The elongation of yeast prion fibers involves separable steps of association and conversion

Area of Science:

• Molecular Biology
• Biochemistry
• Yeast Genetics

Background:

• The prion [PSI+] in Saccharomyces cerevisiae is a protein-based genetic element driven by self-perpetuating conformational changes in the translation termination factor Sup35p.
• The conversion of soluble Sup35p (NM region) into amyloid fibers occurs via nucleated conformational conversion, involving nucleus formation and subsequent polymerization.

Purpose of the Study:

• To investigate the molecular mechanisms of the polymerization step during nucleated conformational conversion of Sup35p NM into amyloid fibers.
• To characterize the kinetics and intermediate species involved in amyloid fiber elongation.

Main Methods:

• Creation of single-cysteine substitution mutants in the NM region of Sup35p for probe attachment.
• In vivo and in vitro characterization of mutant protein behavior, including assembly kinetics and fiber morphology.
• Fluorescent labeling of mutants (NMT158C, NME167C) to monitor amyloid assembly.
• Biotinylation and streptavidin capture of a specific mutant (NMK184C) to isolate conversion intermediates.

Main Results:

• Mutants behaved like wild-type Sup35p in vivo and assembled into amyloid fibers with similar kinetics and morphologies in vitro.
• Fluorescent mutants showed changes correlating with amyloid assembly, enabling sensitive kinetic measurements of fiber elongation and lag phases.
• The NMK184C mutant, with an exposed cysteine, facilitated the capture of a detergent-susceptible intermediate during fiber elongation.

Conclusions:

• Amyloid fiber elongation in Sup35p conversion involves at least two steps: association of soluble protein with existing fibers to form an intermediate, followed by conformational conversion.
• The identification of a detergent-susceptible intermediate refines our understanding of the prion propagation process at a molecular level.