Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions

Abstract

The nuclear-encoded Sup35p protein is responsible for the prion-like [PSI(+)] determinant of yeast, with Sup35p existing largely as a high molecular weight aggregate in [PSI(+)] strains. Here we show that the five oligopeptide repeats present at the N-terminus of Sup35p are responsible for stabilizing aggregation of Sup35p in vivo. Sequential deletion of the oligopeptide repeats prevented the maintenance of [PSI(+)] by the truncated Sup35p, although deletants containing only two repeats could be incorporated into pre-existing aggregates of wild-type Sup35p. The mammalian prion protein PrP also contains similar oligopeptide repeats and we show here that a human PrP repeat (PHGGGWGQ) is able functionally to replace a Sup35p oligopeptide repeat to allow stable [PSI(+)] propagation in vivo. Our data suggest a model in which the oligopeptide repeats in Sup35p stabilize intermolecular interactions between Sup35p proteins that initiate establishment of the aggregated state. Modulating repeat number therefore alters the rate of yeast prion conversion in vivo. Furthermore, there appears to be evolutionary conservation of function of the N-terminally located oligopeptide repeats in prion propagation.

Fig. 1. A plasmid-based assay for [PSI⁺] maintenance by N-terminally truncated forms of Sup35p. Strain MT700/9d contains a sup35::kanMX disruption and harbours a copy of the wild-type SUP35 gene on plasmid pYK810. Derivatives of plasmid pUKC1512, carrying N-terminally modified alleles of SUP35, are then transformed into this strain and cells lacking the pYK810 plasmid subsequently selected on 5-FOA-containing medium. The [PSI] phenotype of the resulting haploid strain is then assessed qualitatively using the suppression of the ade2-1 allele by the SUQ5-encoded tRNA suppressor. In plasmid pUKC1512, the N-terminal domain variants are introduced via a BamHI (B)–EcoRV (RV) fragment.

Fig. 2. The ability of N-terminally truncated Sup35p to maintain the [PS1⁺] determinant in the absence of endogenous wild-type Sup35p synthesis. (A) Cell suspensions of individual transformants post-5-FOA selection were plated onto either YEPD (to determine colony colour) or defined medium lacking adenine (–Ade) to test for suppression of the ade2-1 marker. Three independent transformants (a–c) are shown on the –Ade plates. (B) Western blot analysis of either total cell-free extracts (upper panel) or a soluble protein fraction (lower panel; prepared as described in Materials and methods) using an anti-Sup35p polyclonal antibody. Details of the various Sup35p derivatives tested are given in Figure 3.

Fig. 3. Construction of a series of N-terminally truncated alleles of the SUP35 gene. (A) Sequences of the oligopeptide repeats within the N-terminal domain of Sup35p (R1–R6) and comparison with the consensus sequence of the mammalian PrP ‘octarepeat’. The conserved residues between the Sup35p and PrP repeats are underlined. (B) Deletion series encompassing the oligopeptide repeats of Sup35p (R1–R6). The construct numbers are shown on the left while the amino acids deleted are indicated on the right.

Fig. 4. The incorporation of N-terminally truncated Sup35p derivatives into aggregates containing wild-type Sup35p. (A) Cell suspensions of three independent transformants of BSC783/4a were plated on YEPD to determine qualitatively the degree of SUQ5-mediated ade2-1 suppression. The three different phenotypes of the deletants are indicated as follows: SS, strong suppression; WS, weak suppression; NS, no suppression. (B) Western blot analysis of either total cell-free extracts (upper panel) or a soluble protein fraction (lower panel; prepared as described in Materials and methods) using an anti-Sup35p polyclonal antibody. The positions of the full-length wild-type Sup35p (Sup35p) and the co-expressed, N-terminally truncated form (Sup35pdel) are indicated. Details of the various derivatives tested are given in Figure 3.

Fig. 5. Quantification of the degree of nonsense suppression in the BSC783/4a transformants co-expressing N-terminally truncated and wild-type Sup35p. The various transformants were transformed with the PGK–lacZ fusion plasmids pUKC815 and pUKC817, and the levels of expressed β-galactosidase were used to determine the percentage nonsense suppression, as described in Materials and methods. The level of nonsense suppression in the pSUP transformant was taken as 100% and all other transformants were compared with that strain. For each transformant, four samples were assayed independently and the error bars represent standard deviation.

Fig. 6. Replacement of the R5 repeat in the Sup35p-PrD with a copy of the mammalian PrP octarepeat allows for propagation of the [PSI⁺] determinant. (A) The various constructs shown were generated as described in Materials and methods. (B) Analysis of two independent transformants (a and b) in which repeat R5 is replaced by the PrP octarepeat (R-PrP/R-PrP-R6) (see the legend to Figure 2). (C) Analysis of two independent transformants (a and b) in which repeat R5 is replaced by an octapeptide from the S.pombe Sup35p N-terminus (R-Sp).

Fig. 7. The nucleated conformational conversion model of Serio et al. (2000). By increasing the number of oligopeptide repeats (+R), Sup35p–Sup35p interactions are favoured over Sup35p–Sup45p interactions. Similarly, deletion of oligopeptide repeats (–R) leads to reduced efficiency of Sup35p–Sup35p interactions. For further discussion of the model, refer to the text.