Prion species barrier between the closely related yeast proteins is detected despite coaggregation

Abstract

Prions are self-perpetuating and, in most cases, aggregation-prone protein isoforms that transmit neurodegenerative diseases in mammals and control heritable traits in yeast. Prion conversion requires a very high level of identity of the interacting protein sequences. Decreased transmission of the prion state between divergent proteins is termed "species barrier" and was thought to occur because of the inability of divergent prion proteins to coaggregate. Species barrier can be overcome in cross-species infections, e.g., from "mad cows" to humans. We studied the counterparts of yeast prion protein Sup35, originated from three different species of the Saccharomyces sensu stricto group and exhibiting the range of prion domain divergence that overlaps with the range of divergence observed among distant mammalian species. All three proteins were capable of forming a prion in Saccharomyces cerevisiae, although prions formed by heterologous proteins were usually less stable than the endogenous S. cerevisiae prion. Heterologous Sup35 proteins coaggregated in the S. cerevisiae cells. However, in vivo cross-species prion conversion was decreased and in vitro polymerization was cross-inhibited in at least some heterologous combinations, thus demonstrating the existence of prion species barrier. Moreover, the barrier between the S. cerevisiae protein and its Saccharomyces paradoxus and Saccharomyces bayanus counterparts was asymmetric both in vivo and in vitro. Our data show that a decreased cross-species prion transmission does not necessarily correlate with a lack of cross-species coaggregation, suggesting that species-specificity of prion transmission is controlled at the level of conformational transition rather than coaggregation.

Fig. 1.

S. paradoxus and S. bayanus Sup35 proteins retain prion-forming abilities despite divergence of PrD sequences. (A) Structural and functional organization of the S. sensu stricto Sup35 proteins. N, M, C, QN, and OR refer to the Sup35N, Sup35M, Sup35C, QN-rich stretch, and ORs, respectively. Numbers correspond to amino acid positions; the percentage of amino acid identity to S. cerevisiae is shown for each region of S. paradoxus and S. bayanus proteins individually. For sequence alignment of the Sup35N regions, see SI Fig. 5. Data are from refs. – and the Yeast Genome Database (www.yeastgenome.org), confirmed by sequencing of our PCR-amplified clones. (B and C) Transient overproduction of S. paradoxus Sup35 protein (Sup35_SP) or the Sup35NM_SP-GFP fusion protein (B), or transient overproduction of S. bayanus Sup35 protein (Sup35_SB) (C) induces prion formation in the [psi⁻ PIN⁺] S. cerevisiae strains bearing the SUP35_SP (B) or SUP35_SB (C) gene instead of SUP35_SC. Empty plasmids pmCUP-GFP (B) or pRS316GAL (C) were used as controls. Prion formation was detected by growth on −Ade medium after induction on P_CUP1-SUP35_SP or P_CUP1-SUP35NM_SP-GFP constructs on the medium with 100 μM CuSO₄ (B) or P_GAL-SUP35_SB construct on Gal medium (C). (D) Sup35_SP generates both strong and weak prion variants, whereas Sup35_SB generates only weak prion variants in S. cerevisiae, as judged from the efficiency of ade1–14 suppression reflected by growth on −Ade. Note that both strong and weak variants of the Sup35_SP prion show low mitotic stability (see SI Table 2). Plates were photographed after 5 (B), 8 (C), and 7 (D) days of incubation.

Fig. 2.

Coaggregation of heterologous Sup35 proteins in S. cerevisiae. (A) The S. cerevisiae [PSI⁺] strain simultaneously expressing both endogenous (Sup35_SC) and heterologous (Sup35_SP or Sup35_SB) proteins shows all of the Sup35-reacting material in the pellet (P) after centrifugation at 39,000 × g, whereas the isogenic [psi⁻] strain retains a fraction of Sup35 in the supernatant (S). Shift of Sup35_SB to pellet can be monitored directly because of its lower molecular weight, compared with Sup35_SC. T, total lysate. (B) The chimeric Sup35NM_SP-GFP protein, expressed from the P_CUP1 promoter in the presence of background levels (2 μM) of CuSO₄, shifts to pellet together with the endogenous Sup35_SC protein in the [PSI⁺] extract, in contrast to the [psi⁻] extract. Designations are as in A. (C–F) The GFP-tagged NM fragments of Sup35_SC (C), Sup35_SP (D), and Sup35_SB (E), but not the GFP-tagged Sup35NM fragment of P. methanolica (Sup35NM_PM-GFP, F), colocalize with the aggregated clumps of RFP-tagged Sup35_SC in the S. cerevisiae [PSI⁺] cells. GFP- and RFP-tagged constructs were expressed from the P_CUP1 and P_GAL promoters, respectively, in the Gal+Raf medium supplemented with 150 μM CuSO₄. In each case, >100 cells containing both GFP and RFP aggregates were scored, and the percentage of cells with colocalization is shown. Scale bars are indicated.

Fig. 3.

Prion species barrier between the closely related Sup35 proteins in the plasmid shuffle assay. Designations SC, SP, and SB refer to the SUP35_SC, SUP35_SP, and SUP35_SB genes, respectively. Donor [PSI⁺] sup35Δ strain with SC gene on the CEN plasmid, which grew on −Ade before the experiment (stage I), was transformed individually with CEN plasmids either bearing the intact SC, SP, or SB genes (A) or containing the chimeric constructs composed of the SUP35MC region of S. cerevisiae (MC_SC) and SUP35N regions (N) of various origins (B). In B, reconstructed SUP35 gene with SUP35N_SC origin was used as a control. In contrast to intact or reconstructed SUP35_SC, heterologous genes (A) or chimeric genes with the heterologous N regions (B) inhibited suppression of ade1–14 by [PSI⁺], as judged by decreased growth on −Ade medium selective for both plasmids (stage II). After elimination of the original SC plasmid (stage III), all colonies with the new SC plasmid or reconstructed N_SC-MC_SC plasmid retained [PSI⁺], whereas most or all colonies with SP, SB, or chimeric plasmids containing N_SP or N_SB lost [PSI⁺], as seen by growth/no growth on −Ade medium, respectively. Numbers of [PSI⁺] and [psi⁻] colonies obtained are given in each case.

Fig. 4.

In vitro polymerization of the S. sensu stricto Sup35NM protein fragments. The purified (His)₆-tagged Sup3NM regions of S. cerevisiae (Sup35NM_SC or SC, A), S. paradoxus (Sup35NM_SP or SP, B), and S. bayanus (Sup35NM_SB or SB, C) spontaneously polymerize in the nondenaturing conditions after a certain lag period, as detected by a decrease of the monomeric fraction remaining soluble in SDS and capable of entering the SDS/PAGE gel without boiling. Boiled samples where all protein enters the gel are shown in each case as controls. Addition of preformed polymers at the ratio of 1:20 leads to the following results: Sup35NM_SC promotes polymerization of Sup35NM_SC (A) and Sup35NM_SP (B) but delays polymerization of Sup35NM_SB (C); Sup35NM_SP promotes polymerization of Sup35NM_SP (B) but delays polymerization of both Sup35NM_SC (A) and Sup35NM_SB (C); Sup35NM_SB promotes polymerization of both Sup35NM_SC (A) and Sup35NM_SB (C) but delays polymerization of Sup35NM_SP (B).