Fig 3. Coiled-coil region of Rpt subunits is required for proteasome function.

(A) We constructed a series of deletion mutants (upper figures) in the pAUR123 vector and expressed them in the Rpt tet-off strains. Yeast cultures were grown to early log phase (OD₆₀₀ of approximately 0.6–0.8). Ten-fold serial dilutions of these cultures were spotted on YPDA medium agar plates or on YPDA medium agar plates containing 10 μg/ml doxycycline (Dox). Plates were incubated at 30°C for 2 days and then photographed (lower panels). (B) Deletion of N-terminal coiled-coil region of Rpt subunits induces the accumulation of polyubiquitinated proteins. Accumulation of polyubiquitinated proteins in the Rpt tet-off yeast cells expressing wild-type and deletion mutants (Rpt1Δ65, Rpt2Δ65, Rpt3Δ65, Rpt4Δ65, Rpt5Δ40, and Rpt6Δ50) were analyzed using western blot with an anti-polyubiquitin antibody. After yeast cells at early log phase were treated with 20 μg/mL Dox for 3 h, cells were harvested and lysed with glass beads in the presence of 10% trichloroacetic acid (TCA) to preserve ubiquitination patterns. PGK1 was used as a loading control.