Abstract
Metabolic pathways play an indispensable role in supplying cellular systems with energy and molecular building blocks for growth, maintenance and repair and are tightly linked with lifespan and systems stability of cells. For optimal growth and survival cells rapidly adopt to environmental changes. Accumulation of acetic acid in stationary phase budding yeast cultures is considered to be a primary mechanism of chronological aging and induction of apoptosis in yeast, which has prompted us to investigate the dependence of acetic acid toxicity on extracellular conditions in a systematic manner. Using an automated computer controlled assay system, we investigated and model the dynamic interconnection of biomass yield- and growth rate-dependence on extracellular glucose concentration, pH conditions and acetic acid concentration. Our results show that toxic concentrations of acetic acid inhibit glucose consumption and reduce ethanol production. In absence of carbohydrates uptake, cells initiate synthesis of storage carbohydrates, trehalose and glycogen, and upregulate gluconeogenesis. Accumulation of trehalose and glycogen, and induction of gluconeogenesis depends on mitochondrial activity, investigated by depletion of the Hap2-3-4-5 complex. Analyzing the activity of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK), and glucose-6-phosphate dehydrogenase (G6PDH) we found that while high acetic acid concentration increased their activity, lower acetic acids concentrations significantly inhibited these enzymes. With this study we determined growth and functional adjustment of metabolism to acetic acid accumulation in a complex range of extracellular conditions. Our results show that substantial acidification of the intracellular environment, resulting from accumulation of dissociated acetic acid in the cytosol, is required for acetic acid toxicity, which creates a state of energy deficiency and nutrient starvation.
Highlights
Basic metabolic pathways provide energy and molecular building blocks required for growth, maintenance and repair
response surface models (RSMs) of cell growth dynamics confirmed a strong shift of optimal pH conditions from low pH values toward higher ones in presence of increasing acetic acid concentrations (Figure1)
The growth of yeast cells under high glucose concentration does not require mitochondrial activity, our results indicate that functional mitochondria and the transcriptional complex Hap 2-3-4-5 are indispensable for optimal growth rates and biomass production
Summary
Basic metabolic pathways provide energy and molecular building blocks required for growth, maintenance and repair. Acetic acid is a normal end product of alcoholic fermentation in S. cerevisiae that cannot be metabolized by glucose-repressed yeast cells. In undissociated form acetic acid is freely membrane permeable and enters the cell by simple diffusion. At higher extracellular pH acetic acid will dissociate to the acetate anion, a form that is relatively membrane-impairment. Acetic acid was shown to induce apoptosis in yeast cells involving release of cytochrom c in a mitochondria dependent apoptotic pathway (Ludovico et al, 2002; Pereira et al, 2007). Acetic acid was identified as a cell-extrinsic mediator of cell death during chronological aging in S. cerevisiae (Burtner et al, 2009)
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