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The role of intracellular pH in the regulation of cation exchanges in yeast.

ABSTRACT: 1. When yeast oxidizes propan-2-ol in the presence of KCl no uptake of K(+) occurs. 2. When propionate is added to suspensions containing propan-2-ol, or if the suspensions are bubbled with CO(2), a considerable uptake of K(+) occurs. 3. Maximum K(+) uptake occurs at a propionate concentration of 2mm. 4. The addition of 20mm-propionate to the suspension lowers the intracellular pH of the yeast from a resting value in the region of 6.2 to approx. 5.6. 5. When K(+) uptake is measured in the presence of 20mm-propionate, progressive changes in the rate of K(+) uptake and intracellular pH occur. The optimum rate of K(+) uptake occurs at an intracellular pH of 5.70. 6. The effect of both intra- and extra-cellular pH on K(+)-K(+) exchange was studied and an optimum rate was found at an extracellular pH of 5.35, the corresponding intracellular pH being 6.44. 7. When a Na(+)-loaded yeast oxidizes propan-2-ol in the presence of KCl, a steady efflux of Na(+) and influx of K(+) occurs. The addition of 10mm-propionate to the suspension markedly inhibited the Na(+) efflux but only slightly decreased the K(+) influx. 8. The effect of both extra- and intra-cellular pH on Na(+) efflux was studied with propan-2-ol and with glucose. The results can be best interpreted in terms of intracellular pH changes, and an optimum was obtained at approx. pH6.40.

SUBMITTER: Ryan JP

PROVIDER: S-EPMC1173578 | biostudies-other | 1972 Jun

REPOSITORIES: biostudies-other

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Project description:BackgroundThe metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering the cell and is again exported or even catabolized. Here, a quantitative approach for the identification of product recycling fluxes is developed.ResultsThe metabolic flux distributions at two time-points of the fermentation process were analyzed. 13C labeled succinic acid was added to the extracellular space and intracellular enrichments were measured and subsequently used for the estimation of metabolic fluxes. The labeling was introduced by a labeling switch experiment, leading to an immediate labeling of about 85% of the acid while keeping the total acid concentration constant. Within 100 s significant labeling enrichment of the TCA cycle intermediates fumarate, iso-citrate and α-ketoglutarate was observed, while no labeling was detected for malate and citrate. These findings suggest that succinic acid is rapidly exchanged over the cellular membrane and enters the oxidative TCA cycle. Remarkably, in the oxidative direction malate 13C enrichment was not detected, indicating that there is no flux going through this metabolite pool. Using flux modeling and thermodynamic assumptions on compartmentation it was concluded that malate must be predominantly cytosolic while fumarate and iso-citrate were more dominant in the mitochondria.ConclusionsAdding labeled product without changing the extracellular environment allowed to quantify intracellular metabolic fluxes under high producing conditions and identify product degradation cycles. In the specific case of succinic acid production, compartmentation was found to play a major role, i.e. the presence of metabolic activity in two different cellular compartments lead to intracellular product degradation reducing the yield. We also observed that the flux from glucose to succinic acid branches at two points in metabolism: (1) At the level of pyruvate, and (2) at cytosolic malate which was not expected.

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The role of intracellular pH in the regulation of cation exchanges in yeast.

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