The elongation of yeast prion fibers involves separable steps of association and conversion.
A self-perpetuating change in the conformation of the translation termination factor Sup35p is the basis for the prion [PSI+], a protein-based genetic element of Saccharomyces cerevisiae. In a process closely allied to in vivo conversion, the purified soluble, prion-determining region of Sup35p (NM) converts to amyloid fibers by means of nucleated conformational conversion. First, oligomeric species convert to nuclei, and these nuclei then promote polymerization of soluble protein into amyloid fibers. To elucidate the nature of the polymerization step, we created single-cysteine substitution mutants at different positions in NM to provide unique attachment sites for various probes. In vivo, the mutants behaved like wild-type protein in both the [psi-] and [PSI+] states. In vitro, they assembled with wild-type kinetics and formed fibers with the same morphologies. When labeled with fluorescent probes, two mutants, NMT158C and NME167C, exhibited a change in fluorescence coincident with amyloid assembly. These mutants provided a sensitive measure for the kinetics of fiber elongation, and the lag phase in conversion. The cysteine in the mutant NMK184C remained exposed after assembly. When labeled with biotin and bound to streptavidin beads, it was used to capture radiolabeled soluble NM in the process of conversion. This process established the existence of a detergent-susceptible intermediate in fiber elongation. Thus, the second stage of nucleated conformational conversion, fiber elongation, itself contains at least two steps: the association of soluble protein with preformed fibers to form an assembly intermediate, followed by conformational conversion into amyloid.
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA.
Thomas
T
Scheibel
Jesse
J
Bloom
Susan L
SL
Lindquist
D007700:Kinetics; D011328:Prions / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D011328:Prions