Alternative assembly pathways of the amyloidogenic yeast prion determinant Sup35-NM

Area of Science:

• Biochemistry and Molecular Biology
• Prion Biology
• Yeast Genetics

Background:

• The translation termination factor Sup35 in Saccharomyces cerevisiae is linked to prion phenomena.
• The prion-determining region (NM) of Sup35 is known to assemble into amyloid-like fibers in vitro via nucleated conformational conversion.

Purpose of the Study:

• To investigate alternative assembly pathways of the Sup35 NM region.
• To characterize novel filamentous structures formed by Sup35 NM and their relationship to amyloid fiber formation.

Main Methods:

• In vitro assembly of the Sup35 NM region.
• Characterization of filament structure using biophysical techniques (e.g., diameter, staining properties).
• Assessment of filament stability (SDS and protease resistance) and seeding potential.
• Analysis of the role of oligopeptide repeats in assembly pathway selection.

Main Results:

• An alternative pathway produces NM filaments with smaller diameters than amyloid fibers, characterized by beta-strands and random coils.
• These filaments are not detected by standard amyloid stains (Thioflavin T, Congo Red) and lack SDS/protease resistance.
• Filaments do not self-convert or seed fiber formation, indicating a stable off-pathway structure.
• The number of oligopeptide repeats in Sup35 NM dictates the preference for filament or amyloid fiber formation; fewer repeats favor filaments, more repeats favor fibers.

Conclusions:

• Sup35 NM can adopt a stable, non-amyloid filamentous conformation distinct from amyloid fibers.
• This filamentous form represents an off-pathway assembly product.
• Oligopeptide repeat content is a critical determinant of Sup35's conformational fate, influencing the balance between filament and amyloid fiber formation.