Anthony S. Kowal

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 assembles with these nuclei to form amyloid fibers. Whether fiber growth is bidirectional or unidirectional has been determined for two amyloids. In these cases, bidirectional growth was established by time lapse atomic-force microscopy. Here, we investigated the growth of amyloid fibers formed by NM, the prion-determining region of the yeast protein Sup35p. The conformational changes in NM that lead to amyloid formation in vitro serve as a model for the self-perpetuating conformational changes in Sup35p that allow this protein 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 after fiber formation. The mutant protein assembled in vitro with kinetics indistinguishable from those of the wild-type protein and propagated the heritable 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.

Yeast prion [psi +] and its determinant, Sup35p.

Publisher Summary [ PSI + ] and [ URE3 ] are two non-Mendelian genetic elements of the yeast Saccharomyces cerevisiae that appear to be inherited through an unusual mechanism—the continued propagation of an alternate protein conformation. The protein determinants of these elements, Sup35p for [ PSI + ] and Ure2p for [ URE3 ], have the unique ability to exist in at least two different, stable conformations in vivo . Although the spontaneous generation of one conformer is rare, this alternate form, once acquired, becomes predominant, influencing the other conformer to change states. This self-perpetuation of protein conformation is the key to the non-Mendelian inheritance of both [ PSI + ] and [ URE3 ]. In addition, the [ Het-S ] phenotype of Podospora anserina, another fungus, may be inherited by a similar mechanism. This chapter focuses on both in vivo and in vitro methods used to analyze [ PSI + ], the most extensively studied member of this group. The study of amyloidogenic proteins is complex both in vivo and in vitro. Each assay presented in this chapter provides unique information about the physical state of Sup35p, but these techniques also have inherent pitfalls.