In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth The specific pH values of cellular compartments affect virtually all biochemical processes, including enzyme activity, protein folding and redox state. Accurate, sensitive and compartmentspecific measurements of intracellular pH (pH i ) dynamics in living cells are therefore crucial to the understanding of stress response and adaptation. We used the pH-sensitive GFP derivative 'ratiometric pHluorin' expressed in the cytosol and in the mitochondrial matrix of growing Saccharomyces cerevisiae to assess the variation in cytosolic pH (pH cyt ) and mitochondrial pH (pH mit ) in response to nutrient availability, respiratory chain activity, shifts in environmental pH and stress induced by addition of sorbic acid. The in vivo measurement allowed accurate determination of organelle-specific pH, determining a constant pH cyt of 7.2 and a constant pH mit of 7.5 in cells exponentially growing on glucose. We show that pH cyt and pH mit are differentially regulated by carbon source and respiratory chain inhibitors. Upon glucose starvation or sorbic acid stress, pH i decrease coincided with growth stasis. Additionally, pH i and growth coincided similarly in recovery after addition of glucose to glucose-starved cultures or after recovery from a sorbic acid pulse. We suggest a relation between pH i and cellular energy generation, and therefore a relation between pH i and growth. INTRODUCTIONMicrobes are able to adapt to a wide range of stressful environments such as deviant temperature, high or low osmotic pressure, oxidative stress and exposure to weak organic acids. The mechanisms by which they adapt to these environments are often poorly understood. To study these adaptive responses we rely on techniques that focus on various levels of cellular regulation, such as transcription profiles, protein levels and metabolic flux analysis. However, global physiological parameters such as intracellular pH (pH i ) affect nearly all processes in a living cell. pH i directly influences the redox state of the cell by influencing the NAD + /NADH equilibrium (Veine et al., 1998) and determines pH gradients necessary for transport over membranes (Goffeau & Slayman, 1981;Wohlrab & Flowers, 1982). Additionally, the effect of pH is very prominent in enzyme kinetics, as pH influences ionization states of acidic or basic amino acid side-chains and thereby influences the structure, solubility and activity of most, if not all, enzymes.The different organelles in the cell all maintain their own specific pH, which is used to define and maintain the processes associated with each organelle. Yeast vacuoles, for instance, are reported to have an acidic pH (Preston et al., 1989;Brett et al., 2005; Martínez-Muñoz & Kane, 2008). The proton gradient across the vacuolar membrane has been shown to be essential for the transport of various compounds (Nishimura et al., 1998;Ohsumi & Anraku, ...

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 V(max) 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.

A major challenge in systems biology lies in the integration of processes occurring at different levels, such as transcription, translation, and metabolism, to understand the functioning of a living cell in its environment. We studied the high temperatureinduced glycolytic flux increase in Saccharomyces cerevisiae and investigated the regulatory mechanisms underlying this increase. We used glucose-limited chemostat cultures to separate regulatory effects of temperature from effects on growth rate. Growth at increased temperature (38°C versus 30°C) resulted in a strongly increased glycolytic flux, accompanied by a switch from respiration to a partially fermentative metabolism. We observed an increased flux through all enzymes, ranging from 5-to 10-fold. We quantified the contributions of direct temperature effects on enzyme activities, the gene expression cascade and shifts in the metabolic network, to the increased flux through each enzyme. To do this we adapted flux regulation analysis. We show that the direct effect of temperature on enzyme kinetics can be included as a separate term. Together with hierarchical regulation and metabolic regulation, this term explains the total flux change between two steady states. Surprisingly, the effect of the cultivation temperature on enzyme catalytic capacity, both directly through the Arrhenius effect and indirectly through adapted gene expression, is only a moderate contribution to the increased glycolytic flux for most enzymes. The changes in flux are therefore largely caused by changes in the interaction of the enzymes with substrates, products, and effectors.Microorganisms encounter environmental changes, which they have to withstand and adapt to in order to survive. Changes in ambient temperature are common to almost every ecological niche. Temperature influences the structural and functional properties of cellular components, both physically and chemically. Physically, temperature affects membrane fluidity (1, 2) and diffusion rates, as well as protein folding and stability (3). Chemically, temperature directly affects reaction rates in the cell. This study focuses on the adaptation of cells to temperatures higher than that optimal for growth.Microbes adapt to high temperature by altering their cellular make-up such as lipid composition, membrane fluidity, and the induction of large numbers of heat shock genes (3-11), which have a wide variety of functions. Many encode protein chaperones involved in protein (un)folding (12, 13) or degradation of damaged proteins (14). Others are involved in the synthesis of the thermoprotecting disaccharide trehalose, which is known to be involved in stabilization of membranes and proteins (15, 16) as well as in storage of free energy (17). Many of these adaptive responses put a significant additional energy burden on the cells (18).There still is little clarity on the actual mechanisms by which cells maintain a balance between the energy needs for adaptive responses to stress survival and those for processes indispensable for grow...

SummaryWe investigated the interaction between FtsZ and the cytoplasmic membrane using inside-out vesicles. Comparison of the trypsin accessibility of purified FtsZ and cytoplasmic membrane-bound FtsZ revealed that the protruding loop between helix 6 and helix 7 is protected from trypsin digestion in the latter. This hydrophobic loop contains an arginine residue at position 174. To investigate the role of R174, this residue was replaced by an aspartic acid, and FtsZ-R174D was fused to green fluorescent protein (GFP). FtsZ-R174D-GFP could localize in an FtsZ and in an FtsZ84(Ts) background at both the permissive and the non-permissive temperature, and it had a reduced affinity for the cytoplasmic membrane compared with wild-type FtsZ. FtsZ-R174D could also localize in an FtsZ depletion strain. However, in contrast to wildtype FtsZ, FtsZ-R174D was not able to complement the ftsZ84 mutation or the depletion strain and induced filamentation. In vitro polymerization experiments showed that FtsZ-R174D is able to polymerize, but that these polymers cannot form bundles in the presence of 10 mM CaCl 2 . This is the first description of an FtsZ mutant that has reduced affinity for the cytoplasmic membrane and does not support cell division, but is still able to localize. The mutant is able to form protofilaments in vitro but fails to bundle. It suggests that neither membrane interaction nor bundling is a requirement for initiation of cell division.

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.