α-Methyl glucoside (α-MG) transport in Saccharomyces cerevisiae was reported previously to be an active transport, a H+-symport mediated by the AGT1 permease.
Strain AP77-11B (a strain obtained in our laboratory) takes up 14C-α-MG by a mechanism which was ascribed to be facilitated diffusion since there is no H+-cotransport during the α-MG uptake. The HXT1-HXT17 genes belong to a family of hexose transporters in Saccharomyces cerevisiae. Therefore, we decided to investigate the possibility that α-MG transport could be mediated by hexose transporters. We demonstrated that strains MC966A (w.t.), KY73 (isogenic to MC966A but hxt1-hxt7-null), BSY08 (isogenic to KY73 with AGT1 deleted), BSY09 (isogenic to MC966A with AGT1deleted) and even strain EBY.VW4000 (hxt1-hxt17 agt1 gal2-null), were not able to grow on α-MG as the sole carbon source. Moreover, none of them presented α-MG transport by facilitated diffusion when the strains were grown on maltose leading us to conclude that the HXT glucose transporters were not involved in α-MG transport.
We found that strain AP77-11B displayed a high periplasmic α-methylglucosidase activity when cells were grown on α-MG. This enzymatic activity was assayed using a method first described for periplasmic invertase in which cells were incubated with sodium fluoride, an inhibitor of enolase, prior to the incubation with α-MG. Then the glucose produced during α-MG hydrolysis could be accurately measured. This extracellular activity was present only in cells grown on α-MG. Glucose derepressed cells did not show periplasmic α-methylglucosidase activity.
α-Methylglucosidase kinetic parameters indicated that this enzyme has a low affinity for the α-MG substrate. Moreover, the specific activity increased during growth on α-MG.
The results reported here showed that there are two distinct pathways for α-MG assimilation in Saccharomyces cerevisiae. One pathway is mediated by AGT1 permease, responsible for active α-MG transport. In the other pathway α-methylglucosidase is secreted to the cell periplasmic space. Thus, the glucose produced there by α-MG hydrolysis is transported to the cytoplasm via the hexose transporters by facilitated diffusion.
Support – FAPESP grant 04/10067-6