Figure 2. Prion form of Sup35 is deposited to the age-associated protein deposit.
(A, B) Co-localization of Sup35-GFP and Hsp104-mCherry in diploid [psi−] (A) and [PSI+] (B) cells, where a single locus of the respective gene was tagged with the fluorescent marker indicated. White arrowhead indicates the age-associated protein deposit (Hsp104 positive) and the red arrowhead Sup35-aggregates not associated with the age-associated protein deposit. (C) Percentage of age-associated protein deposits in [psi–] and [PSI+] cells with enriched Sup35, (N = 130). (D) Percentage of cells with large Sup35-foci in cells of indicated age groups (N = 157–184 cells pre age group, average age young [psi–] 1.1, young [PSI+] 1.1, aged [psi–] 6.9, aged [PSI+] 5.9 generations). (E) Percentage of [psi–] and [PSI+] cells of the indicated age group (see D) containing an age-associated protein deposit, (N = 157–184 cells pre age group). (F) Time-lapse images of a [PSI+] cell co-expressing Sup35-GFP (green) Hsp104-mCherry (red) at the indicated time points. Arrowheads point at the age-associated protein deposit as observed in the different channels. The newborn daughters are indicated in the bottom row. (G) Percentage of divisions where the Sup35-GFP-labeled age-associated protein deposit is retained in the aging mother cell lineage, (N = 204 divisions). (H) Fluorescent images of a cell co-expressing the P-body protein Dcp2 tagged with GFP and Hsp104 tagged with mCherry. (I) Percentages of cells of indicated age groups that contain the indicated fluorescent foci, (N = 18–71 per group). (J) Time-lapse, fluorescent images of Dcp2-GFP and Hsp104-mCherry expressing cells at the indicated time points after switching the cells to 0.1% glucose. Scale bars (A, B, H, J) 5 μm, (F) 2 μm. Graphs display mean ± SEM.