Fig. 1.
Substrate specificity of the CO3H5.2 transporter. (A) CO3H5.2-encoded nucleotide sugar transport into Golgi vesicles from S. cerevisiae. Transport activity is shown as the amount of nucleotide sugar within vesicles at 25°C minus the corresponding value at 0°C. S. cerevisiae was transformed with pG426 (gray bars) or pG426-FLAG-CO3H5.2 (black bars). Results are the average of two independent determinations and standard error. (B) Western blot analyses of proteins derived from Golgi vesicles after SDS/PAGE. S. cerevisiae was transformed with the empty vector, pG426, or the vector encoding FLAG-CO3H5.2 wild-type and mutations V192F and G267E. Proteins were visualized with an anti-FLAG monoclonal antibody. (C) Nucleotide sugar transport with UDP-[3H]GalNAc (Left), UDP-[3H]GlcNAc (Center), and UDP-[3H]Glc (Right) into Golgi vesicles of cells transformed with wild-type CO3H5.2 and the two above-described point mutants. Concentration of nucleotide sugar was 1 μM. Results are expressed as averages of two independent determinations and the standard error. (D) Separation of K. lactis by FACS after cell-surface labeling with fluorescent lectin from G. simplicifolia II. K. lactis Golgi apparatus UDP-GlcNAc transporter mutants KL3 were transformed with pE4 or pE4-CO3H5.2. Cells were then incubated with the fluorescent lectin and separated in a FACS. (Upper Left) KL8 wild type. (Upper Right) KL3 mutant. (Lower Left) KL3 transfected with empty plasmid pE4. (Lower Right) KL3 transfected with pE4-C03H5.2.