Figure 4
Heterologous expression of gat-1 in yeast confers the ability to uptake D-galacturonic acid (D-GalA) with high affinity. The cDNA of gat-1 was fused to the super folder-green fluorescent protein (sfGFP) under the control of the phosphoglycerate kinase 1 (PGK1) promoter and transformed into yeast (S. cerevisiae strain BY4742). (A) The incorporation of the construct into the plasma membrane was followed by confocal microscopy (100 × oil; upper panels), whereas no GFP fluorescence could be observed in the vector-only transformed control cells (lower panels). (B) D-GalA transport by the S. cerevisiae strains as described above. Shown is D-GalA transport by yeast with (closed circles) or without (open circles) GAT-1. The initial concentration of D-GalA was 50 μM. All values are the mean of two measurements. (C) Kinetics of D-GalA transport by GAT-1. The transport rate was determined as a function of D-GalA concentration (ranging from 0.1 μM to 10 μM) by yeast strains expressing gat-1-sfGFP and was normalized by total protein concentration.