Biochemical and genetic methods for characterization of [PIN+]...

The glutamine- and asparagine-rich Rnq1p protein in Saccharomyces cerevisiae can exist in the cell as a soluble monomer or in one of several aggregated, infectious, prion forms called [PIN(+)]. Interest in [PIN(+)] is heightened by its ability to promote the conversion of other proteins into a prion or an aggregated amyloid state. However, little is known about the function of Rnq1p, which makes it difficult to assay the phenotypes associated with its normal vs. prion forms. In this chapter, we describe methods used to detect [PIN(+)] and distinguish between different variations of the prion. Genetic methods are based on the ability of the [PIN(+)] prion to facilitate the appearance of another yeast prion, [PSI(+)], which has an easily detectable phenotype. Biochemical methods exploit the fact that the [PIN(+)] prion exists in the yeast cytosol in the form of large aggregates, composed of SDS-stable subparticles. Sucrose gradient centrifugation, agarose SDS electrophoresis and GFP fusions are used to distinguish between aggregates and subparticles from different [PIN(+)] variants.