Direct proof of the amyloid nature of yeast prions [PSI+] and [PIN+] by the method of immunoprecipitation of native fibrils

Abstract

Prions are proteins that can exist in several structurally and functionally distinct states, one or more of which is transmissible. Yeast proteins Sup35 and Rnq1 in prion state ([PSI+] and [PIN+], respectively) form oligomers and aggregates, which are transmitted from parents to offspring in a series of generations. Several pieces of indirect evidence indicate that these aggregates also possess amyloid properties, but their binding to amyloid-specific dyes has not been shown in vivo. Meanwhile, it is the specific binding to the Congo Red dye and birefringence in polarized light after such staining that is considered the gold standard for proving the amyloid properties of a protein. Here we used immunoprecipitation to extract native fibrils of the Sup35 and Rnq1 proteins from yeast strains with different prion status. These fibrils are detected by electron microscopy, stained with Congo Red and exhibit yellow-green birefringence after such staining. All these data show that the Sup35 and Rnq1 proteins in prion state form amyloid fibrils in vivo. The technology of fibrils extraction in combination with standard cytological methods can be used to identify new pathological and functional amyloids in any organism and to analyze the structural features of native amyloid fibrils.

Amyloid Properties of the FXR1 Protein Are Conserved in Evolution of Vertebrates

“…Then, immunoprecipitated material was washed, eluted without boiling, and concentrated by centrifugation. This method makes it possible to isolate native, nondenatured fibrils [ 5 , 21 ]. The results presented in Figure 4 show that FXR1 isolated from different animals forms fibrils that are detected by TEM (transmission electron microscopy).…”

Beyond Amyloid Fibers: Accumulation, Biological Relevance, and Regulation of Higher-Order Prion Architectures

“…Evidence for such a higher-order Sup35 complex in vivo, as well as for other prion-forming proteins including the HET-s prion of Podospora anserina [ 6 ], has been revealed by electron microscopy (EM) methods. For example, morphological size measurements of Sup35 aggregates immunocaptured from [ PSI + ] yeast lysates are consistent with the bundling of fibrils even in the presence of SDS [ 5 ], and morphologically distinct filaments and bundles of them have been immunoprecipitated from [ PSI + ] yeast lysates in the presence of Tween-20 [ 96 ]. Such architecture has also been observed in vivo where Sup35 aggregates are visualized as dot-like by fluorescence microscopy in yeast strains expressing a Sup35 fusion to the green fluorescent protein (GFP) and as bundles of laterally associated fibrils by rapid-freeze EM [ 8 ], where interfibrillar structure has been attributed to possible chaperone binding [ 12 ].…”

“…Thus, it is unclear if these structures are the product of biologically significant regulated events or an artifact of driving cells too far outside of their proteostatic capacity. Similarly, they have only been observed in lysates treated with detergent and/or concentrated by centrifugal force [ 5 , 34 , 56 , 96 ], which may promote association by stripping away partner proteins that shield interaction surfaces or increasing the concentration of fibrils, respectively. To add further evidence to these early observations, there is a clear need for supplementary methods that allow the biophysical characterization of in vivo [ PSI + ] aggregates under endogenous expression conditions, the direct comparison of the species observed under native and detergent conditions, and the correlation of these observations to biological outcomes.…”

“…Upon cell fusion, the prion phenotype emerges concomitantly with Sup35 incorporation into foci at the single-cell level [ 101 , 102 ]. These foci bind to the amyloid-selective dyes thioflavinS and Congo red both in vivo [ 103 ] and ex vivo [ 96 ], providing direct evidence of their amyloid character.…”

Differential Interactions of Molecular Chaperones and Yeast Prions

“…The most studied of these include [PSI + ], [URE3], and [PIN + ] (or [RNQ + ]), the prion forms of the yeast Sup35, Ure2, and Rnq1 proteins, respectively [16,17]. All three of these prions are characterized by the presence of the amyloid aggregates of the corresponding protein [18][19][20][21]. Presence of the [PSI + ] prion causes nonsense suppression, i.e., the ability to read through premature termination codons [11].…”