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 non-Mendelian genetic factor [PSI], which enhances the efficiency of tRNA-mediated nonsense suppression in Saccharomyces cerevisiae, is thought to be an abnormal cellular isoform of the Sup35 protein. Genetic studies have established that the N-terminal part of the Sup35 protein is sufficient for the genesis as well as the maintenance of [PSI]. Here we demonstrate that the N-terminal polypeptide fragment consisting of residues 2-114 of Sup35p, Sup35pN, spontaneously aggregates to form thin filaments in vitro. The filaments show a -sheet-type circular dichroism spectrum, exhibit increased protease resistance, and show amyloid-like optical properties. It is further shown that filament growth in freshly prepared Sup35pN solutions can be induced by seeding with a dilute suspension of preformed filaments. These results suggest that the abnormal cellular isoform of Sup35p is an amyloid-like aggregate and further indicate that seeding might be responsible for the maintenance of the [PSI] element in vivo.Self-propagating protein conformational changes have been proposed to be the cause of transmission of mammalian transmissible spongiform encephalopathies (1, 2), as well as for two yeast non-Mendelian inheritance elements, [PSI] and [URE3] (3-7). In the case of [PSI], an altered conformation of the Sup35 protein (Sup35p), which is the yeast homolog of the eukaryotic translation termination factor eRF3, is thought to be the determinant (3,8). Consistent with this proposal it was observed that the maintenance of [PSI] only requires the N-terminal 114-amino acid domain of Sup35p, and that overproduction of Sup35p or its N-terminal fragment in yeast induces the de novo appearance of [PSI] (9-11). Differential sedimentation and fluorescence microscopic studies further established a correlation between Sup35p coalescence and the appearance of [PSI], suggesting that ordered aggregation converts newly synthesized Sup35p into its like and is thus responsible for the propagation of the [PSI] element (12,13). In this study, we investigate the properties of the Sup35p polypeptide fragment consisting of residues 2-114, Sup35pN. It is found that Sup35pN aggregates to form amyloid-like filaments in vitro. We then show that seeding with preexisting filaments can speed up the formation of filaments from freshly prepared Sup35pN solutions. EXPERIMENTAL PROCEDURESProtein Purification and Filament Preparation. A DNA fragment encoding the N-terminal 114-residue segment of Sup35p, with an extra Met-Gly-Ser 2 -His 6 -Ser 2 -Gly 2 -Ser segment at the N terminus and a stop codon at the C terminus, was obtained by PCR from yeast genomic DNA, using appropriate oligonucleotides. The fragment was inserted into the expression vector pMW172 (14) and the protein was overexpressed in the BLR21(DE3)͞pLysS Escherichia coli strain (Novagen). Cell lysate was prepared in 20 mM Tris⅐HCl buffer (pH 7.9) containing 0.5 M NaCl, 5 mM imidazole, and 6 M guanidine hydrochloride (GdmCl), and loaded onto a Ni 2ϩ -NTA affinity column (Qiagen, Cha...
(1,2) indicates that the differences between strains must somehow be embodied in distinguishable, self-propagating structural features of the infectious amyloid fibrils formed by seeded growth in vitro with recombinant Sup35 prion protein. Induction of prion disease in transgenic mice by injection of amyloid aggregates of recombinant mouse prion protein (3) supports the hypothesis (4) that strains of the mammalian transmissible spongiform encephalopathies are caused by self-propagating misfolded forms of the prion protein.Considering all of the evidence associating both mammalian and yeast prions with infectious amyloid fibers, and the many atomic models proposed for amyloid structures (cf. refs. 5 and 6) since the cross- polypeptide folding pattern (7, 8) was recognized as characteristic of pathological amyloid fibers (9, 10), it is surprising that molecular explanations of how amyloid fibers are actually constructed and why they are so stable remain elusive.Seeded amyloid fibril growth from soluble protein interacting with nucleating amyloid fragments (which is the presumed mechanism of prion propagation in vivo) was first demonstrated in vitro with fibrous insulin (11) before its cross- conformation was identified (12). Now such self-nucleated growth is considered a defining characteristic of amyloid fiber assembly (cf. refs. , which we have determined, are comparable. Correlation of thermal instability with dominance and suppressor strength implies that greater structural lability of infectious amyloid fibrils allows their more efficient fragmentation by cellular machinery to generate the nuclei that specifically sop up Sup35 molecules, thereby selectively propagating the more frangible fibrils.The molecular architecture of the amyloid fibrils formed in our studies (1) by the recombinant Sup35 prion domain fused with GFP is similar to that of in vitro self-assembled Ure2p constructs that have been analyzed in a detailed electron microscopy study by Baxa et al. (23). They established that the N-terminal 70-residue prion-defining domain of this yeast prion protein (17) forms the amyloid core and the globular C-terminal portion could be replaced in fusion constructs by four other globular proteins, including GFP, with no evident effect on the amyloid core structure. Mass per length (mpl) measurements by scanning transmission electron microscopy (STEM) (23) indicated that, within the experimental uncertainty, there might be just one prion molecule for each 4.7-Å cross- repeat period of the amyloid core, independently of the size of the attached C-terminal domain. These results suggested a simple model for the amyloid core in which segments of a sinuously folded N-terminal domain should form a single layer of regularly Abbreviations: STEM, scanning transmission electron microscopy; TEM transmission electron microscope; mpl, mass per length; TMV, tobacco mosaic virus; X- ply, cross- ply.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.