Structural definition is important for the propagation of the yeast [PSI+] prion.

Prions are propagated in Saccharomyces cerevisiae with remarkable efficiency, yet we know little about the structural basis of sequence variations in the prion protein that support or prohibit propagation of the prion conformation. We show that certain single-amino-acid substitutions in the prion protein Sup35 impact negatively on the maintenance of the associated prion-based [PSI(+)] trait by combining in vivo phenotypic analysis with solution NMR structural studies. A clear correlation is observed between mutationally induced conformational differences in one of the oligopeptide repeats (R2) in the N terminus of Sup35 and the relative ability to propagate [PSI(+)]. Strikingly, substitution of one of a Gly-Gly pair with highly charged residues that significantly increase structural definition of R2 lead to a severe [PSI(+)] propagation defect. These findings offer a molecular explanation for the dominant-negative effects of such psi-no-more (PNM) mutations and demonstrate directly the importance of localized structural definition in prion propagation.

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions.

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive 'toolbox' available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP (Sc) . Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.

Development of a novel yeast cell-based system for studying the aggregation of Alzheimer's disease-associated Abeta peptides in vivo.

Alzheimer's disease is the most common neurodegenerative disease, affecting approximately 50% of humans by age 85. The disease process is associated with aggregation of the Abeta peptides, short 39-43 residue peptides generated through endoproteolytic cleavage of the Alzheimer's precursor protein. While the process of aggregation of purified Abeta peptides in vitro is beginning to be well understood, little is known about this process in vivo. In the present study, we use the yeast Saccharomyces cerevisiae as a model system for studying Abeta-mediated aggregation in an organism in vivo. One of this yeast's endogenous prions, Sup35/[PSI+], loses the ability to aggregate when the prion-forming domain of this protein is deleted. We show that insertion of Abeta peptide sequences in place of the original prion domain of this protein restores its ability to aggregate. However, the aggregates are qualitatively different from [PSI+] prions in their sensitivity to detergents and in their requirements on trans-acting factors that are normally needed for [PSI+] propagation. We conclude that we have established a useful new tool for studying the aggregation of Abeta peptides in an organism in vivo.