Figure 5

(A-E) Visualization of protein aggregates from GAF-Q domains fused to GFP. 582q-GFP (A) and 519q-GFP (B) clearly show aggregates similar to (C) NM-GFP i.e. prion-forming domain of Sup35 (NM region of SUP35) fused to GFP, which was used as a positive control. (D) Z-GFP, which contains GFP fused to polyglutamine domain of Drosophila Zeste protein, acts as a negative control together with yeast cells containing GFP alone (E), which does not show any aggregation. (F) Sedimentation analysis of 519Q-SupC, 582Q-SupC expressing cells which exhibit stable non-sense suppression phenotype. Total extracts from GuHCl treated (after GuHCl) or non-treated (before GuHCl) cells expressing 519Q-SupC, 582Q-SupC, SupC∆NM and stable [PSI⁺] strain were subject to 50 K x g ultacentrifugation for 15 minutes. Total (T), supernatant (S) and pellet (P) fractions were resolved by SDS-PAGE and Western blot was performed using anti-SUP35 serum. Extracts from SupC∆NM and [PSI+] strains were used as controls. As with [PSI+], both 519Q-SupC, 582Q-SupC exhibit sedimentation in pellet before GuHCl treatement and 519Q-SupC, 582Q-SupC became soluble in supernatant (S) fraction after GuHCl treatement. SupC∆NM lacking prion domain remains soluble regardless of GuHCl treatment. (G) Non-mendelian inheritance of 582Q+. A diploid (2n) made by mating 582q + MATa strain with 582Q- MATα displayed 582Q + phenotype. Tetrad dissection of diploid (2n) shows 4 meiotic progeny (1n) which stably exhibit non-sense suppression visible by growth on -ADE and all this progeny was curable when grown on YPD containing 5 mM GuHCl.