Integrity of the Saccharomyces cerevisiae Rpn11 Protein Is Critical for Formation of Proteasome Storage Granules (PSG) and Survival in Stationary Phase
Figure 6
Overexpression of the Rpn11 carboxyl terminus.
(A) Survival during starvation-induced stationary phase of wild type or rpn11-m1 strains containing the plasmid overexpressing the 45 amino acids of the Rpn11 carboxyl terminus (C-R11(45)) or its mutated form (C-R11m1(25)) on rich medium (YPD) at 26°C after growth for 1 and 15 days in rich medium at 26°C. Comparable number of cells was spotted at 10-fold dilutions on YPD medium. (B) Wild type and rpn11-m1 cells expressing Rpn1-GFP and overproducing C-R11(45) or C-R11m1(25) were grown in rich medium (YPDA) at 26°C and examined by fluorescence microscopy in the exponential growth phase (EP) and after 5 days in stationary phase (SP). Typical images of Rpn1-GFP localization are shown. (C) Wild type and rpn11-m1 cells expressing Rpn1-GFP and overproducing C-R11(45) or C-R11m1(25) were grown in YPDA medium at 26°C. For each time point (day), the localization of Rpn1-GFP fluorescence was scored as nuclear (blue bar), at the nuclear periphery (red bar) or as cytosolic dots (green bar; n>100 cells for each time point; two independent experiments; error bars report the differences between the two experiments) and (*) indicate that the difference in the distribution of the Rpn1-GFP signal in the mutant cells are significant relative to the wild-type cells both overproducing C-R11(45) after statistical analyses (Pearson’s chi-squared test, P values <0.05). (D) Comparison of Rpn1-GFP-cytosolic foci apparition between the wild type (grey) and the rpn11-m5 mutant (black) both overexpressing C-R11(45) for each day. Error bars represent the difference observed between the two experiments and (*) indicates that the difference between the two strains is significant (Fisher’s exact test, P values <0.05).