The role of Stl1p in glycerol accumulation in osmotically stressed Icewine yeast Saccharomyces cerevisiae K1V1116

The high sugar concentration in Icewine juice exerts hyperosmotic stress in the wine yeast causing water loss and cell shrinkage. To counteract the dehydration, yeast synthesize and accumulate glycerol as an internal osmolyte. In a laboratory strain of S. cerevisiae, STL1 encodes for Stl1p, an H+/glycerol symporter that is glucose inactivated, but induced upon hyperosmotic stress.;We proposed that Stl1p is (1) active during Icewine fermentation and is not glucose inactivated and (2) its activity contributes to the limited cell growth observed during Icewine fermentation as a result of the dissipation of the plasma membrane proton gradient.;To measure the contribution of Stl1p in active glycerol transport (energy dependent) during Icewine fermentation, we first developed an Stl1p-dependent [14C]glycerol uptake assay using a laboratory strain of S. cerevisiae (BY4742 and DeltaSTL1) that was dependent on the plasma membrane proton gradient and therefore energy-dependent. Wine yeast K1-V1116 was also shown to have this energy dependent glycerol uptake induced under salt stress.;The expression of STL1 and Stl1p activity were compared between yeast cells harvested from Icewine and diluted Icewine fermentations. Northern blot analysis revealed that STL1 was expressed in cells fermenting Icewine juice but not expressed under the diluted juice conditions. Glycerol uptake by cells fermenting Icewine juice was not significantly different than cells fermenting diluted Icewine juice on day 4 and day 7 of Vidal and Riesling fermentations respectively, despite encountering greater hyperosmotic stress. Furthermore, energy- dependent glycerol uptake was not detected under either fermentation conditions.;STL1, was found to be a highly upregulated gene in Icewine fermenting cells and its expression was 25-fold greater than in yeast cells fermenting diluted Icewine juice, making it one of the most differentially expressed genes between the two fermentation conditions. In addition, Icewine fermenting cells showed a two-fold higher glycerol production in the wine compared to yeast fermenting diluted Icewine juice.;Because our findings show that active glycerol uptake was not detected in yeast cells harvested from Icewine fermentation, it is likely that Stl1p was glucose inactivated despite the hyperosmotic stress induced by the Icewine juice and therefore did not playa role in active glycerol uptake during Icewine fermentation.