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

Abstract

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, ...

Consequences of Loss of Vph1 Protein-containing Vacuolar ATPases (V-ATPases) for Overall Cellular pH Homeostasis

“…Cells grown in selective medium to retain the pHluorin plasmid were deprived of glucose for ϳ30 min and then monitored before and after the addition of glucose to 50 mM final concentration. As shown by others using pHluorinbased and other methods (22,23), Fig. 2A illustrates that the addition of glucose to glucose-deprived wild-type cells results in a transient cytosolic acidification within a minute of glucose addition followed by a rapid rise in cytosolic pH that is complete within 3 min of glucose addition.…”

Heme dynamics and trafficking factors revealed by genetically encoded fluorescent heme sensors

“…S3), which is the pH of the yeast cytosol (24). Given the relatively reducing cellular environment, E m Cytosol ∼ −320 mV vs. normal hydrogen electrode (NHE) (25), which is governed by the ratio of oxidized-to-reduced glutathione, and the reduction potential of aqueous heme, estimated to be between −50 mV and −220 mV vs. NHE (26,27), we propose that LH is biased toward the reduced state and assume herein that LH is 100% reduced.…”

Study on the effects of near-future ocean acidification on marine yeasts: a microcosm approach

“…In general, alkaline pH is considered to be a stress factor that yeasts have to cope with in the marine environment. The intracellular pH of yeasts was reported to be around 7 when growing on glucose in laboratory studies and to remain stable between extracellular pH values from 3.0 to 7.5 (Orij et al 2009). At lower external pH values, protons can be efficiently exported out of the cytoplasm, either to the exterior of the cell or into the vacuole, involving different ATPases (Carmelo et al 1997;Diakov and Kane 2010;Orij et al 2011).…”