Heterologous cross-seeding mimics cross-species prion conversion in a yeast model - PubMed (original) (raw)

Heterologous cross-seeding mimics cross-species prion conversion in a yeast model

Namitha Vishveshwara et al. BMC Biol. 2009.

Abstract

Background: Prions are self-perpetuating, infectious, aggregated proteins that are associated with several neurodegenerative diseases in mammals and heritable traits in yeast. Sup35p, the protein determinant of the yeast prion [PSI+], has a conserved C terminal domain that performs the Sup35p function and a prion domain that is highly divergent. Prions formed by chimeras of the prion domain of various species fused to the C domain of Saccharomyces cerevisiae exhibit a 'species barrier', a phenomenon first observed in mammals, and often fail to transmit the prion state to chimeras with prion domains of other species.

Results: We focus on the chimera containing the prion domain of Pichia methanolica and examine how tight the 'species barrier' is between the chimera and S. cerevisiae. Although either of two Q/N-rich prions, [PSI+] or [PIN+], enhances the formation of the chimeric prion, [CHI+PM], neither a non-Q/N-rich prion nor a non-prion Q-rich aggregate promotes the formation of [CHI+PM]. [CHI+PM] has many features characteristic of yeast prions: aggregation, cytoplasmic transmission and a two-level protein structure. [CHI+PM] formed in the presence of [PSI+] can propagate independently of [PSI+] and forms at least two different variants of the prion, suggesting the generation and not transmission of new prion seeds.

Conclusion: Although the sequence similarity between the S. cerevisiae Q/N-rich prion determinants and the P. methanolica prion domain is low, we find that the chimera containing the prion domain of P. methanolica can occasionally be cross-seeded by [PSI+] to mimic crossing the species barrier, to form the [CHI+PM] prion. Our data suggests that crossing the barrier occurs by a de novo formation of the foreign chimeric prion. Thus, the species barrier appears to be crossed by a heterologous seeding mechanism, wherein the infected prion protein uses the pre-existing seed as an inefficient template.

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Figures

Figure 1

Figure 1

NMPM-CSC but not NMCA-CSC is inactivated in the presence of [PSI+]. A. NMPM-CSC and NMCA-CSC provide translation termination function. [PSI+] or sup35 cells ectopically expressing prion domains of either Pichia methanolica (NMPM) or Candida albicans (NMCA) fused to the C domain of Saccharomyces cerevisiae (NMSC) expressed on a URA3 plasmid on media containing low adenine-Ura and -Ade-Ura. -Ura is used to maintain the plasmid. B. NMPM-CSC is inactivated in [PSI+] cells and not in a sup35 mutant. Around 107 [PSI+] cells expressing either NMPM-CSC or NMCA-CSC, or sup35 mutant yeast expressing NMPM-CSC were plated on -Ade-Ura media.

Figure 2

Figure 2

Ade+ colonies have prion properties. A. NMPM-CSC is mainly in the pellet fraction in Ade+ colonies. [CHI+PM] and [_chi_-PM] lysates subjected to high-speed centrifugation and probed for the chimeric protein using an anti-HA tag antibody. T: total; S: supernatant; P: pellet. B. [CHI+PM] colonies have SDS-stable sub-particles. [CHI+PM] and [_chi_-PM] lysates analyzed on an agarose gel and probed for the chimeric protein (anti-HA tag). C. NMPM-GFP forms puncta in [CHI+PM] colonies. NMPM-GFP under a copper-inducible promoter overexpressed in [CHI+PM] or [_chi_-PM] cells for 4 hours and examined under a fluorescence microscope. The two kinds of NMPM-GFP puncta are sometimes observable in the same [CHI+PM] culture and are not representative of different [CHI+PM] colonies.

Figure 3

Figure 3

NMPM-CSC is not incorporated into [PSI+] sub-particles. [_chi_-PM][PSI+] and [CHI+PM][PSI+] lysates resolved on an agarose gel and probed for the chimeric protein (anti-HA) and reprobed for endogenous Sup35p (_anti-NM_SC).

Figure 4

Figure 4

More than one variant of [CHI+PM] forms in the presence of [PSI+]. A. Some [CHI+PM] variants have differing amounts of SDS-stable sub-particles. Lysates from [CHI+PM] colonies differing in color on low adenine media were resolved on an agarose gel and probed with an anti-HA tag antibody that detects NMPM-CSC. Stained chicken pectoralis muscle extract provided molecular weight markers. The difference in amount of SDS-stable sub-particles was reproducible for this and another pair (not shown) isolated on the basis of color difference. B. Some [CHI+PM] variants have different sized SDS-stable sub-particles. Colonies formed on -Ade-Ura could be distinguished phenotypically on low adenine media by differences in color. Lysates from these variants were resolved on an agarose gel and probed with anti-HA tag antibody, which detects NMPM-CSC. The difference in size of SDS-stable sub-particles was reproducible for this and another pair (not shown) isolated on the basis of color difference.

Figure 5

Figure 5

[PIN+] enhances [CHI+PM] formation. A. NMPM-CSC provides translation termination function to a sup35 mutant containing the medium [PIN+] variant. sup35 medium [PIN+] or [_pin_-] yeast ectopically expressing NMPM-CSC on media containing low or no adenine without uracil (to maintain the plasmid). Cells with the medium [PIN+] variant are shown here. NMPM-CSC is also functional in sup35 mutant yeast with the low [PIN+] variant. B. NMPM-CSC is inactivated in sup35 mutant cells containing the medium or low [PIN+] variant and not in a [_pin_-] sup35 mutant Around 107 sup35 mutant cells containing either medium [PIN+] or [_pin_-] expressing NMPM-CSC were plated onto -Ade-Ura. The frequency of Ade+ colonies was estimated in sup35 yeast with medium and low [PIN+] variants. The Ade+ frequency in sup35 yeast with high and very high [PIN+] variants was not determined as suppression in these strains is enhanced compared with sup35 [_pin_-] yeast.

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