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Transport of maltose in Saccharomyces cerevisiae. Effect of pH and potassium ions.

ABSTRACT: The transport of maltose in Saccharomyces cerevisiae has been generally accepted as a H+-sugar symport, with a stoichiometrical ratio of 1:1. A simultaneous exit of K+ from the cells with the initial uptake of maltose has been reported previously. By using a K+-selective electrode and radioactive maltose, we were able to measure the exit of 1 mol of K+/mol of maltose taken up by the cells in the first 10-15 s. This stoichiometrical ratio is pH-independent. So, uptake of protons in a non-buffered cell suspension or exit of K+ in a buffered one can be used to measure initial rates of maltose uptake. We have used a K+ electrode and a pH electrode to study the effect of external pH and K+ respectively on the kinetic parameters of maltose transport. The following results were obtained: the apparent half-saturation constant for maltose (Km) increased from 5.2 mM at pH 5.8 to 38.0 mM at pH 7.8; the same increase in pH halved the apparent maximum uptake rate (Vmax); K+ had an inhibitory effect, decreasing Vmax. and increasing Km at pH values above 5; K+ had a stimulating effect at pH values below or equal to 4. Under physiological conditions, i.e. lower pH outside, neutral pH inside and much higher [K+] inside the cell, and assuming symmetry of the system, a higher affinity for maltose is to be expected in the outer face of the plasma membrane. This behaviour of the system could explain, by itself, the maintenance of the high concentration of free maltose inside the cell (necessary because of the low affinity of the maltase), without significant back transport to the outside.

SUBMITTER: Loureiro-Dias MC

PROVIDER: S-EPMC1144178 | biostudies-other | 1984 Sep

REPOSITORIES: biostudies-other

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Project description:?-Ketoglutarate (?KG) is a metabolite of the tricarboxylic acid cycle, important for biomass synthesis and a precursor for biotechnological products like 1,4-butanediol. In the eukaryote Saccharomyces cerevisiae ?KG is present in different compartments. Compartmentation and (intra-)cellular transport could interfere with heterologous product pathways, generate futile cycles and reduce product yields. Batch and chemostat cultivations at low pH (??5) showed that ?KG can be transported, catabolized and used for biomass synthesis. The uptake mechanism of ?KG was further investigated under ?KG limited chemostat conditions at different pH (3, 4, 5, and 6). At very low pH (3, 4) there is a fraction of undissociated ?KG that could diffuse over the periplasmic membrane. At pH 5 this fraction is very low, and the observed growth and residual concentration requires a permease/facilitated uptake mechanism of the mono-dissociated form of ?KG. Consumption of ?KG under mixed substrate conditions was only observed for low glucose concentrations in chemostat cultivations, suggesting that the putative ?KG transporter is repressed by glucose. Fully 13C-labeled ?KG was introduced as a tracer during a glucose/?KG co-feeding chemostat to trace ?KG transport and utilization. The measured 13C enrichments suggest the major part of the consumed 13C ?KG was used for the synthesis of glutamate, and the remainder was transported into the mitochondria and fully oxidized. There was no enrichment observed in glycolytic intermediates, suggesting that there was no gluconeogenic activity under the co-feeding conditions. 13C based flux analysis suggests that the intracellular transport is bi-directional, i.e. there is a fast exchange between the cytosol and mitochondria. The model further estimates that most intracellular ?KG (88%) was present in the cytosol. Using literature reported volume fractions, the mitochondria/cytosol concentration ratio was 1.33. Such ratio will not require energy investment for transport towards the mitochondria (based on thermodynamic driving forces calculated with literature pH values). Growth on ?KG as sole carbon source was observed, suggesting that S. cerevisiae is not fully Krebs-negative. Using 13C tracing and modelling the intracellular use of ?KG under co-feeding conditions showed a link with biomass synthesis, transport into the mitochondria and catabolism. For the engineering of strains that use cytosolic ?KG as precursor, both observed sinks should be minimized to increase the putative yields.

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Transport of maltose in Saccharomyces cerevisiae. Effect of pH and potassium ions.

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