Pin4C overexpression leads to larger [<i>PSI</i>⁺] aggregates.

<p>(A) Sup35-GFP aggregates become larger upon overexpression of Pin4C. Upper panels: Representative GFP images of strong [<i>PSI</i>⁺][<i>PIN</i>⁺] <i>SUP35-GFP</i> expressing strains (GF657) after an overnight induction of <i>PIN4C</i>; empty vector control was incubated in galactose medium for the same amount of time; [<i>psi</i>⁻] culture shown is after 4-days of induction of <i>PIN4C</i>. Lower panels: Lysates prepared from cultures shown above were treated with 2% SDS at room temperature and analyzed for the presence of Sup35 by SDD-AGE with anti-Sup35C-GFP. (B) Overexpression of Pin4C-DsRed leads to the formation of single dot-like aggregates concomitant with enlargement of Sup35-GFP aggregates. Representative DsRed and GFP images are of strong [<i>PSI</i>⁺][<i>PIN</i>⁺] <i>SUP35-GFP</i> (GF657) cells after induction of pHR81<i>GAL</i>-<i>PIN4C-DsRED</i> for the times indicated. Cells were grown in liquid plasmid selective galactose media. (C) Pin4C-DsRed aggregates are composed of SDS-resistant polymers. Lysates from cultures where ∼60% of cells contain large single Pin4-DsRed dots were treated with 2% SDS at room temperature and analyzed by SDD-AGE. The blot was probed with anti-DsRed antibody (left lane), stripped and re-probed with anti-Sup35C antibody (right lane). (D) Increased size of visible [<i>PSI</i>⁺] aggregates requires continuous synthesis of the Sup35 protein. Single cells of strong [<i>PSI</i>⁺][<i>PIN</i>⁺] expressing <i>SUP35ΔNM</i> at its endogenous locus and harboring extrachromosomal p<i>TET^r</i>-<i>SUP35-GFP</i> (L3126) were transformed with pHR81<i>GAL</i>-<i>PIN4C</i>-<i>DsRED</i>, or vector, pHR81<i>GAL</i>-<i>DsRED</i>. Cells were grown in 2% raffinose +2% galactose +0.025 µg/ml doxcycline for 6 hrs, which induced <i>PIN4C-DsRED</i> and allowed the Sup35-GFP level to be close to the normal Sup35 level. Single cells with diffuse DsRed were then micromanipulated and grown for 18 hrs dividing ∼3 times on 2% raffinose +2% galactose +10 µg/ml doxcycline medium where new synthesis of Sup35-GFP is repressed. GFP and DsRed images of a representative part of a growing microcolony are shown. The arrows point to mother cells, and the arrowheads point to two daughter cells with and without a large DsRed dot.