Analysis and modification of trehalose 6-phosphate levels in the yeast Saccharomyces cerevisiae with the use of Bacillus subtilis phosphotrehalase

In the yeast Saccharomyces cerevisiae, trehalose is synthesized by the treh alose synthase complex in two steps. The Tps1 subunit catalyses the formati on of trehalose 6-phosphate (Tre6P), which is dephosphorylated by the Tps2 subunit, Tps1 also controls sugar influx into glycolysis; a tps1 deletion s train is therefore unable to grow on glucose. It is unclear whether this re gulatory 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 glu cose formed is measured with the glucose-oxidase/peroxidase method. The Tre 6P 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 muM. To modify Tre6P levels 61 vi vo, we expressed B. subtilis phospho-trehalase in yeast. The enzyme is func tional because it rescues the temperature-sensitive growth defect of a tps2 Delta strain and drastically lowers Tre6P levels in this strain. However, phospho trehalase expression remains without effect on TreBP levels in wild -type strains, as opposed to overexpression of Tps2. Because Tps2 is part o f the TreBP synthase (TPS) complex and because this complex is destabilized in tps2 deletion strains, these results can be explained if Tre6P is seque stered within the TPS complex in wild-type cells. The very low levels of Tr e6P in cells overexpressing Tps2 have a limited effect on sugar phosphate a ccumulation and do not prevent growth on glucose. Taken together, our resul ts support a model in which the regulatory function of Tps1 on sugar influx is mediated both by the Tps1 protein and by Tre6P.