Protein-only transmission of three yeast prion strains

Abstract

Key questions regarding the molecular nature of prions are how different prion strains can be propagated by the same protein and whether they are only protein. Here we demonstrate the protein-only nature of prion strains in a yeast model, the [PSI] genetic element that enhances the read-through of nonsense mutations in the yeast Saccharomyces cerevisiae. Infectious fibrous aggregates containing a Sup35 prion-determining amino-terminal fragment labelled with green fluorescent protein were purified from yeast harbouring distinctive prion strains. Using the infectious aggregates as 'seeds', elongated fibres were generated in vitro from the bacterially expressed labelled prion protein. De novo generation of strain-specific [PSI] infectivity was demonstrated by introducing sheared fibres into uninfected yeast hosts. The cross-sectional morphology of the elongated fibres generated in vitro was indistinguishable from that of the short yeast seeds, as visualized by electron microscopy. Electron diffraction of the long fibres showed the 4.7 A spacing characteristic of the cross-beta structure of amyloids. The fact that the amyloid fibres nucleated in vitro propagate the strain-specific infectivity of the yeast seeds implies that the heritable information of distinct prion strains must be encoded by different, self-propagating cross-beta folding patterns of the same prion protein.

The yeast prion protein Ure2: Structure, function and folding

“…In addition, [Het-s] has been identified as a prion of the filamentous fungus Podospora anserina [16,17]. The relative simplicity and tractability of yeast and other fungal systems has allowed convincing proof of the protein-only hypothesis for Het-s [18], Sup35 [19,20] and Ure2 [21].…”

Interplay between Heat Shock Proteins HSP101 and HSA32 Prolongs Heat Acclimation Memory Posttranscriptionally in Arabidopsis

“…PCR products were introduced into pGEM-T Easy vector (Promega) for sequencing confirmation. The cDNA and promoter were ligated in the intermediate plasmid and then subcloned to the YEp195 expression vector by replacing the HindIII/NotI fragment from YEp-CUP1-N1-GFP-T (King and Diaz-Avalos, 2004) to yield plasmids Yep-HSP104p-HSP101-T, Yep-HSP104p-DLT1-1-T, and Yep-HSP104p-DLT1-2-T for complementation analysis.…”

“Prion-proof” for [PIN+]: Infection with In Vitro-made Amyloid Aggregates of Rnq1p-(132–405) Induces [PIN+]

“…pID116, which contains the wild-type RNQ1 sequence, was also used as template for PCR amplification of a C-terminal fragment of RNQ1 encoding a Q/N rich region (aa: 132-405) using primers 1: CGTCATATGGGCCAAAG TATG GGTGCTTCC and 2: CTTTGGGGCCCTACCGCGGGTAGCGGTT. The amplified product was then cloned as an NdeI+ApaI restriction fragment into a recombinant expression vector, pJC45 48 (a kind gift of Joachim Clos, Bernhard Nocht Institute) to obtain a plasmid named pHis 10 …”