Rick Orij

Phosphatidic acid is a pH biosensor that links membrane biogenesis to metabolism

Intracellular pH and Lipid Metabolism Intracellular pH regulates metabolism by poorly understood mechanisms, but biosensors are likely to be important in this process. Young et al. (p. 1085) took a systems-biology approach in yeast to identify in excess of 200 genes that regulate phospholipid metabolism. They found that the signaling lipid, phosphatidic acid, appeared to act as a cytosolic biosensor via the pH-dependent binding of protein effectors to phosphatidic acid. This pH-dependent mechanism directly affects gene expression and is involved in a pathway in which nutrient availability regulates phospholipid metabolism to control production of membranes. Lipid signaling in yeast is regulated by intracellular pH. Recognition of lipids by proteins is important for their targeting and activation in many signaling pathways, but the mechanisms that regulate such interactions are largely unknown. Here, we found that binding of proteins to the ubiquitous signaling lipid phosphatidic acid (PA) depended on intracellular pH and the protonation state of its phosphate headgroup. In yeast, a rapid decrease in intracellular pH in response to glucose starvation regulated binding of PA to a transcription factor, Opi1, that coordinately repressed phospholipid metabolic genes. This enabled coupling of membrane biogenesis to nutrient availability.

Measuring enzyme activities under standardized in vivo-like conditions for systems biology

Realistic quantitative models require data from many laboratories. Therefore, standardization of experimental systems and assay conditions is crucial. Moreover, standards should be representative of the in vivo conditions. However, most often, enzyme–kinetic parameters are measured under assay conditions that yield the maximum activity of each enzyme. In practice, this means that the kinetic parameters of different enzymes are measured in different buffers, at different pH values, with different ionic strengths, etc. In a joint effort of the Dutch Vertical Genomics Consortium, the European Yeast Systems Biology Network and the Standards for Reporting Enzymology Data Commission, we have developed a single assay medium for determining enzyme–kinetic parameters in yeast. The medium is as close as possible to the in vivo situation for the yeast Saccharomyces cerevisiae, and at the same time is experimentally feasible. The in vivo conditions were estimated for S. cerevisiae strain CEN.PK113‐7D grown in aerobic glucose‐limited chemostat cultures at an extracellular pH of 5.0 and a specific growth rate of 0.1 h−1. The cytosolic pH and concentrations of calcium, sodium, potassium, phosphorus, sulfur and magnesium were determined. On the basis of these data and literature data, we propose a defined in vivo‐like medium containing 300 mm potassium, 50 mm phosphate, 245 mm glutamate, 20 mm sodium, 2 mm free magnesium and 0.5 mm calcium, at a pH of 6.8. The Vmax values of the glycolytic and fermentative enzymes of S. cerevisiae were measured in the new medium. For some enzymes, the results deviated conspicuously from those of assays done under enzyme‐specific, optimal 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 cells 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

ABSTRACT 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 (pHi) 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 cells ability to restore pHi, was a determinant for growth inhibition. This pHi 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 pHi, 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 pHi upon lipophilic (sorbic and benzoic) acid stress, probably through a charge interaction of anion and proton transport.

Genome-wide analysis of intracellular pH reveals quantitative control of cell division rate by pHc in Saccharomyces cerevisiae

BackgroundBecause protonation affects the properties of almost all molecules in cells, cytosolic pH (pHc) is usually assumed to be constant. In the model organism yeast, however, pHc changes in response to the presence of nutrients and varies during growth. Since small changes in pHc can lead to major changes in metabolism, signal transduction, and phenotype, we decided to analyze pHc control.ResultsIntroducing a pH-sensitive reporter protein into the yeast deletion collection allowed quantitative genome-wide analysis of pHc in live, growing yeast cultures. pHc is robust towards gene deletion; no single gene mutation led to a pHc of more than 0.3 units lower than that of wild type. Correct pHc control required not only vacuolar proton pumps, but also strongly relied on mitochondrial function. Additionally, we identified a striking relationship between pHc and growth rate. Careful dissection of cause and consequence revealed that pHc quantitatively controls growth rate. Detailed analysis of the genetic basis of this control revealed that the adequate signaling of pHc depended on inositol polyphosphates, a set of relatively unknown signaling molecules with exquisitely pH sensitive properties.ConclusionsWhile pHc is a very dynamic parameter in the normal life of yeast, genetically it is a tightly controlled cellular parameter. The coupling of pHc to growth rate is even more robust to genetic alteration. Changes in pHc control cell division rate in yeast, possibly as a signal. Such a signaling role of pHc is probable, and may be central in development and tumorigenesis.

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.

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.