van Vaeck C, Wera S, van Dijck P, Thevelein J M
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium.
Biochem J. 2001 Jan 1;353(Pt 1):157-162.
In the yeast Saccharomyces cerevisiae, trehalose is synthesized by the trehalose synthase complex in two steps. The Tps1 subunit catalyses the formation of trehalose 6-phosphate (Tre6P), which is dephosphorylated by the Tps2 subunit. Tps1 also controls sugar influx into glycolysis; a tps1 deletion strain is therefore unable to grow on glucose. It is unclear whether this regulatory function of Tps1 is mediated solely by Tre6P or also involves the Tps1 protein. We have developed a novel sensitive and specific assay method for Tre6P. It is based on the conversion of Tre6P into glucose and glucose 6-phosphate with purified phosphotrehalase from Bacillus subtilis. The glucose formed is measured with the glucose-oxidase/peroxidase method. The Tre6P assay is linear in the physiological concentration range. The detection limit, including the entire extraction procedure, is 15 nmol, corresponding to an intracellular concentration of 100 microM. To modify Tre6P levels in vivo, we expressed B. subtilis phosphotrehalase in yeast. The enzyme is functional because it rescues the temperature-sensitive growth defect of a tps2Delta strain and drastically lowers Tre6P levels in this strain. However, phosphotrehalase expression remains without effect on Tre6P levels in wild-type strains, as opposed to overexpression of Tps2. Because Tps2 is part of the Tre6P synthase (TPS) complex and because this complex is destabilized in tps2 deletion strains, these results can be explained if Tre6P is sequestered within the TPS complex in wild-type cells. The very low levels of Tre6P in cells overexpressing Tps2 have a limited effect on sugar phosphate accumulation and do not prevent growth on glucose. Taken together, our results support a model in which the regulatory function of Tps1 on sugar influx is mediated both by the Tps1 protein and by Tre6P.
在酿酒酵母中，海藻糖由海藻糖合酶复合体分两步合成。Tps1亚基催化6-磷酸海藻糖（Tre6P）的形成，然后由Tps2亚基将其去磷酸化。Tps1还控制糖进入糖酵解的过程；因此，tps1缺失菌株无法在葡萄糖上生长。目前尚不清楚Tps1的这种调节功能是仅由Tre6P介导，还是也涉及Tps1蛋白。我们开发了一种针对Tre6P的新型灵敏且特异的检测方法。该方法基于用来自枯草芽孢杆菌的纯化磷酸海藻糖酶将Tre6P转化为葡萄糖和6-磷酸葡萄糖。生成的葡萄糖用葡萄糖氧化酶/过氧化物酶法进行测定。Tre6P检测在生理浓度范围内呈线性。包括整个提取过程在内的检测限为15 nmol，对应于100 microM的细胞内浓度。为了在体内改变Tre6P水平，我们在酵母中表达了枯草芽孢杆菌磷酸海藻糖酶。该酶具有功能，因为它挽救了tps2Δ菌株的温度敏感型生长缺陷，并大幅降低了该菌株中的Tre6P水平。然而，与Tps2的过表达相反，磷酸海藻糖酶的表达对野生型菌株中的Tre6P水平没有影响。由于Tps2是Tre6P合酶（TPS）复合体的一部分，并且该复合体在tps2缺失菌株中不稳定，如果Tre6P在野生型细胞中被隔离在TPS复合体内，那么这些结果就可以得到解释。过表达Tps2的细胞中Tre6P水平非常低，对磷酸糖积累的影响有限，并且不会阻止在葡萄糖上的生长。综上所述，我们的结果支持一个模型，其中Tps1对糖流入的调节功能由Tps1蛋白和Tre6P共同介导。
