Figure 4. Uracil-induced Fur4 sorting is perturbed in ubp2Δ mutant cells.

A FUR4-GFP fusion reporter was transformed into cells, which were then grown in raffinose overnight. Galactose was added (OD₆₀₀ = 0.6) for 2 hours to induce synthesis, followed by glucose for 10 min to chase Fur4-GFP to the plasma membrane, and then uracil (40 µg/ml) to trigger transporter internalization. (A) Fur4-GFP signal viewed by fluorescence microscopy at the indicated time points (min) after uracil addition. T0 indicates cells immediately before addition. (B) Fur4-GFP protein levels as measured by Western blotting. Total protein extracts were generated from cells harvested at the indicated time points following uracil addition. Fur4-GFP was probed with anti-GFP antibodies. 3-phosphoglycerate kinase (PGK) was used as a loading control. The asterisk indicates an unknown background band.