Strain-specific sequences required for yeast [ PSI ⁺ ] prion propagation

Abstract

[PSI] strains ͉ amyloid ͉ Sup35 A myloid is a generic class of ordered protein aggregates self-assembled by many unrelated proteins (1). X-ray diffraction studies reveal that amyloid fibers are rich in ␤-strands, which run perpendicular to the fiber axis, forming the characteristic cross-␤ pattern (2). One of the most intriguing features of the amyloid is structural polymorphism, whereby the same protein polypeptide can adopt distinct chain-folding patterns to give rise to a variety of cross-␤ structures (3). Structural plasticity of the amyloid underlies the prion strain phenomenon. Selfnucleating amyloid conformers faithfully maintain their distinct folds in the host but sometimes can adjust their structures when encountering different sequences (such as during interspecies transmission), thus giving rise to novel prion strains (4, 5). It is not well understood how distinct cross-␤ folding patterns are determined and what is the mechanism for their interconversion.The yeast prion [PSI] is a self-propagating amyloid aggregate of Sup35, the yeast translation-termination factor whose aggregation in the [PSI ϩ ] state results in enhanced read-through of nonsense mutations (6, 7). Structural polymorphism of Sup35 amyloids gives rise to [PSI] strains that exhibit distinctive nonsense-suppression efficiencies and differential compatibility with Sup35 mutations (8-11). With the ease of biochemical and genetic manipulation, [PSI] strains provide excellent experimental systems to study amyloid polymorphism. However, because in vitro assembled amyloid samples often contain multiple fiber morphologies, great care must be taken to ensure correct interpretation of experimental results. Attributing genetic observations to pure structural causes, however, is complicated by the interplay between amyloid structures and cellular machineries. Here, we design a synthesized approach to avoid these difficulties, combining biochemistry and yeast genetics to examine the amyloid structures of three [PSI] strains: [VH], [VK], and [VL] (11). By individually substituting residues 5-55 of Sup35 with proline and inserting glycine in front of every other residue in this segment, we identify strain-specific Sup35 sequences in vivo. The necessity of these sequences for encoding infectious prion structures is then investigated by infecting yeast with amyloid fibers of Sup35 fragments assembled in vitro. Our goal is to define the minimally required Sup35 sequences that form core structures of the three [PSI] strains to understand how the same polypeptides are recruited in different ways to assemble polymorphic amyloid structures. ResultsProline Substitutions. We employ proline substitutions to map possible ␤-structures along the polypeptide chain. Proline residues with the unique cyclic structure generate bulges in ␤-strands and destabilize periodic amyloid structures. A panel of 51 [psi Ϫ ] haploid mutants, each having a single proline substitution in the amino acid residues 5-55 of the SUP35 allele, was created by homologous recombination. ...

The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies

“…Substitution of proline at amino acid 25 of Sup35 has been suggested to abolish the propagation and infectivity of all three [PSI] variants 33 . To confirm that impairing the cellular properties of the 'prion domain' leads to pathological degradation of TDP-43 proteins, we generated a mTDPSupN mutant, GFP-mTDPSupN*, which contained a G25P substitution in SupN, and assessed its localisation and ability to skip exon 9 of CFTR.…”

Prions in Yeast

“…These stretches could correspond to sequences that initiate intermolecular interactions, resulting in the formation of the cross-b amyloid core. Indeed, stronger [PSI + ] variants need shorter portions of PrD for their propagation, both in vivo (Shkundina et al 2006) and in vitro (Chang et al 2008). The locations of the specificity stretches could control the variant-specific prion patterns by determining the position and size of the amyloid core(s).…”

Prion Variants and Species Barriers Among Saccharomyces Ure2 Proteins

“…Dal5p is the allantoate transporter and ureidosuccinate (USA), the product of aspartate transcarbamylase (Ura2p), is structurally similar to allantoate (Turoscy and Cooper 1987). The presence of the [URE3] prion is thus assayed by the uptake by the Ure2-controlled Dal5p transporter of USA or by activity of 1 ADE2 driven by the DAL5 promoter (Lacroute 1971;Schlumpberger et al 2001 (Shewmaker et al 2006;Baxa et al 2007;Wickner et al 2008a), and different prion variants of [PSI 1 ] differ at least in the extent of the b-sheet structure (Toyama et al 2007;Chang et al 2008). …”