Fig. 2. Internalization and degradation of uracil permease are impaired in yckts cells. Wild-type, apm3-
, yckts and yckts apm3- cells transformed with pFL38gF-GFP were grown to the exponential growth phase at 24°C with galactose as a carbon source. Cells were incubated at 37°C for 20 minutes. Adding glucose and incubating for 10 minutes at the same restrictive temperature stopped the synthesis of Fur4p-GFP. Cycloheximide (100 µg/ml) was then added. (A) Uracil uptake (permease activity) was measured at 37°C at various times after the addition of cycloheximide. The results are expressed as a percentage of the initial activity. (B) Protein extracts were prepared at the times indicated after the addition of cycloheximide. Aliquots were analyzed for uracil permease by western immunoblotting using anti-GFP antibody. *, blots were reprobed with anti-Pgk antibody to provide loading controls. I, mostly permease conjugated with ubiquitin. Open, gray and black circles corresponded to different levels of phosphorylation of the permease with the faster migrating bands corresponding to the lowest level of phosphorylation of the permease.
