Figure 5.

HSP104 and RNQ1 are required for formation of the microscopically visible aggregates. Wild-type, hsp104Δ, and rnq1Δ cells were grown in a raffinose-containing medium to log phase before addition of galactose to induce expression of the GPF^C and GFP^N104 proteins. Cells were harvested at the 6-hour time point after induction and their GFP patterns were categorized as described in Figure 2 legend. (A) Percentage of cells containing mature aggregates. (B) Percentage of cells containing diffuse GFP signals. (C) Comparison of levels of SDS-insoluble aggregates between the wild-type and the hsp104Δ and rnq1Δ mutant cells by a dot blot filter trap assay. Induced expression of NLS-GFP^N104 and NES-GFP^N104 and supernatant preparation from total cell lysates were as described in Figure 3 legend. The number of cells loaded in the first well was empirically determined to result in similar Western blot signals between strains on the nitrocellulose membrane, and the extract was subjected to two-fold serial dilutions in the subsequent wells. **p value ≤ 0.005, ***p value ≤ 0.001.