FIGURE 2.

Degradation of FBPase is impaired in all GID1 deletion mutants. A, cells were grown overnight in YP-ethanol (YPE), transferred to YPD, and harvested at the indicated time points. Cell extracts were prepared, and FBPase degradation was analyzed via Western blotting using FBPase or 3-phosphoglycerate kinase (Pgk) antibodies. 3-Phosphoglycerate kinase served as a loading control. B, shown is ubiquitination of FBPase in the GID1 mutants. Cells expressing FBPase-TAP from a plasmid were incubated overnight in YPE (sample E) and then transferred to YPD for 25 min to induce FBPase ubiquitination. After cell lysis, FBPase-TAP was pulled down using IgG-Sepharose beads. Ubiquitination was detected with ubiquitin antibodies after immunoblotting.