FIGURE 4:

Htt103QP degradation after a short period of induction (1 h) is independent of Dsk2. (A) The degradation of Htt103QP after a short induction in the absence and presence of proteasome inhibitor. WT cells with Htt103QP plasmid growing in raffinose medium containing 0.1% l-proline at 30°C were pretreated with 0.003% SDS. After 3 h, dimethyl sulfoxide (DMSO) or 75 μM MG132 was added into the cultures. After incubation for 30 min, galactose (2%) was added into the medium to induce Htt103QP expression for 1 h. Finally, glucose (2%) was added into the cultures to shut off Htt103QP expression. The cells were collected over time to examine Htt103QP protein levels by Western blotting. Pgk1 levels were used as a loading control. Right, ratio change of Htt103QP to Pgk1 after Htt103QP expression shut-off. (B) Cell cycle protein Clb5 is stabilized in the presence of MG132. Yeast cells with Clb5-HA were treated as described. The Clb5 proteins levels were examined at the indicated time points after glucose addition. Right, ratio change. (C) dsk2Δ mutants show similar Htt103QP degradation kinetics as WT cells after a short induction. WT and dsk2Δ, rad23∆, and ddi1∆ mutant cells with Htt103QP plasmid were grown in 30°C raffinose medium to log phase, and then galactose (2%) was added to induce Htt103QP expression. After 1 h of induction, glucose (2%) was added into the medium to shut off Htt103QP expression. Cells were collected at the indicated times, and Htt103QP protein levels were determined by Western blotting with anti-GFP antibody. Pgk1, loading control. Right, ratio change of Htt103QP to Pgk1 after Htt103QP expression shut-off.