Bidirectional amyloid fiber growth for a yeast prion determinant

The polymerization of many amyloids is a two-stage process initiated by the formation of a seeding nucleus or protofibril. Soluble protein then assemb les with these nuclei to form amyloid fibers. Whether fiber growth is bidir ectional or unidirectional has been determined for two amyloids, In these c ases, bidirectional growth was established by time lapse atomic-force micro scopy, Here, we investigated the growth of amyloid fibers formed by NM, the prion-determining region of the yeast protein Sup35p, The conformational c hanges in NM that lead to amyloid formation in vitro serve as a model for t he self-perpetuating conformational changes in Sup35p that allow this prote in to serve as an epigenetic element of inheritance in vivo. To assess the directionality of fiber growth, we genetically engineered a mutant of NM so that it contained an accessible cysteine residue that was easily labeled a fter fiber formation. The mutant protein assembled in vitro with kinetics i ndistinguishable from those of the wild-type protein and propagated the her itable genetic trait [PSI+] with the same fidelity. In reactions nucleated with prelabeled fibers, unlabeled protein assembled at both ends. Thus, NM fiber growth is bidirectional. (C) 2001 Elsevier Science Ltd. All rights re served.