Colorimetric determination of active α-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 (pNP alphaG) as substrate. Our results show that pNP alphaG is actively transported by S. cerevisiae cells by a H+-symport mechanism. which depends on the electrochemical proton gradient across the plasma membrane. pNP alphaG uptake is mediated by the AGT1 alpha -glucoside permease, which has a high affinity (K-m = 3 mM) for this chromogenic substrate. This simple colorimetric uptake assay can he used to analyze the expression and regulation of the AGT1 permease in S. cerevisiae cells. (C) 2001 Elsevier Science B.V. All rights reserved.