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 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.
van Vaeck
C
C
Laboratorium voor Moleculaire Celbiologie, Katholieke Universiteit Leuven, Kardinaal Mercierlaan 92, B-3001 Leuven-Heverlee, Flanders, Belgium.
Wera
S
S
van Dijck
P
P
Thevelein
J M
JM
D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D004186:Disaccharidases / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal / Q000235:genetics; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism; D006019:Glycolysis; D009097:Multienzyme Complexes; D021122:Protein Subunits; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D013403:Sugar Phosphates / Q000032:analysis / Q000378:metabolism*; D014199:Trehalose / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism*
D005656:Fungal Proteins; D005960:Glucosides; D009097:Multienzyme Complexes; D021122:Protein Subunits; D013403:Sugar Phosphates; C070512:2',4'-dinitrophenylglucopyranoside; C082722:trehalose-6-phosphate; D014199:Trehalose; D004186:Disaccharidases; C040631:alpha, alpha-phosphotrehalase; D005947:Glucose