During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

Abstract

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (GGS1FDP1BYP1CIF1GLC6TSS1)‐encoded trehalose‐6‐phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50‐fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild‐type cells. However, it causes higher levels of glucose‐6‐phosphate, fructose‐6‐phosphate and also faster accumulation of fructose‐1,6‐bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild‐type strain. Apparently, during the start‐up of fermentation hexokinase is more rate‐limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d‐glucose and an equal concentration of radiolabelled l‐glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d‐glucose measured (intracellular+periplasmic/extracellular) and the total l‐glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm‐glucose to 0·5–2 mm in the wild‐type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2–3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1‐dependent control system is apparently able to restrict properly up to 50‐fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.

Mitochondrial Oxidative Phosphorylation Is Regulated by Fructose 1,6-Bisphosphate

“…In yeast cells, the addition of large amounts of glucose results in a metabolic shift toward fermentation and in the accumulation of glycolysis hexose phosphates (25,26). Since accumulation of hexose phosphates occurs concomitantly with an inhibition of respiration, we tested the possibility of a direct role of these intermediates in the regulation of mitochondrial oxidative phosphorylation.…”

“…In the presence of F6P, this increase was less important, and for physiological concentrations (less than 1 mM), there was no effect. On the other hand, F16bP, at physiological concentrations (2-10 mM) (25,26,34) induced an inhibition of the respiratory rate that reached 25%. This effect is not dependent on the respiratory substrate, since the same effect was also observed when using ethanol as substrate instead of NADH (data not shown).…”

“…A strain overexpressing phosphofructokinase has lower JO 2 values (48). Furthermore, the ⌬tps1 strain, well known for accumulating hexose phosphates (24,26,41,42), has an enhanced Crabtree effect (see Tables 2 and 3). FIGURE 9.…”

“…In S. cerevisiae, cytoplasmic levels of glycolysis hexose phosphates increase after glucose addition to the culture medium (25,26). Given the possible role of glycolysis intermediates as metabolic messengers, we investigated whether some of these could contribute to the short term Crabtree effect (i.e.…”

Novel alleles of yeast hexokinase PII with distinct effects on catalytic activity and catabolite repression of SUC2

“…Apparently these mutants lack a feedback control of glycolysis on hexokinase activity. The precise regulatory mechanism is not well understood (Blazquez et al, 1993;Ernandes et al, 1998). Reduction of hexokinase activity by deletion of H X K 2 restores growth of the tpslA mutant on glucose, but not on fructose .…”