Figure 1.
Insertion of hydrophobic residues increases prion formation.
(A) Schematic of Sup35. The sequence of the nucleation domain (amino acids 1–40) is shown. (B) Sequences of the nucleation domains of each of the hydrophobic-addition constructs. Inserted hydrophobic residues are indicated in bold. For each, the remainder of the protein is the same as wild-type Sup35. (C) Prion formation by each construct. Strains expressing the indicated Sup35 mutants as the sole copy of Sup35 were transformed either with an empty vector (left) or with a plasmid expressing the matching Sup35 mutant under control of the GAL1 promoter (right). All strains were cultured for three days in galactose/raffinose dropout medium, and then 10-fold serial dilutions were plated onto medium lacking adenine to select for [PSI+]. (D) Western blot of wild-type and mutant Sup35.
Figure 2.
In vitro amyloid aggregation of the mutant prion forming domains.
Aggregation of purified PFDs was monitored using thioflavin T. Reactions were incubated with intermittent shaking for 48 h. Fluorescent readings were taken approximately every 90 min. Error bars represent the standard deviations of three samples.
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.
Figure 4.
Deletion of tyrosine residues reduces prion formation.
(A) Amino acid sequences of constructs in which tyrosines were deleted from various positions within the Sup35 nucleation domain. Tyrosines are indicated in bold. (B) Western blot of expression levels of wild-type and mutant Sup35s. (C) Prion formation by each construct. Strains expressing the indicated Sup35 mutants as the sole copy of Sup35 were transformed either with an empty vector (left) or with a plasmid expressing the matching Sup35 mutant under control of the GAL1 promoter (right). All strains were cultured for three days in galactose/raffinose dropout medium, and then 10-fold serial dilutions were plated onto medium lacking adenine to select for [PSI+].
Figure 5.
Tyrosine and hydrophobic residues promote foci formation.
(A) The Sup35 PFD promotes formation of fluorescent foci. GFP or the NM domain from wild-type Sup35 fused to GFP were expressed under control of the GAL1 promoter. Cells were grown in galactose/raffinose dropout medium for 24 h, and then visualized by confocal microscopy. (B) The hydrophobic insertion constructs each support formation of fluorescent foci. Conditions were as described in (A). (C) The −5Try and −2TyrA constructs fail to form fluorescent foci. (D) The −2TyrB construct forms foci in a fraction of cells.
Figure 6.
Aromatic residues are not required in the Sup35 nucleation domain.
(A) The five tyrosines in the Sup35 nucleation domain (amino acids 1–40) were replaced with either leucines, isoleucines or valines. Strains were transformed either with an empty vector (left) or with a plasmid expressing the matching Sup35 mutant under control of the GAL1 promoter (right). All strains were cultured for three days in galactose/raffinose dropout medium, and then 10-fold serial dilutions were plated onto medium lacking adenine to select for [PSI+]. (B–E) Stability and curability of the Ade+ phenotype in cells expressing wild-type Sup35 (B), or Sup35 in which the five tyrosines in the nucleation domain were replaced with leucine (C), valine (D) or isoleucine (E). For each mutant, eight individual Ade+ isolates were grown on YPD (−) and YPD plus 4 mM guanidine HCl (+). Cells were then restreaked onto YPD to test for loss of the Ade+ phenotype.
Figure 7.
Amino acid composition of prion and non-prion Q/N-rich domains.
(A) Histogram of the prevalence of strongly prion-promoting residues (FYWIMV) among Q/N-rich proteins (open bars) and among peptide fragments from the yeast proteome (black bars). For the Q/N-rich proteins, each of the regions of the yeast proteome identified by Harrison and Gerstein [16] as having high Q/N-bias were scored for the fraction of strongly prion-promoting amino acids. For the proteomic data, the yeast proteome was scanned using a 100 amino acid window size; each 100-amino-acid window was scored for the fraction of strongly prion-promoting amino acids. (B) Histogram of the prevalence of strongly prion-promoting amino acids among yeast prion and non-prion Q/N-rich domains. The black bars include Q/N-rich regions (as identified by Harrison and Gerstein) from yeast proteins shown to act as prions, as well as from proteins containing domains shown by Alberti et al. to have prion-like activity in four independent assays [19]. Open bars represent all other yeast Q/N-rich regions identified by Harrison and Gerstein. (C) Amino acid prevalence in Q/N-rich domains. Grey bars represent the prevalence of different groups of amino acids in the yeast proteome. Black bars represent the average frequency of these amino acids among Q/N-rich regions from both proteins shown to act as prions and proteins containing domains shown by Alberti et al. to have prion-like activity in four independent assays. Open bars represent the average frequency of these amino acids among all other yeast Q/N-rich domains identified by Harrison and Gerstein. (D) The prevalence of different groups of amino acids, plotted as a fraction of non-Q/N residues.