Increasing Prion Propensity by Hydrophobic Insertion
Figure 3
Effects of primary sequence on prion formation.
(A) Amino acid sequences of constructs in which two additional hydrophobic residues were added at various positions within the Sup35 nucleation domain. For each, the remainder of the protein is the same as wild-type Sup35. Amyloid stretches, as predicted by Lopez de la Paz and Serrano [62], are underlined. The inserted hydrophobic residues are indicated in bold. (B) Prion formation by each of the constructs. Strains expressing the indicated Sup35 mutants as the sole copy of Sup35 were grown in YPAD medium for two days, and then 10-fold serial dilutions were plated onto medium lacking adenine to select for [PSI+]. For each construct, the position and scores of amyloid stretches predicted by Waltz [28], as well as the minimum ZipperDB score [63], are indicated. Individual Ade+ colonies we picked from each plate and tested for stability and curability, as in Figure S2. Colonies were considered stable and curable if they maintained a white/pink phenotype on YPD, but were red after treatment with guanidine HCl. (C) Western blot of expression levels of wild-type and mutant Sup35s.