Propagation of Mammalian Prions in Yeast.

摘要

The focus of this grant is on development of a novel model system for propagation and quantitation of mammalian prions: the budding yeast Saccharomyces cerevisiae. This unicellular organism offers a number of potential advantages for the study of prion biology, including rapid generation time, ease of culturing, and facile genetics. There is a strong conservation of cellular mechanisms between yeast and mammalian cells, particularly as regards the biogenesis, trafficking and localization of membrane proteins. Thus, although yeast do not express an endogenous PrP-like molecule, there are strong reasons to believe that they possess the molecular machinery to allow propagation of mammalian prions. We hypothesize that the only additional requirement is the provision of a source of membrane-anchored PrPc, the essential substrate for conversion into PrPSc. Using S. cerevisiae strains that express engineered forms of PrPC, we propose to: (1) Determine whether mammalian prions can be propagated in yeast; (2) Develop methods for titering prions in yeast; (3) Characterize the phenotype of prion-infected yeast; and (4) Identify genes that modulate prion propagation in yeast. During years 2 and 3 of the grant, we initiated a new line of investigation that makes use of PrP- expressing yeast (Task 5). This new project is aimed at investigating an intriguing and potentially important hypothesis concerning the normal, physiological function of PrPc.