance or growth on glucose. Neither the 90%-reduced T6P content observed in a tps1 mutant expressing the Tps1 protein from Yarrowia lipolytica, nor the hyperaccumulation of T6P in the tps2 mutant had significant effects on fermentation rates, growth on fermentable carbon sources or plasma membrane H⁺-ATPase activation. However, intracellular metabolite dynamics and pH homoeostasis were strongly affected by changes in T6P concentrations. Hyperaccumulation of T6P in the tps2 mutant caused an increase in cytosolic pH and strongly reduced growth rates on non-fermentable carbon sources, emphasizing the crucial role of the trehalose pathway in the regulation of respiratory and fermentative metabolism.

Intracellular pH homeostasis in Candida glabrata in infection-associated conditions.

Candida glabrata is an opportunistic fungal pathogen which is a growing concern for immunocompromised patients. It is ranked as the second most common cause of candidiasis after Candida albicans. For pathogenic yeasts, intracellular pH (pHi) has been implicated in proliferation, dimorphic switching and virulence. We expressed the pH-sensitive green fluorescent protein variant ratiometric pHluorin in the cytosol of C. glabrata to study pHi dynamics in living cells. We evaluated the response of pHi to the various growth and stress conditions encountered during interaction with the host and during antifungal treatment. C. glabrata maintained a pHi higher than that of Saccharomyces cerevisiae in all growth conditions. The pHi of S. cerevisiae cells appeared better controlled than the pHi in C. glabrata when the cells were exposed to food and fermentation-associated conditions. C. glabrata in turn maintained its pHi better when exposed to host-associated conditions.

Intracellular pH is a tightly controlled signal in yeast.

BACKGROUND: Nearly all processes in living cells are pH dependent, which is why intracellular pH (pH(i)) is a tightly regulated physiological parameter in all cellular systems. However, in microbes such as yeast, pH(i) responds to extracellular conditions such as the availability of nutrients. This raises the question of how pH(i) dynamics affect cellular function. SCOPE OF REVIEW: We discuss the control of pH(i,) and the regulation of processes by pH(i), focusing on the model organism Saccharomyces cerevisiae. We aim to dissect the effects of pH(i) on various aspects of cell physiology, which are often intertwined. Our goal is to provide a broad overview of how pH(i) is controlled in yeast, and how pH(i) in turn controls physiology, in the context of both general cellular functioning as well as of cellular decision making upon changes in the cell's environment. MAJOR CONCLUSIONS: Besides a better understanding of the regulation of pH(i), evidence for a signaling role of pH(i) is accumulating. We conclude that pH(i) responds to nutritional cues and relays this information to alter cellular make-up and physiology. The physicochemical properties of pH allow the signal to be fast, and affect multiple regulatory levels simultaneously. GENERAL SIGNIFICANCE: The mechanisms for regulation of processes by pH(i) are tightly linked to the molecules that are part of all living cells, and the biophysical properties of the signal are universal amongst all living organisms, and similar types of regulation are suggested in mammals. Therefore, dynamic control of cellular decision making by pH(i) is therefore likely a general trait. This article is part of a Special Issue entitled: Systems Biology of Microorganisms.

Quantitative analysis of the modes of growth inhibition by weak organic acids in Saccharomyces cerevisiae.

Weak organic acids are naturally occurring compounds that are commercially used as preservatives in the food and beverage industries. They extend the shelf life of food products by inhibiting microbial growth. There are a number of theories that explain the antifungal properties of these weak acids, but the exact mechanism is still unknown. We set out to quantitatively determine the contributions of various mechanisms of antifungal activity of these weak acids, as well as the mechanisms that yeast uses to counteract their effects. We analyzed the effects of four weak organic acids differing in lipophilicity (sorbic, benzoic, propionic, and acetic acids) on growth and intracellular pH (pH(i)) in Saccharomyces cerevisiae. Although lipophilicity of the acids correlated with the rate of acidification of the cytosol, our data confirmed that not initial acidification, but rather the cell's ability to restore pH(i), was a determinant for growth inhibition. This pH(i) recovery in turn depended on the nature of the organic anion. We identified long-term acidification as the major cause of growth inhibition under acetic acid stress. Restoration of pH(i), and consequently growth rate, in the presence of this weak acid required the full activity of the plasma membrane ATPase Pma1p. Surprisingly, the proposed anion export pump Pdr12p was shown to play an important role in the ability of yeast cells to restore the pH(i) upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.

Isoenzyme expression changes in response to high temperature determine the metabolic regulation of increased glycolytic flux in yeast.

Qualitative phenotypic changes are the integrated result of quantitative changes at multiple regulatory levels. To explain the temperature-induced increase of glycolytic flux in fermenting cultures of Saccharomyces cerevisiae, we quantified the contributions of changes in activity at many regulatory levels. We previously showed that a similar temperature increase in glucose-limited cultivations lead to a qualitative change from respiratory to fermentative metabolism, and this change was mainly regulated at the metabolic level. In contrast, in fermenting cells, a combination of different modes of regulation was observed. Regulation by changes in expression and the effect of temperature on enzyme activities contributed much to the increase in flux. Mass spectrometric quantification of glycolytic enzymes revealed that increased enzyme activity did not correlate with increased protein abundance, suggesting a large contribution of post-translational regulation to activity. Interestingly, the differences in the direct effect of temperature on enzyme kinetics can be explained by changes in the expression of the isoenzymes. Therefore, both the interaction of enzyme with its metabolic environment and the temperature dependence of activity are in turn regulated at the hierarchical level.

Development of the pacemaker tissues of the heart.

Pacemaker and conduction system myocytes play crucial roles in initiating and regulating the contraction of the cardiac chambers. Genetic defects, acquired diseases, and aging cause dysfunction of the pacemaker and conduction tissues, emphasizing the clinical necessity to understand the molecular and cellular mechanisms of their development and homeostasis. Although all cardiac myocytes of the developing heart initially possess pacemaker properties, the majority differentiates into working myocardium. Only small populations of embryonic myocytes will form the sinus node and the atrioventricular node and bundle. Recent efforts have revealed that the development of these nodal regions is achieved by highly localized suppression of working muscle differentiation, and have identified transcriptional repressors that mediate this process. This review will summarize and reflect new experimental findings on the cellular origin and the molecular control of differentiation and morphogenesis of the pacemaker tissues of the heart. It will also shed light on the etiology of inborn and acquired errors of nodal tissues.

Dynamic regulation of mitochondrial respiratory chain efficiency in Saccharomyces cerevisiae.

To adapt to changes in the environment, cells have to dynamically alter their phenotype in response to, for instance, temperature and oxygen availability. Interestingly, mitochondrial function in Saccharomyces cerevisiae is inherently temperature sensitive; above 37 °C, yeast cells cannot grow on respiratory carbon sources. To investigate this phenomenon, we studied the effect of cultivation temperature on the efficiency (production of ATP per atom of oxygen consumed, or P/O) of the yeast respiratory chain in glucose-limited chemostats. We determined that even though the specific oxygen consumption rate did not change with temperature, oxygen consumption no longer contributed to mitochondrial ATP generation at temperatures higher than 37 °C. Remarkably, between 30 and 37 °C, we observed a linear increase in respiratory efficiency with growth temperature, up to a P/O of 1.4, close to the theoretical maximum that can be reached in vivo. The temperature-dependent increase in efficiency required the presence of the mitochondrial glycerol-3-phosphate dehydrogenase GUT2. Respiratory chain efficiency was also altered in response to changes in oxygen availibility. Our data show that, even in the absence of alternative oxidases or uncoupling proteins, yeast has retained the ability to dynamically regulate the efficiency of coupling of oxygen consumption to proton translocation in the respiratory chain in response to changes in the environment.

Subunits Rip1p and Cox9p of the respiratory chain contribute to diclofenac-induced mitochondrial dysfunction.

The widely used drug diclofenac can cause serious heart, liver and kidney injury, which may be related to its ability to cause mitochondrial dysfunction. Using Saccharomyces cerevisiae as a model system, we studied the mechanisms of diclofenac toxicity and the role of mitochondria therein. We found that diclofenac reduced cell growth and viability and increased levels of reactive oxygen species (ROS). Strains increasingly relying on respiration for their energy production showed enhanced sensitivity to diclofenac. Furthermore, oxygen consumption was inhibited by diclofenac, suggesting that the drug inhibits respiration. To identify the site of respiratory inhibition, we investigated the effects of deletion of respiratory chain subunits on diclofenac toxicity. Whereas deletion of most subunits had no effect, loss of either Rip1p of complex III or Cox9p of complex IV resulted in enhanced resistance to diclofenac. In these deletion strains, diclofenac did not increase ROS formation as severely as in the wild-type. Our data are consistent with a mechanism of toxicity in which diclofenac inhibits respiration by interfering with Rip1p and Cox9p in the respiratory chain, resulting in ROS production that causes cell death.

Extreme Low Cytosolic pH Is a Signal for Cell Survival in Acid Stressed Yeast.

Yeast biomass is recycled in the process of bioethanol production using treatment with dilute sulphuric acid to control the bacterial population. This treatment can lead to loss of cell viability, with consequences on the fermentation yield. Thus, the aim of this study was to define the functional cellular responses to inorganic acid stress. Saccharomyces cerevisiae strains with mutation in several signalling pathways, as well as cells expressing pH-sensitive GFP derivative ratiometric pHluorin, were tested for cell survival and cytosolic pH (pHc) variation during exposure to low external pH (pHex). Mutants in calcium signalling and proton extrusion were transiently sensitive to low pHex, while the CWI slt2Δ mutant lost viability. Rescue of this mutant was observed when cells were exposed to extreme low pHex or glucose starvation and was dependent on the induced reduction of pHc. Therefore, a lowered pHc leads to a complete growth arrest, which protects the cells from lethal stress and keeps cells alive. Cytosolic pH is thus a signal that directs the growth stress-tolerance trade-off in yeast. A regulatory model was proposed to explain this mechanism, indicating the impairment of glucan synthesis as the primary cause of low pHex sensitivity.

pH Biosensing by PI4P Regulates Cargo Sorting at the TGN.

Phosphoinositides, diacylglycerolpyrophosphate, ceramide-1-phosphate, and phosphatidic acid belong to a unique class of membrane signaling lipids that contain phosphomonoesters in their headgroups having pKa values in the physiological range. The phosphomonoester headgroup of phosphatidic acid enables this lipid to act as a pH biosensor as changes in its protonation state with intracellular pH regulate binding to effector proteins. Here, we demonstrate that binding of pleckstrin homology (PH) domains to phosphatidylinositol 4-phosphate (PI4P) in the yeast trans-Golgi network (TGN) is dependent on intracellular pH, indicating PI4P is a pH biosensor. pH biosensing by TGN PI4P in response to nutrient availability governs protein sorting at the TGN, likely by regulating sterol transfer to the TGN by Osh1, a member of the conserved oxysterol-binding protein (OSBP) family of lipid transfer proteins. Thus, pH biosensing by TGN PI4P allows for direct metabolic regulation of protein trafficking and cell growth.

Role of cell cycle-regulated expression in the localized incorporation of cell wall proteins in yeast.

The yeast cell wall is an essential organelle that protects the cell from mechanical damage and antimicrobial peptides, participates in cell recognition and adhesion, and is important for the generation and maintenance of normal cell shape. We studied the localization of three covalently bound cell wall proteins in Saccharomyces cerevisiae. Tip1p was found only in mother cells, whereas Cwp2p was incorporated in small-to-medium-sized buds. When the promoter regions of TIP1 and CWP2 (responsible for transcription in early G1 and S/G2 phases, respectively) were exchanged, the localization patterns of Tip1p and Cwp2p were reversed, indicating that the localization of cell wall proteins can be completely determined by the timing of transcription during the cell cycle. The third protein, Cwp1p, was incorporated into the birth scar, where it remained for several generations. However, we could not detect any role of Cwp1p in strengthening the birth scar wall or any functional interaction with the proteins that mark the birth scar pole as a potential future budding site. Promoter-exchange experiments showed that expression in S/G2 phase is necessary but not sufficient for the normal localization of Cwp1p. Studies of mutants in which septum formation is perturbed indicate that the normal asymmetric localization of Cwp1p also depends on the normal timing of septum formation, composition of the septum, or both.

Caloric restriction controls stationary phase survival through Protein Kinase A (PKA) and cytosolic pH.

Calorie restriction is the only physiological intervention that extends lifespan throughout all kingdoms of life. In the budding yeast Saccharomyces cerevisiae, cytosolic pH (pHc ) controls growth and responds to nutrient availability, decreasing upon glucose depletion. We investigated the interactions between glucose availability, pHc and the central nutrient signalling cAMP-Protein Kinase A (PKA) pathway. Glucose abundance during the growth phase enhanced acidification upon glucose depletion, via modulation of PKA activity. This actively controlled reduction in starvation pHc correlated with reduced stationary phase survival. Whereas changes in PKA activity affected both acidification and survival, targeted manipulation of starvation pHc showed that cytosolic acidification was downstream of PKA and the causal agent of the reduced chronological lifespan. Thus, caloric restriction controls stationary phase survival through PKA and cytosolic pH.

On the origin of the extremely different solubilities of polyethers in water.

The solubilities of polyethers are surprisingly counter-intuitive. The best-known example is the difference between polyethylene glycol ([-CH2-CH2-O-]n) which is infinitely soluble, and polyoxymethylene ([-CH2-O-]n) which is completely insoluble in water, exactly the opposite of what one expects from the C/O ratios of these molecules. Similar anomalies exist for oligomeric and cyclic polyethers. To solve this apparent mystery, we use femtosecond vibrational and GHz dielectric spectroscopy with complementary ab initio calculations and molecular dynamics simulations. We find that the dynamics of water molecules solvating polyethers is fundamentally different depending on their C/O composition. The ab initio calculations and simulations show that this is not because of steric effects (as is commonly believed), but because the partial charge on the O atoms depends on the number of C atoms by which they are separated. Our results thus show that inductive effects can have a major impact on aqueous solubilities.

Syntaxin 5 Is Required for the Formation and Clearance of Protein Inclusions during Proteostatic Stress.

Spatial sorting to discrete quality control sites in the cell is a process harnessing the toxicity of aberrant proteins. We show that the yeast t-snare phosphoprotein syntaxin5 (Sed5) acts as a key factor in mitigating proteotoxicity and the spatial deposition and clearance of IPOD (insoluble protein deposit) inclusions associates with the disaggregase Hsp104. Sed5 phosphorylation promotes dynamic movement of COPII-associated Hsp104 and boosts disaggregation by favoring anterograde ER-to-Golgi trafficking. Hsp104-associated aggregates co-localize with Sed5 as well as components of the ER, trans Golgi network, and endocytic vesicles, transiently during proteostatic stress, explaining mechanistically how misfolded and aggregated proteins formed at the vicinity of the ER can hitchhike toward vacuolar IPOD sites. Many inclusions become associated with mitochondria in a HOPS/vCLAMP-dependent manner and co-localize with Vps39 (HOPS/vCLAMP) and Vps13, which are proteins providing contacts between vacuole and mitochondria. Both Vps39 and Vps13 are required also for efficient Sed5-dependent clearance of aggregates.

Transcriptome analysis of sorbic acid-stressed Bacillus subtilis reveals a nutrient limitation response and indicates plasma membrane remodeling.

The weak organic acid sorbic acid is a commonly used food preservative, as it inhibits the growth of bacteria, yeasts, and molds. We have used genome-wide transcriptional profiling of Bacillus subtilis cells during mild sorbic acid stress to reveal the growth-inhibitory activity of this preservative and to identify potential resistance mechanisms. Our analysis demonstrated that sorbic acid-stressed cells induce responses normally seen upon nutrient limitation. This is indicated by the strong derepression of the CcpA, CodY, and Fur regulon and the induction of tricarboxylic acid cycle genes, SigL- and SigH-mediated genes, and the stringent response. Intriguingly, these conditions did not lead to the activation of sporulation, competence, or the general stress response. The fatty acid biosynthesis (fab) genes and BkdR-regulated genes are upregulated, which may indicate plasma membrane remodeling. This was further supported by the reduced sensitivity toward the fab inhibitor cerulenin upon sorbic acid stress. We are the first to present a comprehensive analysis of the transcriptional response of B. subtilis to sorbic acid stress.

Picosecond orientational dynamics of water in living cells.

Cells are extremely crowded, and a central question in biology is how this affects the intracellular water. Here, we use ultrafast vibrational spectroscopy and dielectric-relaxation spectroscopy to observe the random orientational motion of water molecules inside living cells of three prototypical organisms: Escherichia coli, Saccharomyces cerevisiae (yeast), and spores of Bacillus subtilis. In all three organisms, most of the intracellular water exhibits the same random orientational motion as neat water (characteristic time constants ~9 and ~2 ps for the first-order and second-order orientational correlation functions), whereas a smaller fraction exhibits slower orientational dynamics. The fraction of slow intracellular water varies between organisms, ranging from ~20% in E. coli to ~45% in B. subtilis spores. Comparison with the water dynamics observed in solutions mimicking the chemical composition of (parts of) the cytosol shows that the slow water is bound mostly to proteins, and to a lesser extent to other biomolecules and ions.The cytoplasm's crowdedness leads one to expect that cell water is different from bulk water. By measuring the rotational motion of water molecules in living cells, Tros et al. find that apart from a small fraction of water solvating biomolecules, cell water has the same dynamics as bulk water.

Stress tolerance in fungi -- to kill a spoilage yeast.

The fungal spoilage of ingredients of food manufacture is an economic problem, often causes product loss and may constitute a health hazard. To effectively combat fungal food spoilage, a mechanistic understanding of tolerance for, and adaptation to, the preservation method used is crucial. Both are dependent on the genetic make-up and growth history of the organism. In the post-genomic era we are arriving at a situation in which, in the model organism Saccharomyces cerevisiae, physiological data, classical molecular biology and whole-genome responses can be combined to obtain explanatory and predictive models for growth. For food spoilage fungi we have not yet reached such a level of understanding, but we may use the knowledge gained for S. cerevisiae for the prevention of spoilage.

Yeast adaptation to weak acids prevents futile energy expenditure.

Weak organic acids (WOAs) are widely used preservatives to prevent fungal spoilage of foods and beverages. Exposure of baker's yeast Saccharomyces cerevisiae to WOA leads to cellular acidification and anion accumulation. Pre-adaptation of cultures reduced the rate of acidification caused by weak acid exposure, most likely as a result of changes in plasma membrane or cell wall composition. In order to adapt to sublethal concentrations of the acids and grow, yeast cells activate ATP consuming membrane transporters to remove protons and anions. We explored to what extent ATP depletion contributes to growth inhibition in sorbic or acetic acid treated cells. Therefore, we analyzed the effect of the reduction of proton and anion pumping activity on intracellular pH (pHi), growth, and energy status upon exposure to the hydrophilic acetic acid (HA) and the lipophilic sorbic acid (HS). ATP concentrations were dependent on the severity of the stress. Unexpectedly, we observed a stronger reduction of ATP with growth reducing than with growth inhibitory concentrations of both acids. We deduce that the not the ATP reduction caused by proton pumping, but rather the cost of sorbate anion pumping contributes to growth inhibition. A reduction of proton pumping activity may reduce ATP consumption, but the resulting decrease of pHi affects growth more. ATP utilization was differentially regulated during moderate and severe stress conditions. We propose that the energy depletion alone is not the cause of growth inhibition during HA or HS stress. Rather, the cells appear to reduce ATP consumption in high stress conditions, likely to prevent futile cycling and maintain energy reserves for growth resumption in more favorable conditions. The mechanism for such decision making remains to be established.