In Sup35p filaments (the [PSI+] prion), the globular C‐terminal domains are widely offset from the amyloid fibril backbone

Abstract

Summary In yeast cells infected with the [PSI+] prion, Sup35pforms aggregates and its activity in translation termination is downregulated. Transfection experiments have shown that Sup35p filaments assembled in vitro are infectious, suggesting that they reproduce or closely resemble the prion. We have used several EM techniques to study the molecular architecture of filaments, seeking clues as to the mechanism of downregulation. Sup35p has an N-terminal 'prion' domain; a highly charged middle (M-)domain; and a C-terminal domain with the translation termination activity. By negative staining, cryo-EM and scanning transmission EM (STEM), filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse 65-nm-wide cloud of globular C-domains. In diameter (~8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield~1 subunit per 0.47 nm for N-fibrils, NM-filaments and Sup35p filaments, further supporting the fibril backbone model. The 30 nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations, offering an explanation for the residual Sup35p activity in infected cells, whereby the C-domains remain free enough to interact with ribosomes.

Heritable yeast prions have a highly organized three-dimensional architecture with interfiber structures

“…An alternative explanation for the observed variable spacing is that the apparent separation distance depends on the oblique orientations of the fibrils in these 50-to 80-nm-thick sections. The core fibril diameter was consistent with EM studies of full-length, NM-and N-domain fibrils in vitro, which showed that the middle and Cterminal domains are flexible and can extend considerably from the fibril backbone (49). By analogy, it is likely that the globular GFP domains are also mobile in our fibrils.…”

“…NM fibers have never been observed to form such arrays in vitro (22,27,49,52). Therefore, we reasoned that other cellular factors must dictate the arrangement of the fibril arrays.…”

Prions in Yeast

“…Extended and flexible M domains connect the core to the C domains that have enough space to interact with ribosomes (shown to scale). Adapted from Baxa et al (2011). loops. These non-b-sheet loops can account for the residues that are not within the 4.7-Å distance ( Figure 7B).…”

Functions of Yeast Hsp40 Chaperone Sis1p Dispensable for Prion Propagation but Important for Prion Curing and Protection From Prion Toxicity

“…The lack of suppression of toxicity by Sup35MC argues against such a burden being caused by possible production of proteins with C-terminal extensions due to the read through of stop codons in [PSI 1 ] cells. Recent data suggest that the globular C termini of the Sup35p monomers within amyloid fibers is accessible to interact with ribosomes, which might explain why the considerable depletion of soluble Sup35p in [PSI 1 ] cells does not reduce growth (Baxa et al 2011). It is possible that a function of the Sis1p CTD facilitates such an interaction.…”