[<i>PSI</i>⁺] requirements for Sis1 are prion-variant dependent.

<p>(A and B) Sis1-plasmid shuffling experiments to examine the potential effects of Sis1 domain deletions on [<i>PSI</i>⁺] propagation. [<i>PSI</i>⁺] bearing cells were transformed by plasmids expressing Sis1 truncations or deletions and subjected to plasmid shuffling. Color phenotype assays are shown for representative transformants (<i>n</i>≥6 for each plasmid) following loss of the [<i>SIS1</i>-Sis1, <i>URA3</i>] plasmid and bearing either the strong [<i>PSI⁺</i>] variant [<i>PSI⁺</i>]^Sc4 (A), or the weak [<i>PSI⁺</i>] variant [<i>PSI⁺</i>]^Sc37 (B). Parental [<i>PSI</i>⁺] cells for each variant (parent) and cells cured by growth in the presence of GdnHCl (cured) are included as positive and negative controls for colony color. Cells expressing full-length Sis1 (Sis1) from a plasmid were used as a positive control for the stability of the prion throughout the plasmid-shuffling procedure. For clarity, images taken from different parts of the same plate have been arranged in columns. (C) Sis1 protein expression levels in cells containing the weak [<i>PSI⁺</i>] variant [<i>PSI⁺</i>]^Sc37 in the W303 genetic background. Cell extracts from isolates in panel B were subjected to immunoblot analysis using antibody specific for Sis1. A band cross-reacting with the Sis1 antibody is shown as a loading control. Dotted lines separate lanes taken from different parts of the same gel. (D) Maintenance or loss of [<i>PSI</i>⁺] in cells shown in (A) ([<i>PSI⁺</i>]^Sc4, <i>left</i>) and (B) ([<i>PSI⁺</i>]^Sc37, <i>right</i>) was also confirmed by semi-denaturing detergent agarose gel electrophoresis (SDDAGE). Detergent resistant Sup35 aggregates indicative of the presence of [<i>PSI</i>⁺] were resolved by SDDAGE and visualized by immunoblot analysis using an antibody specific for Sup35. Control [<i>PSI</i>⁺] and [<i>psi</i>⁻] cells for each variant were included for comparison.