RESUMO
We detail some of the genetic, biochemical, and physical methods useful in studying amyloids in yeast, particularly the yeast prions. These methods include cytoduction (cytoplasmic mixing), infection of cells with prion amyloids, use of green fluorescent protein fusions with amyloid-forming proteins for cytology, protein purification and amyloid formation, and electron microscopy of filaments.
Assuntos
Proteínas Priônicas/genética , Proteínas Priônicas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Eletrônica de Transmissão , Mutação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Prion strains are different self-propagating conformers of the same infectious protein. Three strains of the [PSI] prion, infectious forms of the yeast Sup35 protein, have been previously characterized in our laboratory. Here we report the discovery of a new [PSI] strain, named W8. We demonstrate its robust cellular propagation as well as the protein-only transmission. To reveal strain-specific sequence requirement, mutations that interfered with the propagation of W8 were identified by consecutive substitution of residues 5-55 of Sup35 by proline and insertion of glycine at alternate sites in this segment. Interestingly, propagating W8 with single mutations at residues 5-7 and around residue 43 caused the strain to transmute. In contrast to the assertion that [PSI] existed as a dynamic cloud of sub-structures, no random drift in transmission characteristics was detected in mitotically propagated W8 populations. Electron diffraction and mass-per-length measurements indicate that, similar to the 3 previously characterized strains, W8 fibers are composed of about 1 prion molecule per 4.7-Å cross-ß repeat period. Thus differently folded single Sup35 molecules, not dimeric and trimeric assemblies, form the basic repeating units to build the 4 [PSI] strains.