title: In Sup35p filaments (the [PSI+] prion), the globular C-terminal domains are widely offset from the amyloid fibril backbone.
abstract: In yeast cells infected with the [PSI+] prion, Sup35p forms 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.
authors.affiliation: Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal, and Skin Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Baxa
authors.forename: Paul W
authors.initials: PW
authors.lastname: Keller
authors.forename: Naiqian
authors.initials: N
authors.lastname: Cheng
authors.forename: Joseph S
authors.initials: JS
authors.lastname: Wall
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D010454:Peptide Termination Factors / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011328:Prions / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000682:Amyloid; D010454:Peptide Termination Factors; D011328:Prions; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins