Figure 7.

Ssa1p and its cochaperone Ydj1p are required for rescue of aggregated ΔssCG*. Cells expressing ΔssCG* were grown at 30°C and shifted to 37°C for 60 min before the solubility assay. The solubility of ΔssCG* was assessed in SSA1, ssa1-45^ts (A), wild-type W303-1C (SSA1, SSA2, SSA3, SSA4), and ydj1-151^ts strains (B). The same amount of total (T), supernatant (S), and pellet (P) fraction was analyzed via SDS-PAGE and immunoblot. Immunoblots were analyzed with CPY antibody and PGK antibody as a control. The fluorescence of ΔssCG* was analyzed in living cells (C) as described in Material and Methods. The cells harboring overexpressed ΔssCG* or an empty plasmid were grown at 30°C and shifted to 37°C for 60 min before analysis. All the cells were visualized by fluorescence microscopy using equal exposure times and conditions. Resolubilization of aggregated ΔssCG* was assessed in SSA1 and ssa1-45^ts cells (D). After temperature shift of cells to 37°C for 1 h, cycloheximide was added to a final concentration of 0.5 mg/ml to block further protein synthesis. Twenty OD₆₀₀ of cells were taken at the indicated time points and treated as indicated for the above solubility assay. Immunoblot of Sec61p served as control. Three independent experiments gave similar results. The fluorescence of GFP-cODC and GFP-cODC-C414A were analyzed in SSA1 cells at 37°C as stated above (E).