Yeast prions form infectious amyloid inclusion bodies in bacteria

Abstract

Background: Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively.

Results: Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold.

Conclusions: An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.

Figure 1

Solubility properties of recombinant Sup35-NM (left panel) and Ure2p (right panel) proteins. (A and C) Western blot of the soluble and insoluble fractions of cells expressing Sup35-NM and Ure2p at 37°C detected with an anti-histag antibody and quantified by densitometry using the Quantity-One software (Bio-Rad). (B and D) Localization of cytoplasmic IBs at the poles of cells expressing Sup35-NM and Ure2p proteins, as imaged by phase contrast microscopy.

Figure 2

Conformational properties of soluble and aggregated Sup35-NM and Ure2p proteins. Secondary structure of Sup35-NM (A) and Ure2p (B) yeast proteins in their soluble forms and inside the IBs formed at 37°C as determined FT-IR spectroscopy in the amide I region of the spectrum. Empty circles, solid thick lines and solid thin line show the absorbance spectra, the sum of individual spectral components and the inter-molecular β-sheet band, respectively; note that whereas Sup35-NM and Ure2p IBs display the typical inter-molecular β-sheet band at 1625–1630 cm^-1, this signal is low or absent in soluble species. (C) Comparative analysis of the secondary structure of Sup35-NM and Ure2p IBs. Empty circles, solid thick lines and solid thin lines show the absorbance spectra, the sum of individual spectral components and the deconvolved component bands, respectively. (D) Stability of yeast prionogenic IBs in front of Gdn·HCl denaturation at equilibrium monitored by changes in turbidity at 350 nm.

Figure 3

Specific amyloid dyes staining of yeast prion IBs. (A) CR spectral changes in the presence of each IB; displaying the characteristic red-shift in λ_max and intensity increase in CR spectra in the presence of IBs. (B) Difference absorbance spectra of CR in presence and absence of IBs showing the characteristic amyloid band at 541 nm for both yeast proteins. (C) Fluorescence emission spectrum of Th-T in the presence of each IB when excited at 445 nm; note the characteristic maximum at ~ 480 nm upon binding to amyloid structures. (D) Yeast prions IBs stained with Th-S and observed at 40x magnification by phase contrast and fluorescence microscopy displaying the green fluorescence characteristic of amyloid structures.

Figure 4

Aggregation kinetics of Sup35-NM and Ure2p. The aggregation reactions of 20 μM yeast prionogenic proteins were carried out under agitation at 37°C. 2 μM of in vitro formed fibrils (representing 10% of the final protein concentration) or IBs (at a final OD_350nm of 0.125) were used for seeding and cross-seeding assays. The fibrillar fraction of Sup35-NM (A) and Ure2p (B) is represented as a function of time. The formation of Sup35-NM and Ure2p amyloid fibrils are accelerated only in the presence of pre-aggregated homologous protein, either fibrils or IBs.

Figure 5

Sup35-NM and Ure2p amyloid fibrils. Morphology of Sup35-NM (A) and Ure2p (B) amyloid-like aggregates observed at the final time point of the aggregation kinetics. Fibrils in un-seeded, seeded and cross-seeded reactions were monitored by transmission electronic microscopy.

Figure 6

Infectivity of Sup35-NM IBs. Induction of different [PSI+] strains upon transformation of a [psi-] yeast strain with the soluble (S), insoluble (I) fractions of E. coli cells expressing Sup35-NM protein at 18 and 37°C or purified Sup35-NM IBs. After PEG transformation with the indicated material, yeast cells were recovered on SD-URA and randomly selected colonies were spotted onto ¼ YPD plates to identify [PSI⁺] converted colonies. [psi-] and [PSI+] columns correspond to the parental negative and positive control strains. Transformation with the bacterial material induced pink (weak) and to white (strong) [PSI+] phenotypes. Representative images of spots corresponding to distinct strains are shown for each transformed material (see Additional file 1: Table S1 for quantitative data).

Figure 7

Curing the Sup35-NM IBs induced [PSI+] phenotype. Comparison of spots of control [psi-] and [PSI+] strains with cells displaying weak and strong [PSI+] phenotypes obtained by infection with Sup35-NM IBs. Cells were spotted on ¼ YPD before (left) and after (right) culture on a medium containing 3 mM Gdn·HCl.

Figure 8

Solubility and conformational properties of Sup35-NM as a function of the temperature. (A) Western blot of the soluble and insoluble fractions of cells expressing Sup35-NM at 18 and 37°C detected with anti-histag antibody and quantified by Quantity One software. (B) Comparative analysis of the secondary structure of Sup35-NM IBs formed at 18°C and 37°C as determined FT-IR spectroscopy in the amide I region of the spectrum. Empty circles, solid thick lines and solid thin lines show the absorbance spectra, the sum of individual spectral components and the deconvolved component bands, respectively.