Ernandes J R, De Meirsman C, Rolland F, Winderickx J, de Winde J, Brandão R L, Thevelein J M
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Flanders, Belgium.
Yeast. 1998 Feb;14(3):255-69. doi: 10.1002/(SICI)1097-0061(199802)14:3<255::AID-YEA228>3.0.CO;2-N.
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 tps1 delta 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 hxk1 delta hxk2 delta glk1 delta and 2-3 mM in a tps1 delta 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.
在酿酒酵母中，由TPS1（= GGS1 = FDP1 = BYP1 = CIF1 = GLC6 = TSS1）编码的海藻糖-6-磷酸合酶施加的一种新型调控，对于限制葡萄糖流入糖酵解过程至关重要，这显然是通过在体内抑制己糖激酶活性来实现的。我们发现，在野生型细胞中，己糖激酶高达50倍的过表达对葡萄糖或果糖上的生长没有明显影响。然而，在发酵开始时，它会导致更高水平的6-磷酸葡萄糖、6-磷酸果糖，并且还会使1,6-二磷酸果糖积累得更快。ATP和Pi的水平与较高的磷酸糖水平呈负相关。在添加葡萄糖后的最初几分钟内，观察到的代谢物模式介于tps1Δ突变体和野生型菌株之间。显然，在发酵启动过程中，己糖激酶在糖酵解的第一阶段比磷酸果糖激酶更具限速作用。我们开发了一种基于同时添加D-葡萄糖和等浓度放射性标记的L-葡萄糖来测量细胞内游离葡萄糖水平的方法。由于后者不被转运，细胞内游离葡萄糖水平可以计算为测得的总D-葡萄糖（细胞内 + 周质/细胞外）与测得的总L-葡萄糖（周质/细胞外）之间的差值。在野生型菌株中，添加100 mM葡萄糖后5分钟内，细胞内葡萄糖水平从0.5 - 2 mM上升，在hxk1Δ hxk2Δ glk1Δ菌株中为±10 mM，在tps1Δ菌株中为2 - 3 mM。在过表达己糖激酶PII的菌株中，细胞内游离葡萄糖水平没有降低。己糖激酶PII的过表达从未对乙醇产生速率和葡萄糖消耗速率产生强烈影响。我们的结果表明，己糖激酶的过表达不会导致与Tps1缺失相同的表型。然而，在发酵开始时它会短暂模拟这种表型。之后，Tps1依赖的控制系统显然能够适当地限制高达50倍的更高己糖激酶活性。
