Colorimetric determination of active alpha-glucoside transport in Saccharomyces cerevisiae.
Fermentation of alpha-glucosides (maltose, maltotriose) by Saccharomyces cerevisiae cells is a critical phase in the processes of brewing and breadmaking. Utilization of alpha-glucosides requires the active transport of the sugar across the cell membrane and, subsequently, its hydrolysis by cytoplasmic glucosidases. Although transport activities are usually assayed using radiolabeled substrates, we have developed a simple, cheap and reliable colorimetric assay for the determination of alpha-glucoside uptake using p-nitrophenyl-alpha-D-glucopyranoside (pNPalphaG) as substrate. Our results show that pNPalphaG is actively transported by S. cerevisiae cells by a H+-symport mechanism, which depends on the electrochemical proton gradient across the plasma membrane. pNPalphaG uptake is mediated by the AGT1 alpha-glucoside permease, which has a high affinity (Km=3 mM) for this chromogenic substrate. This simple colorimetric uptake assay can be used to analyze the expression and regulation of the AGT1 permease in S. cerevisiae cells.
Hollatz
C
C
Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, SC 88040-900, Florianópolis, Brazil.
Stambuk
B U
BU
D001693:Biological Transport, Active; D002151:Calorimetry / Q000379:methods*; D002352:Carrier Proteins / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D005285:Fermentation; D005656:Fungal Proteins / Q000378:metabolism; D005960:Glucosides / Q000378:metabolism*; D008320:Maltose / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008757:Methylglucosides / Q000378:metabolism; D009004:Monosaccharide Transport Proteins; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D027981:Symporters; D014199:Trehalose / Q000378:metabolism; D014312:Trisaccharides / Q000378:metabolism
D002352:Carrier Proteins; D004187:Disaccharides; D005656:Fungal Proteins; D005960:Glucosides; D026901:Membrane Transport Proteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D027981:Symporters; D014312:Trisaccharides; C067575:maltose transport system, S cerevisiae; C019502:4-nitrophenyl alpha-glucoside; C008317:maltotriose; D008320:Maltose; D014199:Trehalose